EXAMINATION OF FACTORS THAT AFFECT THE RECOVERY AND ANALYSIS OF DNA FROM SPENT CARTRIDGE CASINGS By Rebecca Ray A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Criminal Justice Master of Science 2015 ABSTRACT EXAMINATION OF FACTORS THAT AFFECT THE RECOVERY AND ANALYSIS OF DNA FROM SPENT CARTRIDGE CASINGS By Rebecca Ray Crimes involving firearms are extremely common, and it is therefore important that law enforcement be abl e to identify the individual who fired a weapon. Previous researchers have shown that DNA recovered from spent casings can be used to identify the loader of a firearm, however success has been highly variable, largely due to the wide variety of factors tha t have the potential to influence DNA on spent casings. The goal of this research was to test several such factors, including loading/firing order, pre - processing spent casings for fingerprints, cartridge caliber, swabbing strategy, and analysis technique . Forty caliber c artridges loaded by volunteers were fired and the casings were collected, two - thirds of which were fumed with cyanoacrylate to examine the influence of fuming on DNA recovery and analysis. Volunteers also loaded 0.45 and 0.22 caliber cartri dges, which were swabbed individually or cumulatively in sets of three. DNA was extracted, quantified, and STRs were amplified using a MiniFiler a nd/or Fusion amplification kit. The HV1 and HV2 regions of mtDNA were also amplified. Cyanoacrylate fuming wa s found to have a negative effect on DNA recovery and analysis. Significantly more DNA was recovered from 0.45 caliber casings than from 0.22 caliber casings. Cumulative swabbing yielded more DNA and handler alleles than individual swabbing, although it al so resulted in a higher number of mixtures. Fusion outperformed MiniFiler as an STR amplification kit, and m tDNA was successfully sequenced for all casings tested. Loading order was the only factor that did not have a significant effect, and as such all o ther variables should be strongly considered when DNA analysis from spent casings is undertaken. Copyright by REBECCA RAY 2015 iv ACKNOWLEDGEMENTS First, I would like to thank Dr. Foran for all of his guidance and patience throughout my time in graduate school. I would also like to thank Dr. Edmund McGarrell and Dr. Penny Fischer for their willingness to serve on my thesis committee , and for all of their suggestions . I am very grateful to all of the employees at the Michigan State Police Forensic Laborat ory in Lansing, MI who volunteered to load cartridges for this research, especially the firearms unit who not only provided DNA samples but who also supplied the weapons and fired them. I would like to thank Ashley Mottar for optimizing the extraction proc edures used in this research, and for aiding me in both of my sample collections. I also appreciate Tim Antinick, who helped keep us organized provided DNA s amples . The support and encouragement of the current graduate students in the forensic biology program is also appreciated, including Ellen Jesmok, Kait Germain, and Alyssa Badgley. Lastly, I am extremely grateful for the constant support and encouragement from my friends and family throughout the last two years. v TABLE OF CONTENTS LIST OF TABLES ................................ ................................ ................................ ................... vii LIST OF FIGURES ................................ ................................ ................................ ................. xiv INTRODUCTION ................................ ................................ ................................ ...................... 1 Cartridge Casings: Class and Individual Characteristics ................................ ................... 2 Fingerpr ints on Spent Cartridge Casings ................................ ................................ .......... 3 Cyanoacrylate Fuming: Effect on DNA Recovery and Analysis ................................ ....... 5 Touch DNA: Identification of an Indi vidual ................................ ................................ ..... 6 DNA from Spent Cartridge Casings ................................ ................................ ................. 8 Loading and Firing Order ................................ ................................ ................... 11 STR Analysis: Human Identification ................................ ................................ ............. 11 MtDNA Analysis: Human Identification ................................ ................................ ........ 12 Goals of This Study ................................ ................................ ................................ ....... 13 METHODS ................................ ................................ ................................ ............................... 15 Cartridge Casing Collection ................................ ................................ ........................... 15 Cyanoacrylate Fuming of Collection 1 Casings ................................ .............................. 16 DNA Isolation and Digestion ................................ ................................ ......................... 17 DNA Isolation and Digestion: Collection 1 ................................ ........................ 18 DNA Isolation and Digestion: Collection 2 ................................ ........................ 18 DNA Isolation and Digestion: Buccal Swabs ................................ ..................... 18 Organic DNA Extraction ................................ ................................ ............................... 19 DNA Quantitation Using Real - Time PCR ................................ ................................ ...... 19 STR Analysis of Spent Casing DNA ................................ ................................ .............. 21 PowerPlex ® Fusion: STR Amplification ................................ ............................. 21 ................................ ................................ ........ 22 MtDNA Sequencing of Spent Casings ................................ ................................ ........... 22 Amplification of MtDNA from Reagent Blanks ................................ ............................. 25 Statistical Analysis ................................ ................................ ................................ ........ 25 RESULTS ................................ ................................ ................................ ................................ . 26 Loading, Collecting, Cyanoacrylate Fuming, and DNA Isolation of Cartridge Casings .. 26 Reagent Blanks: Quantitation and MtDNA Amplifica tion Results ................................ . 27 Collection 1: Effect of Fuming on DNA Recovery and Analysis from Spent Casings .... 27 Comparison of DNA Yields from Fu med and Non - Fumed Casings .................... 27 .......................... 30 Comparison of Fusion STR Profiles from Fu med and Non - Fumed Casings ........ 31 Influence of Loading/Firing Order on DNA Yields and STRs ............................ 36 Correlation Between DNA Concentration and STR Profiles ............................... 37 Collection 2: Effect of Swabbing Strategy and Cartridge Caliber on DNA Recovery and Analysis ................................ ................................ ................................ ........................ 37 vi Comparison of DNA Yields Based on Swabbing Strategy and Cartridge Caliber 37 Influence of Handling 0.45 or 0.22 Caliber Cartridges First on DNA Yields from Spent Casings ................................ ................................ ................................ .... 40 Comparison of Fusion STR Profiles ................................ ................................ ... 41 Influence of Loading/Firing Order on DNA Yields and STRs ............................ 46 Correla tion Between DNA Concentration and STR Profiling ............................. 47 Comparison of MtDNA Profiles ................................ ................................ ......... 47 Comparison of STR and mtDNA Results ................................ ........................... 52 DISCUSSION ................................ ................................ ................................ ........................... 54 CONCLUSIONS ................................ ................................ ................................ ....................... 80 APPENDICES ................................ ................................ ................................ .......................... 81 APPENDIX A. ASSIGNMENT OF FUMING METHODS AND SWABBING STRATEGIES FOR COLLECTIONS 1 AND 2 ................................ ............................ 82 APPENDIX B. DNA QUANTITIES RECOVERED FROM SPENT CARTRIDGE CASINGS FRO M COLLECTION 1 ................................ ................................ .............. 88 APPENDIX C. COMPARISON OF FUSION AND MINIFILER STR PROFILES ... 93 APPENDIX D. COMPARISON OF HANDLER AND NON - HANDLE R ALLELES AMPLIFIED WITH MINIFILER AND FUSION ................................ .................... 122 APPENDIX E. FUSION STR PROFILES FROM COLLECTION 1. .......................... 124 APPENDIX F. HANDLER AND NON - HANDLER ALLELES AMPLIFIED WITH FUSION FROM COLLECTION 1 ................................ ................................ .............. 142 APPENDIX G. DNA QUANTITIES RECOVERED FROM SPENT CARTRIDGE CASINGS FROM COLLECTION 2 ................................ ................................ ............ 146 APPENDIX H. FUSION STR PROFILES FROM COLLECTION 2 ........................... 153 APPENDIX I. HANDLER AND NON - HANDLER ALLELES AMPLIFIED WITH FUSION FROM COLLECTION 2 ................................ ................................ .............. 194 APPENDIX J. MTDNA PROFILES GENERATED FROM SPENT CARTRIDGE CASINGS ................................ ................................ ................................ ................... 202 APPENDIX K. COMPARISON OF HANDLER AND NON - HANDLER ALLELES AMPLIFIED WITH FUSION AND MTDNA PROFILE CLASSIFICATIONS .......... 212 REFERENCES ................................ ................................ ................................ ....................... 215 vii LIST OF T ABLES Table 1. Primer, probe, and IPC templ ate sequences for real - time PCR. HEX and 6FAM are fluorescent dyes attached to the 5 end of the probes. BHQ1 and IABkFQ (Iowa Black ® FQ) are quenchers attached to the 3 end of the probes. ZEN is an internal quencher. The Alu primers and probe were design ed by Nicklas and Buel (2005). The IPC primers, probe, and template were designed by Lindquist et al. (2011). ................................ ................................ ........................... 20 Table 2. Primers used to amplify and sequence mtDNA from casings and reference samples. All samples were amplified with F15989, R16410, F15, and R499. F16190 and R285 were used when sequences failed or were not suitable for analysis. ................................ ............................ 23 Table 3. Description of categories used in mtDNA analysis. ................................ ..................... 24 Table 4. Descriptive statistics of quantitation results of fumed and non - fumed casings. Data for the non - fumed casings taken from Mottar (2014). ................................ ................................ ..... 29 Table 5. Pairwise comparisons of DNA yields (pg) for fumed and non - fumed casings (Mann - - fumed casings taken from Mottar (2014). ................................ ................................ ................................ ................................ ...... 30 Table 6. M edian number of handler (H) alleles, non - handler (NH) alleles, and percent profiles produced from fumed and non - - fumed casings taken from Mottar (2014). ................................ ................................ .................. 31 Table 7. Mann - Whitney pairwise comparisons between the number of handler (H) alleles, non - - fumed casings taken from Mottar (2014). .................... 31 Table 8. Descriptive statistics of the number of handler alleles (H), non - handler alleles (NH), and percent profile from fumed and non - fumed casings. Data for the non - fumed casings taken from Mottar (2014). ................................ ................................ ................................ .................. 33 Table 9. Pairwise comparisons of the number of handler alleles (H) and non - handler alleles (NH) and the percent profiles using the Mann - Data for the non - fumed casings taken from Mottar (2014). ................................ ........................ 35 Table 10. Median DNA yields (pg) and number of handler (H) alleles from spent casings. Casing number 1 3 refers to casings from the 1 st , 2 nd , and 3 rd cartridges fired, 4 6 refers the 4 th , 5 th , and 6 th , etc. C artridges 10 12 were sometimes loaded into magazine 1 and fired. Data for the non - fumed casings taken from Mottar (2014). ................................ ................................ 36 Table 11. Mann - Whitney pairwise comparisons between DNA yields an d number of handler (H) for the non - fumed casings taken from Mottar (2014). ................................ ................................ 36 viii Table 12. The c handler alleles generated using Fusion for fumed and non - fumed casings. Data for the non - fumed casings taken from Mottar (2014). ................................ ................................ ............................. 37 Table 13. Descriptive statistics of quantitation results based on swabbing strategy and cartridge caliber. Individual/cumulative refers to the swabbing strategy and 0.45/0.22 refers to the caliber. ................................ ................................ ................................ ................................ ................. 39 Table 14. Mann - 0.0083). Individual/cumulative refers to the swabbing strategy, and 0.45/0.22 refers to the caliber. ................................ ................................ ................................ ................................ ...... 40 Table 15 . Mann - Whitney pairwise comparisons between the DNA yields (pg) from casings handled first and second based on swabbing strategy and cartridge caliber (Bonferroni corrected fers to the caliber. ................................ ................................ ................................ ................................ ...... 41 Table 16. Descriptive statistics of the number of handler alleles (H), non - handler alleles (NH), and percent STR profile based on swabbing strategy and cartridge calibe r. Individual/cumulative refers to the swabbing strategy and 0.45/0.22 refers to the caliber. ................................ ............. 43 Table 17. Pairwise comparisons of the number of handler alleles (H), non - handler alleles (NH), and percent profile (Mann - refers to the swabbing strategy and 0.45/0.22 refers to the caliber. ................................ ............. 45 Table 18. Median DNA yiel ds (pg) and number of handler (H) alleles from spent casings. Casing number 1 3 refers to the casings from the 1 st , 2 nd , and 3 rd cartridges fired, 4 6 refers to the 4 th , 5 th , and 6 th , etc. Individual/cumulative refers to the swabbing strategy and 0.45/ 0.22 refers to the caliber. ................................ ................................ ................................ ............................ 46 Table 19. Mann - Whitney pairwise comparisons of DNA yields (pg) and number of handler (H) alleles between casings from the first and last fired cartridges from ea ch magazine based on refers to the swabbing strategy and 0.45/0.22 refers to the caliber. ................................ ............. 46 Tab le 20. handler alleles generated using Fusion for each caliber and swabbing strategy. Individual/cumulative refers to the swabbing strategy, and 0.45/0.22 refers to the cal iber. ......... 47 Table A1. Assignment of fuming methods for Collection 1. ................................ ...................... 82 Table A2 . Assignment of swabbing strategies for Collection 2. ................................ ................ 85 Table B1. Quantitation re sults of casings fu med at MSU from Collection 1. ............................. 88 Table B2. Quantitation results of casings fu med at MSP from Collection 1. .............................. 89 ix Table B3. Quantitation results of non - fum ed casings from Collection 1. ................................ ... 91 Table C1. from spent cartridge ca sings loaded by individual U. ................................ ................................ ................................ ............................. 93 Table C2. ings loaded by individual MM. ................................ ................................ ................................ ......................... 95 Table C3. individual S. ................................ ................................ ................................ .............................. 96 Table C4. ings loaded by individual VV. ................................ ................................ ................................ .......................... 98 Table C5. ings loaded by individual V. ................................ ................................ ................................ ............................. 99 Table C6. Fusion and ngs loaded by individual HH. ................................ ................................ ................................ ........................ 104 Table C7. ings loaded by individual L. ................................ ................................ ................................ ............................ 105 Table C8. pent cartridge casi ngs loaded by individual OO. ................................ ................................ ................................ ........................ 108 Table C9. ngs loaded by individual XX. ................................ ................................ ................................ ........................ 109 Table C10. in div idual N. ................................ ................................ ................................ ........................... 112 Table C11. ings loaded by individual B. ................................ ................................ ................................ ........................... 113 Table C12. ngs loaded by individual WW. ................................ ................................ ................................ ....................... 116 Table C13. Fusion ngs loaded by individual SS. ................................ ................................ ................................ .......................... 117 Table C14. ings loaded by individual Y. ................................ ................................ ................................ ........................... 118 Table C15. rom spent cartridge casi ngs loaded by individual II. ................................ ................................ ................................ ........................... 120 x Table D1. Comparison of the number of handler alleles (H) and percent profile produced by n from casings fumed at MSU. ................................ ............................. 122 Table D2. Comparison of the number of handler alleles ( H) and percent profile produced by usi on from casings fumed at MSP ................................ ............................... 123 Table D3. Comparison of the number of handler alleles (H) and percent profile produced by sion from non - fumed casings. ................................ ................................ ... 123 Table E1. Fusion profiles g enerated from spent cartridge casings loaded by individual U. ...... 125 Table E2. Fusion profiles generated from spent cartridge casings loaded by individual MM. .. 126 Table E3. Fusion profiles generated from spent cartridge casings loaded by individual S. ....... 127 Table E4. Fusion profiles generated from spent cartridge casings loaded by individual VV. ... 128 Table E5. Fusion profiles generated from spent cartridge casings loaded by individual V. ...... 129 Table E6. Fusion profiles generated from spent cartridge casings lo aded by individual HH. ... 130 Table E7. Fusion profiles generated from spent cartridge casings loaded by individual L. ...... 131 Table E8. Fusion profiles generated from spent cartridge casings loaded by individual OO. ... 132 Table E9. Fusion profiles generated fro m spent cartridge casings loaded by individual T. ...... 133 Table E10. Fusion profiles generated from spent cartridge casings loaded by individual XX. . 134 Table E11. Fusion profiles generated from spent cartridge casings loaded by individual N. .... 135 Table E12. Fusion profiles generated from spent cartridge casings loaded by individual B. .... 136 Table E13. Fusion profiles generated from spent cartridge casings loaded by individual D. .... 137 Table E14. Fusion profiles generated from spent cartridge casings loaded by individual WW. 138 Table E15. Fusion profiles generated from spent cartridge casings loaded by individual SS. .. 139 Table E16. Fusion profiles generated from spent cartridge casings loaded by individual Y. .... 140 Table E17. Fusion profiles generated from spe nt cartridge casings loaded by individual II. .... 141 Table F1. Summary of the number of handler (H) alleles, non - handler (NH) alleles, and percent profile produced using Fusion for all samples in Collection 1. ................................ ................. 142 xi Table G1. Quantitation results for individu ally swabbed 0.45 casings from Collection 2. ....... 146 Table G2. Quantitation results of individually swabbed 0.22 casings from Collection 2. ......... 147 Table G3. Quantitation results for cumulatively swabbed 0.45 casings from Collection 2. ...... 149 Table G4. Quantitatio n results for cumulatively swabbed 0.22 casings from Collection 2. ...... 151 Table H1. Fusion profiles generated from spent cartridge casings loaded by individual SSS. .. 154 Table H2. Fusion profiles generated from spent cartridge casings loaded by individual NN . ... 156 Table H3. Fusion profiles generated from spent cartridge casings loaded by individual ZZZ. . 158 Table H4. Fusion profiles generated from spent cartridge casings loaded by individual B. ...... 160 Table H5. Fusion profiles generated from spent cartridge ca sings loaded by individual BBB. . 162 Table H6. Fusion profiles generated from spent cartridge casings loaded by individual C. ...... 164 Table H7. Fusion profiles generated from spent cartridge casings loaded by individual AA. ... 166 Table H8. Fusion profiles gene rated from spent cartridge casings loaded by individual A. ...... 168 Table H9. Fusion profiles generated from spent cartridge casings loaded by individual J. ....... 170 Table H10. Fusion profiles generated from spent cartridge casings loaded by individual KKK. ................................ ................................ ................................ ................................ ...... 172 T able H11. Fusion profiles generated from spent cartridge casings loaded by individual JJJ. .. 174 Table H12. Fusion profiles generated from spent cartridge casings loaded by individual I. ..... 176 Table H13. Fusion profiles generated from spent cartridge casings loaded by individual YYY. ................................ ................................ ................................ ................................ ...... 178 Table H14. Fusion profiles generated from spent cartridge casings loaded by individual EE. .. 180 Table H15. Fusion profiles generated from spent cartridge casings loaded by individual JJ. ... 182 Table H16. Fusion profiles gener ated from spent cartridge casings loaded by individual DDD. ................................ ................................ ................................ ................................ ...... 184 Table H17. Fusion profiles generated from spent cartridge casings loaded by individual VVV. ................................ ................................ ................................ ................................ ...... 186 xii Table H18. Fusion profiles generated from spent cartridge casings loaded by individual XXX. ................................ ................................ ................................ ................................ ...... 18 8 Table H19. Fusion profiles generated from spent cartridge casings loaded by individual R. .... 190 Table H20. Fusion profiles generated from spent cartridge casings loaded by individual OOO. ................................ ................................ ................................ ................................ ...... 192 Table I1. Summary of number of handler (H) alleles, non - handl er (NH) alleles, and percent profile produced using Fusion from the individually swabbed 0.45 casings from Collection 2. 194 Table I2. Summary of number of handler (H) alleles, non - handler (NH) alleles, and percent profile produced using Fusion from the i ndividually swabbed 0.22 casings from Collection 2. 196 Table I3. Summary of number of handler (H) alleles, non - handler (NH) alleles, and percent profile produced using Fusion from the cumulatively swabbed 0.45 casings from Collection 2. ................................ ................................ ................................ ................................ ............. 198 Table I4. Su mmary of number of handler (H) alleles, non - handler (NH) alleles, and percent profile produced using Fusion from the cumulatively swabbed 0.22 casings from Collection 2. ................................ ................................ ................................ ................................ ............. 200 Table J1 . MtDNA profiles generated from spent cartridge casings loaded by individ ual NN. . 202 Table J2. MtDNA profiles generated from spent cartridge casings loaded by individual ZZZ. 203 Table J3. MtDNA profiles generated from spent cartridge casings loaded by individual B. .... 203 Table J4. MtDNA profiles generated from spent cartridge casings loaded by individual BBB. 204 Table J5. MtDNA profiles generated from spent cartridge casings loaded by individual C. .... 204 Table J6. MtDNA profiles generated from spent cartridge casings loaded by individual AA. . 204 Table J7. MtDNA profiles gene rated from spent cartridge casings loaded by individual A. .... 205 Table J8. MtDNA profiles generated from spent cartridge casings loaded by individual J. ..... 206 Table J9. MtDNA profiles generated from spent cartridge casings loaded by individual KKK. ................................ ................................ ................................ ................................ ...... 206 Tabl e J10. MtDNA profiles generated from spent cartridge casings loaded by individual JJJ. . 207 Table J11. MtDNA profiles generated from spent cartridge casings loaded by individual I. .... 207 xiii Table J12. MtDNA profiles generated from spent cartridge casings loade d by individual YYY. ................................ ................................ ................................ ................................ ...... 208 Table J13. MtDNA profiles generated from spent cartridge casings loaded by individual EE. 208 Table J14. MtDNA profiles generated from spent cartridge casings loaded by individual JJ. .. 209 Table J15. MtDNA profiles generated from spent cartridge casings loaded by individual DDD. ................................ ................................ ................................ ................................ ...... 209 Table J16. MtDNA profiles generated from spent cartridge casings loaded by individual VVV. ................................ ................................ ................................ ................................ ...... 209 Table J17. MtDNA profiles generated from spent cartridge casings loaded by individual XXX. ................................ ................................ ................................ ................................ ...... 210 Table J1 8. MtDNA profiles generated from spent cartridge casings loaded by individual R. .. 210 Table J19. MtDNA profiles generated from spent cartridge casings loaded by individual OOO. ................................ ................................ ................................ ................................ ...... 211 Table K1. Number of alleles both consistent with (H) and not consistent wi th (NH) the handler and the corresponding mtDNA profile result. ................................ ................................ .......... 212 xiv LIST OF FIGURES Figure 1 . Example of a non - fumed (left) and fumed (right) casing covered in a white residue. .. 26 Figure 2 . Median DNA (pg) yields among the fumed and non - fumed casings. Data for the non - fumed casings taken from Mottar (2014). ................................ ................................ .................. 28 Figure 3. Box plots displaying the distribution of the DNA yields (pg) of fumed and non - fumed casings. The box encompasses the interquartile range (the distance between the lower and upper quartiles), with the line through the box symbolizing the median . The mean is represented by a red +, extreme outliers are represented by x, mild outliers are represented by °, and maximum/minimum values are represented by blue squares. The whiskers represent the maximum/minimum values that are not outliers. The MSU - fu med casings contained an extreme outlier at 1420.2 pg that is not shown. Data for the non - fumed casings taken from Mottar (2014). ................................ ................................ ................................ ................................ ................. 29 Figure 4. Median number of handler (H) and non - handler (NH) alleles amplified from fumed and non - fumed casings using Fusion. Data for the non - fumed casings taken from Mottar (2014). ................................ ................................ ................................ ................................ ................. 32 Figure 5. Box plots displaying the distribution of the number of h andler alleles from fumed and non - fumed casings. The box encompasses the interquartile range (the distance between the lower and upper quartiles), with the line through the box symbolizing the median. The mean is represented by a red +, extreme outliers ar e represented by x, mild outliers are represented by °, and maximum/minimum values are represented by blue squares. The whiskers represent the maximum/minimum values that are not outliers. Data for the non - fumed casings taken from Mottar (2014). ................................ ................................ ................................ ........................... 34 Figure 6. Box plots displaying the distribution of the number of non - handler alleles from fumed and non - fumed casings. The box encompasses the interquartile range (the distance between the lower and upper quartiles), with the line through the box symbolizing the median. The mean is represented by a red +, extreme outliers are represented by x, mild outliers are represented by °, and maximum/minimum values are represented by blue squares. The whiskers represent the maximum/minimum values that are not outliers. Data for the non - fumed casings taken from Mottar (2014). ................................ ................................ ................................ ........................... 35 Figure 7. Median DNA yield (pg) based on swabbing strategy and cartridge caliber. Individual/cumulative refers to the swabbing strategy and 0.45/0.22 refers to the caliber. .......... 38 Figure 8. Box plots displaying the distribution of the DNA yield (pg) based on swabbing strategy and cartridge caliber. The box encompasses the interquartile range (the distance between the lower and upper quartiles), with the line through the box symbolizing the median. The mean is represented by a red +, extreme outliers are represented by x, mild outliers are represented by °, and maximum/minimum values are represented by blue squares. The whiskers represent the xv maximum/minimum values that are not outliers. Individual/cumulative refers to the swabbing strategy and 0.45/0.22 refers to the ca liber. ................................ ................................ ................ 39 Figure 9. Median DNA yields (pg) of 0.45/0.22 caliber casings based on swabbing strategy. Individual/cumulative refers to the swabbing strategy, and 0.45/0.22 refers to the caliber. ......... 41 Figure 10. Median number of handler (H) and non - handler (NH) alleles based on swabbing strategy and cartridge caliber. Individual/cumulative refers to the swabbing strategy and 0.45/0.22 re fers to the caliber. ................................ ................................ ................................ ... 42 Figure 11. Box plots displaying the distribution of the number of handler alleles based on swabbing strategy and cartridge caliber. The box encompasses the interquartil e range (the distance between the lower and upper quartiles), with the line through the box symbolizing the median. The mean is represented by a red +, mild outliers are represented by °, and maximum/minimum values are represented by blue squares. The whis kers represent the maximum/minimum values that are not outliers. Individual/cumulative refers to the swabbing strategy and 0.45/0.22 refers to the caliber. ................................ ................................ ................ 44 Figure 12. Box plots displaying t he distribution of the number of non - handler alleles based on swabbing strategy and cartridge caliber. The box encompasses the interquartile range (the distance between the lower and upper quartiles), with the line through the box symbolizing the median. T he mean is represented by a red +, extreme outliers are represented by x, mild outliers are represented by °, and maximum/minimum values are represented by blue squares. The whiskers represent the maximum/minimum values that are not outliers. Individual/cu mulative refers to the swabbing strategy and 0.45/0.22 refers to the caliber. ................................ ............. 45 Figure 13. Classification of mtDNA profiles for all samples (n=96). ................................ ........ 48 Figure 14. MtDNA containing a mixture in which a major contributor could be determined (major = ACTTACC, minor = ACTCACC). Similar peak heights were observed in both strands. ................................ ................................ ................................ ................................ ................. 48 Figure 15. MtDNA containing a mixture in which a major contributor could not be determined. Similar peak heights were observed in both strands. ................................ ................................ .. 49 Figure 16. Classification of mtDNA profiles for the high (A), medium (B), and low (C) DNA quantity samples (n = 32 for each chart). ................................ ................................ ................... 50 Figure 17. Percentage of each mtDNA profile category for cumulatively swabbed casings (A) an d individually swabbed casings (B) (n = 48 for each chart). ................................ ................... 51 Figure 18. Percentage of each mtDNA profile category for 0.45 caliber casings (A) and 0.22 caliber casings (B) (n = 48 for each c hart). ................................ ................................ ................ 52 xvi Figure 19. Medium number of handler (H) and non - handler (NH) STR alleles for each classification of mtDNA profile. Only three samples were classified as mixed - inconsistent, which are not included in this graph. ................................ ................................ ......................... 53 1 INTRODUCTION Hundreds of thousands of crimes are committed using firearms every year in the United States (National Institute of Justice, 2013). From 1993 to 2011, approximately 70% of all h omicides and 6 9% of all non - fatal violent crimes were committed using a firearm, the vast majority of which involved a handgun (Planty and Truman, 2013). Because firearm violence is so prevalent, it is crucial that law enforcement be able to identify th e person who loaded and fired a weapon during the commission of a crime. There are several methods for identifying the shooter when the weapon used in a criminal act is recovered from a scene . Fingerprints may be lifted from the firearm, although they are not commonly recovered (Saferstein, 2005). It is also possible to use serial numbers to help identify the shooter or owner of a gun. Every firearm is required by law to have a serial number, which is associated with the name of the owner when it is purcha s ed (Saferstein, 2005), however c riminals will commonly attempt to remove the serial number in an effort to make identifying the weapon more d ifficult. Forensic examiners can restore obliterated serial numbers through a variety of methods, including the us e of magnetic particles and acid etching, although this is not always successful (Maiden, 2009). An additional challenge in using a serial number is that many firearms involved in crimes are not obtained legally, and therefore are not properly registered. A survey of state prison inmates revealed that only about 10% of firearms used to commit a crime were purchased from a legal source, while 40% were illegally obtained and 37% were acquired from family or friends (Planty and Truman, 2013). If the weapon is not recov ered from a crime scene, or if the serial number cannot be used to identify the owner of the weapon, spent cartridge casings collected from the scene may provide valuable information that could be used to link an individual to both the crime and w eapon. 2 Cartridge Casings: Class and Individual Characteristics elements: the projectile, the primer, the gunpowder, and the casing. The projectile for most pistol ammu nition is a single bullet, while i n shotgun ammunition the cartridge, or shell, contain s a number of projectiles called shot , or a single slug. The primer is the component of the cartridge that is struck to generate a spark, which ignites the gunpowder and causes the gun to fire. Black powder was originally used in firearms, but modern weapons use smokeless powder (Saferstein, 2005). The casing is the metal container that surrounds the projectile, primer, and powder. Casings are commonly made of brass, alth ough nickel , aluminum , and steel are also used (Saferstein, 2005). When a pistol or rifle is fired, the casing is ejected. I t might be easy for a criminal to flee a crime scene with the firearm in hand, but locating and collecting cartridge casings after t heir ejection may be d ifficult and time consuming, so casings are often left behind and collected by crime scene investigators as evidence. There are several class and individualizing features of cartridges that are used to associate them with a gun. One class level trait is the caliber of the ammunition, or the measurement of its width. For example, a 0.22 caliber bullet has a diameter of approximately 0.22 in, and the width of the casing may be the same or slightly wider. The caliber of the ammunition c orrelate s with the caliber of the weapon designed to fire it, which help s firearms examiners identify the type of gun used to commit a crime. Other class characteristics of casings that can associate them with a particular make and model include the type o f cartridge (rimfire or centerfire), type of rim (rimmed, semirimmed, rimless, belted, or rebated), shape and location of the firing pin impression, and presence and location of extr actor and ejector marks, which are examined using a low - power microscope ( Saferstein, 2005). 3 Individual characteristics are then examined to connect a casing to a particular firearm, rather than a class of firearms . For example, marks on the casing such as firing pin impressions, breech face marks, and ejector marks can all be used to associate the casing with a pa rticular gun ). A firearm examiner first test fire s the suspected firearm to obtain known car tridge casings for comparison, and t he known and unknown samples are then evaluated using a comparison microscope. However, while this me thod is useful for associating a cartridge casing with a gun, it cannot id entify the person who fired it. Fingerprints on Spent Cartridge Casings Fingerprints are placed on the casing surface when a cartridge is loaded into a magazine, which can be very useful as no two individuals are thought to share the same fingerprints. Such prints are used to identify the individual who loaded t he weapon, if they are recovered. This can provide valuable evidence in a criminal investigation, although the person who l oaded a firearm is not always the one who fires it . Given (1976) first examined the effect that firing had on the recovery of identifiable fingerprints from cartridge casings. Six volunteers handled sets of brass and nickel - plated 0.38 caliber cartridges, half of which were fired using a Smith & Wesson model 19, .357 Combat Magnum. The time between the handling and firing of cartridges varied, as did the time between firing and recovery of prints, which was attempted using black fingerprint powder. Time did not cause substantial degradation of the fingerprints, though it was proposed that the evaporation of water over time resulted in a decrease in the adherence of powder to the prints. Degradation of fingerprints as a result of firing was primarily due to b lowback of hot gasses along surfaces of the casing not tightly sealed against the chamber wall. Another factor that influenced fingerprint detail was friction between the casing and gun barrel. 4 When the gunpowder ignites, internal pressure causes the casin g to expand. Fingerprints were most commonly recovered from near the head of the casing (by the rim), possibly because it is where the metal of the casing is the thickest and therefore experiences less expansion and friction. Additionally, it was noted tha t while prints were recovered from nickel cartridges, they were recovered more successfully from brass cartridges , of which nearly all yielded an identifiable print. l atent fingerprints on spent casings. Fingerprints were rolled onto 0.38 caliber cartridges, which were fired after 1 hour using a 0.38 Webley revolver. Eleven methods were used to attempt to visualize fingerprints on the cartridge casings, including those that react both with the fingerprint and the substrate. The casings were evaluated for fingerprints, which were classified as identifiable if eight or more ridge characteristics were present. Of the 11 methods tested, vacuum cyanoacrylate (with fluorescent staining) and selenious acid treatments were most effective. However , the authors were not able to replicate the success of Given (1976), and noted that of the 104 murder/attempted murder cases in which the vacuum cyanoacrylate - fluorescent staining method was used in Northern Ireland between the years 1992 and 1993, only two yielded useable prints ; one resulted from handling by investigators and the other was not identified. The lack of success in obtaining useable fingerprints from spen t cartridge casin gs was further demonstrated by Spear et al. (2005). Forty eight fingerprints, characterized as bloody, oily, or sweaty, were intentionally placed on cartridges, half of which were fired. Bloody prints were processed using amido black, while sweaty and oily prints were visualized using cyanoacrylate fuming followed by rhodamine 6G dye. Five useable prints were obtained from the unfired cartridges, of which two were bloody and three were oily. Only one bloody print was 5 recovered from the spent casings. Althou gh half of the fingerprints that were recovered were bloody , the authors acknowledged that this type of print is not frequently encountered on casings submitted as evidence. Excluding bloody prints, only 3 out of 32 (9%) cartridges displayed useable prints , all of which were unfired. It was also noted that casings that did display a print were all of the larger caliber sizes used in the study (0.45 or 9 mm as opposed to 0.22). Cyanoacrylate Fuming: Effect on DNA Recovery and Analysis Cyanoacrylate fuming is a common method used to visualize latent fingerprints. First, cyanoacrylate is heated to form a gaseous vapor. When cyanoacrylate monomer s interact with a fingerprint, they polymerize to form a white residue of poly(ethyl cyanoacrylate) (Dadmun, 2010). The speed of this reaction can vary greatly, and depends largely on the concentration of cyanoacrylate and the humidity of the air in the fuming chamber. Von Wurmb et al. (2001) examined the effect of cyanoacrylate fuming on polymerase chain reaction ( PCR ) efficiency. fluid were fumed with cyanoacrylate for 1 hr, whi le the remaining were left untreated. Samples were divided into two groups and extracted using either the Chelex method (Walsh et al., 1991) or an Invisorb Forensic Kit. Pure cyanoacrylate was also extracted and added to known amounts of control DNA to det ermine if it had an inhibitory effect. Short tandem repeats (STRs) were amplified using an AmpFLSTR ® Profiler Plus ® kit. The results showed cyanoacrylate had a negative effect on PCR efficiency. Fumed blood and saliva samples had reduced amounts of PCR pro duct, though the Invisorb kit exhibited a smaller effect, as did control DNA mixed with cyanoacrylate extract, indicating it had an inhibitory effect on PCR. 6 The effect of cyanoacrylate fuming on the recovery of touch DNA from pipe bombs was examined by Gi cale (2011). Twenty four volunteers each assembled two pipe bombs, one of which was fumed with cyanoacrylate for 15 min after deflagration. DNA was isolated using a double swab technique, in which the first swab was wetted with digestion buffer, followed b y organic extraction, quantified using a Quantifiler ® Human DNA Quantification Kit, and amplified using MiniFiler TM . Slightly more DNA was recovered from fumed pipe bombs than from non - fumed bombs (averaging 29 and 19 pg, respectively), though the differen ce was not significant. Complete consensus profiles 1 were produced from 29% of fumed and 17% of non - fumed pipe bombs. Touch DNA: Identification of an Individual In the early years of forensic DNA analysis, large quantities of biological material were r equired to obtain a result. Consequently, most DNA profiling was performed on body fluids such as blood, semen, and saliva for which an ample amount of sample was available for testing. T he amount of DNA needed to produce a profile has decreased greatly a s technology has improved , and crime laboratories have been receiving more and more requests for the analysis of touch samples (Minor, 2013) , or samples resulting from the transfer of cells through touch. DNA is present in all nucleated cells of the body, including skin. Each hum an cell contains approximately 7 pg of DNA ( Tiersch et al., 1989 ). Full STR profiles have been produced from 100 pg or less of DNA ( e.g. Oostdik et al., 2014), corresponding to fewer than 20 cells. Several authors have stated that humans shed approximately 400,000 epithelial cells per day, so skin can be a valuable source of DNA in forensic cases ( e.g. Wickenheiser, 2002; 1 included in the consensus profile. 7 Schiffner et al. , 2005; Jenny , 2010). In addition to transferring shed epithelial cells onto touched objects, ha nds are also able to act as vectors for other cell types. Rubbing of the face, eyes, mouth, and nose deposit s additional cells on the fingers, which can then be conveyed to another surface through touch. The first published success in obtaining genetic i nformation from touch samples was by van Oorschot and Jones (1997). They demonstrated that genetic profiles could be produced from swabbing handled objects including briefcase handles, pens, and car keys. These types of samples quickly became popular submi ssions to forensic laboratories, and DNA evidence was obtained from weapons such as knives, screwdrivers, and ligatures, as well as from door pulls, door bells, and adhesive tape involved in crimes (reviewed by Wickenheiser, 2002). The success of touch sam ple analysis, however, has remained highly variable. Research ers have shown that the amount of DNA transferred t hrough physical contact depends on many variables, including the individual handler, the surface being handled, and on environmental conditions (Phipps and Petricevic, 2007; Daly et al. , 2010 ). For example, rough, porous surfaces are more likely to yield DNA than smooth, non - porous ones ( Daly et al. , 2010 ) . Surprisingly, the amount of time spent handling the substrate has not been found to affect the amount of DNA deposited, and full profiles have been reported from a contact time of 1 s from paper (Balogh et al., 2003 a ) and 5 s from fabrics (Linacre et al., 2010). Several modifications to the procedures used by forensic scientists have been sugge sted to increase the success of DNA analysis from low template samples. The quantity of DNA collected via swabbing can be raised through the use of detergent - based solutions (Thomasma and Foran, 2013), and pre - treatment of centrifugal filtration devices ha s been shown to decrease DNA loss during extraction (Doran and Foran, 2014). Following extraction, t he amplification of 8 STR alleles has been improved by increasing the number of PCR cycles (Gill, 2001) and reducing PCR reaction volumes (Gaines et al., 2002 ). Detection of alleles can be enhanced through post - PCR clean up and increased injection times (Smith and Ballant yne, 2007; Westen et al., 2009 ) , which allows for the production of more complete profiles . Despite these advances, challenges in processing low copy number samples remain, several of which were discussed by Budowle et al. (2009 ). Stochastic sampling, in which alleles are randomly sampled or amplified, may result in heterozygote peak imbalance and/or drop out of one or both alleles at a locus. Stutter peaks, which are generally less than 20% of the associated allele peak height in high template samples ( e.g. Leclair et al., 2004) , can be as tall as their parent allele, and in some instances might exceed the true peak height. Contamination and drop - in can also have an intensity as strong as true alleles in low template samples, making interpretation difficult and unreliable. The most common method for overcoming these challenges is to perform replicate analysis , in wh ich two or more aliquots of the sample are amplified separately (Budowle et al., 2009). A consensus profile can then be generated, including only alleles that are present in multiple profiles . DNA from Spent Cartridge Casings T he analysis of touch DNA is becoming increasingly succe ssful, but samples obtained from spent cartridge casings present addition al challenges. DN A is deposited onto the surface of the casing during the loading process . However, when the cartridge is fired it is subjected to extremely high temperatures (the bar rel of the gun may reach 1,200 °C when fired [ Lawton, 2001 ] ), pressure, and mechanical stress (U.S. Army Materiel Command, 1965 ), which likely have a strong degradative effect on DNA. Additionally, the metal composition of the casing and 9 the gunshot re sidu e expelled during firing might inhibit PCR. Consequently, authors have stated that crime laboratories do not often attempt to recover DNA from spent casings (Horsman - Hall et al., 2009) . The feasibility of recovering DNA profiles from spent cartridge casin gs has been the focus of multiple studies over the past several years. Horsman - Hall et al. (2009) analyzed the effect that firing had on the recovery of DNA from spent casings. A single donor, said to leave behind substantial DNA in touch samples (although how this was determined was not described), handled ten cartridges. Five were loaded into a rifle by a gloved firearms examiner and were fired, while the remaining five were tested unfired. No magazine was used. DNA was recovered using a double swab techn and was extracted using either an organic procedure (followed by Microcon ® purification) or DNA IQ TM with one of three digestion buffers (proteinase K + 20% sarkosyl, DNA IQ TM Lysis Buffer, or prot einase K + SDS). DNA was quantified using a Plexor ® HY System, and STRs were amplified using MiniFiler TM , Identifiler ® , and PowerPlex ® 16 BIO kits. Organic extraction yielded significantly less DNA than the three DNA IQ TM methods. There was no significant difference between the DNA yields of the fired and unfired casings, which produced an average MiniFiler TM profile of 81 ± 20% and 85 ± 12%, respectively , indicating that firing did not affect DNA profiling . MiniFiler TM produced a significantly greater numb er of alleles than either PowerPlex ® 16 BIO or Identifiler ® . Previous research at Michigan State University has shown that not only is the recovery and analysis of DNA from spent cartridge casings feasible (Orlando, 2011), but the optimization of recovery methods greatly increases its success. Mottar (2014) compared swabbing and soaking as means of recovering DNA, as well as three extraction methods . In total, five 10 recovery/extraction methods were compared: double swab /organic extraction, soak/ organic extr action, double swab / QIAamp ® DNA Investigator extraction, soak/ QIAamp ® DNA Investigator extra c tion, and single swab/ Fingerprint DNA Finder ® Kit extraction . Prior to comparing the five methods, pre - digestion treatments, soaking vessel, soak time, shaking dur ing soak or digestion, and digestion time were optimized. Organic extractions were coupled with Amicon ® filtration columns pretreated with yeast RNA (Doran and Foran, 2014). Volunteers loaded 0.40 caliber Smith & Wesson brass cartridges into the magazine o f a firearm. The cartridges were fired and the casings were collected and assigned a recovery method. DNA was quantified using an Alu based rtPCR assay ( Nicklas and Buel , 2005 ) and STRs were amplified using PowerPlex ® Fusion. Organic extraction yielded sig nificantly more DNA and STR alleles than either kit tested, and double swabbing proved superior to soaking. Overall, double swabbing followed by organic extraction was shown to be the most optimal method for recovering DNA from spent cartridge casings. Thi s likely differed from the findings of Horsman - Hall et al. (2009) because Mottar (2014) pre - treated filtration columns with yeast RNA, decreas ing DNA loss during organic extraction . Mottar (2014) used the double swabbing technique developed by Sweet et al . (1997) when comparing swabbing and soaking , in which a surface is swabbed first with a wet swab and second by a dry one. All casings were swabbed individually, meaning one pair of swabs was used for each casing . However, some crime laboratories swab all casings from a crime scene which appear to have been fired from the same gun using the same pair of swabs (MSP, personal communication). This saves time and effort, as only a single pair of swabs has to be processed , instead of many. T his cumulative swabbi ng approach has the potential to create mixtures if DNA from a different source is present on each casing, though, which has not previously been tested. 11 Loading and Firing Order One variable that has the potential to influence the amount of DNA recovered from spent cartridge casings is the order in which the cartridges are loaded and subsequently fired. It is deposited on the first cartridges loaded, with the number of cells decreasing wi th each subsequent cartridge. Conversely, the last cartridge loaded requires more force to load into the magazine, which might result in the transfer of a greater number of cells . Additionally, the temperature of the gun when it is fired may alter the amou nt of DNA that is present on a spent casing. The temperature inside of a gun will increase as more cartridges are fired, thus the first loaded (last fired) cartridge is exposed to the most heat, potentially having a degradative effect on DNA. STR Analysis : Human Identification The use of PCR to amplify DNA prior to analysis greatly reduce s the amount of cellular material needed to produce a result. While previous techniques, such as restriction fragment length polymorphism analysis, required both high qua ntity ( 50 ng or more) and high quality (at least 12 kb) DNA, techniques employing PCR can amplify fragments less than 100 bp from as little as a single cell (Findlay et al., 1997). STR analysis is a PCR - based technique that is commonly performed in forensi c biology ( reviewed by Jobling and Gill, 2004 ). An STR is a short repeated sequence, which for forensic purposes typically has a repeat unit of four bases . The number of repeat units in a DNA strand is variable among individuals, which makes them useful ta rgets for identity testing. A forensic scientist will analyze multiple STR loci, which c ombined have extremely high discrimination power among individuals. 12 Today, there is a wide variety of STR kits commercially available. Typical kits target amplicons b etween 100 and 450 bp ( e.g. AmpFLSTR ® Identifiler ® , PowerPlex ® 16 ). However, moving PCR primers closer to the STR region to reduce amplicon size allows for more successful amplification from degraded samples (Wiegand and Kleiber, 2001). As a result, new ® Amplification Kit , which targets nine loci all smaller than 300 bp and is advertised as being been developed, such as the Promega PowerPlex ® Fusion System that amplifies 24 loci, 14 of which are smaller than 300 bp, and Promega claims it is highly sensitive and inhibitor - tolerant, working well with low template samples. MtDNA Analysis: Human Iden tification Human mitochondrial DNA (mtDNA) is a circular genome of approximately 16,569 bp. Most of the genome comprises 37 essential genes, and is therefore not highly polymorphic (and consequently not useful for identity testing) (Anderson et al., 1981; Holland and Parsons, 1999). There is, however, a 1,122 bp non - includes two hypervariable regions, HV1 and HV2. These regions are 341 and 267 bp in size, and are commonly targeted by forensic scientists ( reviewed by Holland an d Parsons, 1999). MtDNA analysis is of great valu e to forensic science because mtDNA is often still recove rable after nuclear DNA has degraded. While nuclear DNA is present in only two copies, there are hundreds of mtDNA copies per cell (Robin and Wong, 1 988) , making it more likely that a profile can be obtained from low template samples. Multiple characteristics of mtDNA also protect it from degradation (Foran, 2006). It is possible that the circular nature of mtDNA 13 prevents exonucleases from digesting it . Additionally, m tDNA is located in the mitochondria of the cell, rather than in the nucleus, and is protected by the mitochondria themselves. Due to these factors, mtDNA profiling has been highly successful when working with ancient and degraded sample s ( There are, however, limitations to the use of mtDNA analysis. MtDNA it is maternally inherited, so it is not unique to the individual and therefore cannot be used for positive identification. This maternal inheritance can be us eful, though, when a reference sample for an individual is not available but a sample can be obtained from a maternal relative. Metchikian (2013) previously studied the feasibility of mtDNA analysis from spent cartridges casings at Michigan State Universi ty . DNA extracts were used from a separate study (Orlando, 2011) in which volunteers loaded cartridges into a magazine, the cartrid ges were fired, the casings were collected , and DNA was extracted. HV1 was amplified and sequenced as two pieces (HV1a and HV 1b), and the first half of HV2 was analyzed. Haplotypes were obtained from all casings, about two thirds of which w ere consistent with the handler (although most were mixed profiles) , indicating that mtDNA analysis can potentially be used to identify the l oader of a firearm. Goals of This Study Despite improvements in the recovery of DNA from spent cartridge casings, STR profiling success remains highly variable ( Horsman - Hall et al., 2009; Branch, 2010; Mottar, 2014). Therefore, it is important to unders tand the many factors that may affect both the quantity and quality of genetic information produced from spent casings . The purpose of this research was to examine several such variables to determine what, if any, effect they have on DNA 14 recovery and analy sis . Two different collections of casings were used for this study . The goal of the first collection was to examine the effect of cyanoacrylate fuming on the recovery and analysis of DNA from spent casings, in order to determine if it is advisable to fume casings prior to processing , and whether fuming casing s immediately upon collection i s superior to transporting them back to the laboratory prior to fuming. Collection 1 was also used to compare two commercial STR kits ( ® and PowerPlex ® Fusion) . The goals of the second collection included determining if cartridge caliber has an effect on recoverable DNA from spent casings and examining if swabbing multiple casings with a single pair of swabs is advantag eous or disadvantageous . Finally, the second collection was used to compare the success of mtDNA sequencing with STR profiling. The influence of cartridge loading/firing order was examined using both collections. All of these variables were examined in an attempt to improve the success of identifying the loader of a firearm using DNA from spent casings. 15 METHODS Cartridge Casing Collection C artridges were fired and c asings collected on two occasions. American Eagle ® Federal Premium Ammunition and Blazer ® Brass 0 .40 S&W ammunition was used during Collection 1, while Federal ® American Eagle ® 0.45 Auto center fire pistol cartridges and Federal Premium ® Champion Target 0 .22 LR rim fire cartridges were used during Collection 2. One to four cartridges fr om each box of ammunition were randomly selected and DNA was extracted and quantified to determine if background DNA was present. The resulting quantities were low, so ammunition was not cleaned prior to handling. Cartridges were placed in paper bags in s ets of 21 for Collection 1. Researchers wore lab coats, sleeves, gloves, face masks, and hair nets during this pro cess, and cartridges were transferred using hemostats which h ad been UV irradiated for 5 min on each side ( approximately 5 J/cm 2 ) in a Spect ro linker XL - 1500 UV Crosslinker and rinsed with 70% ethanol. Each volunteer at the Michigan State Police (MSP) Lansing Forensic Laboratory shooting range loaded one set of cartridges into two magazines of a 0.40 caliber handgun (12 in one magazine and 9 in t he other). The volunteers did not wear gloves during the loading process. The magazines were loaded into the firearm by a MSP firearms examiner wearing gloves and a lab coat, and fired. The gun was fired through a denim microscope cover with a hole cut in the corner so that the casings could be collected without falling to the floor. Casings were collected in sets of three using hemostats, and were placed in new paper bags which were assigned different treatments (Appendix A). Fifty percent bleach was place d on a Kimwipe ( Kimberly - Clark Corporation, Irving, TX ) and used to wipe the hemostats before cartridges loaded by each new volunteer were fired. 1 6 Prior to Collection 2, cartridges of each caliber were divided into sets of 12 in separate paper bags. Resear chers wore the same personal protective equipment as in Collection 1, and hemostats used to transfer the cartridges were UV irradiated for 5 min on each side ( approximately 5 J/cm 2 total ) in a Spectrolinker XL - 1500 UV Crosslinker (Spectronics Corporation) and rinsed with 70% ethanol. Volunteers loaded sets of cartridges into two magazines for each caliber. Half of the volunteers load ed the 0.45 caliber cartridges first and half loaded the 0.22 caliber cartridges first. The magazines containing the 0.45 cali ber cartridges were loaded into a 0.45 caliber handgun and the magazines containing the 0.22 caliber cartridges were loaded into a 0.22 caliber rifle . The weapons were fired through a pop - up mesh laundry hamper so that casings could easily be collected in sets of three. Casings were transferred to paper bags using hemostats (which were wiped with 50% bleach before cartridges loaded by each new volunteer were fired) , and were assigned different swabbing methods (Appendix A). Each volunteer provided two bucc al swabs, which were stored in 12 x 75 mm polypropylene culture tubes (Fisher Scientific, Waltham, MA). Volunteers were randomly assigned both a letter and a number, so as to keep the samples de - identified. Bags of spent casings were labeled with the volun teer number, while buccal swabs were labeled with the volunteer letter. Buccal swabs and casings were stored at - 20 °C. The use of human volunteers was approved by the MSU Institutional Review Board (IRB 12 - 770). Cyanoacrylate Fuming of Collection 1 Casin gs Three casings from each volunteer in Collection 1 were taken to the MSP fingerprint unit and fumed the humidity inside the chamber . This wa s followed by a fuming step and a ven tilation step. T he 17 entire process took 1 1.5 hr . A nother three casings from each volunteer were returned to the MSU Forensic Biology Laboratory to be fumed. The MSU fuming chamber consisted of an electric candle warmer (Rimports USA LLC, Provo, UT) in th e center of a 24 x 16 x 13 in, 15 - gallon plastic storage container (Incredible Plastics, Warren, Ohio). A beaker containing 200 mL of water was placed on the candle warmer. Casings were positioned in the chamber on weigh paper surrounding the candle warmer and the container was closed. After 15 min, a tea - light foil container holding approximately 4 mL of cyanoacrylate was added to the candle warmer, and the casings were fumed for 20 min. The cyanoacrylate was removed from the container, which was left slig htly open to ventilate for 10 min before the casings were removed and placed back in their corresponding paper bags. Sets of 15 18 casings were placed in the fuming chamber at a time, and this process was repeated three times. The casings were stored at - 20 °C. The remaining casings were not fumed, and were used for a separate study. All data from non - fumed casings used in this research were taken from Collection 2 of Mottar (2014). DNA Isolation and Digestion Cotton swabs (860 - PPC, Puritan Medical Pr oducts, Guilford, ME) and 1.5 mL microcentrifuge tube s were autoclaved prior to use. Microcentrifuge tubes and other supplies (e.g. scissors, hemostats, pipettes/pipette tips, reagents, etc.) used in all pre - amplification procedures were UV irradiated for 10 min (approximately 5 J/cm 2 ), or 5 min on each side, in a Spectrolinker XL - 1500 UV Crosslinker. All water was purified through a Milli - Q ® Water Purification System ( Millipore Corporation , Billerica, Massachusetts ) and filtered using a 0.2 2 Millex ® - GS syringe driven filter unit (Millipore Corporation) . A lab coat, face mask, sleeves, and two pairs of gloves were worn whenever handling casings or pre - amplified DNA. 18 DNA Isolation and Digestion: Collection 1 DNA was recovered from each casing using a do uble swabbing method (Sweet et al., Tris [pH 7.5], 50 mM EDTA) and the second swab was dry. Casings were held using hemostats during swabbing. The cotton tip of each swab was cut from the shaft using a pair of scissors and onto a swab and placing it and a second dr y swab directly into a 1.5 mL tube containing the same reagents. The tube was vortexed for 10 s and placed in an incubator at 55 °C , shaking at 900 rpm, for 1 h. Hemostats and s cissors were cleaned with 70% ethanol between each casing. DNA Isolation and D igestion: Collection 2 DNA was isolated as described above for the individually swabbed casings. Three casings were used for ea ch cumulatively swabbed sample, and each casing was held by a separate pair of hemostats. The first wetted swab was used to swab the outside surface of each of the three casings (from a single paper bag), followed by a dry swab. Both swabs were placed in a single tube and digested as above. DNA Isolation and Digestion: Buccal Swabs Buccal swabs were placed in 1.5 mL tubes containi proteinase K. The tubes were vortexed for 10 s and incubated at 55 °C for 1 hr. 19 Organic DNA Extraction Amicon ® Ultra - 0.5 mL, 30 kDa filtration columns (Millipore Corporation) were pre - treated using yeast ( Saccharomyce s cerevisiae ) RNA . One microliter RNA (Alfa Tris [pH 7.5], 0.1 mM EDTA) were applied to the columns, which were centrifuged at 14,000 g for 10 min (Doran and Foran, 2014). The lysate from each swab was collected using a spin basket (Fitzco, Spring Park, MN) at 20,000 g for 4 min. The flow through was combined with the remaining solution from the digestion tube. Five hundred microliters of phenol was added to the digestion tube, which was vortexed fo r 10 s and centrifuged at maximum speed for 5 min. The aqueous layer was removed at maximum speed for 5 min. The aqueous layer was removed and transferred to a p re - treated Amicon ® column . The column was centrifuged at 14,000 g for 10 min, and the flow through was discarded. Three hundred microliters TE (10 mM Tris [pH 7.5], 1 mM EDTA) was added, and the column was centrifuged at 14,000 g for 10 min. This step was repeated two more times using for 3 min. The volume recovered was measured and DNA extracts were stored at - 20 °C. DNA Quantitation Using Real - Time PCR Quanti tation standards were created at concentrations of 2000, 200, 20, 2, 0.2, and 0.02 ® 2372 Human DNA Quantitation Standard Standa Alu and IPC 20 primer and probe sequences can be found in Table 1. Both forward and reverse Alu primers were filtered through Microcon YM - 100 membranes (Millipore Corporation) prior to us e. Table 1 . Primer, probe, and IPC template sequences for real - time PCR. HEX and 6FAM are fluorescent dyes attached to the 5 end of the probes. BHQ1 and IABkFQ (Iowa Black ® FQ) are quenchers attached to the 3 end of the probes. Z EN is an internal quencher. The Alu primers and probe were designed by Nicklas and Buel (2005). The IPC primers, probe, and template were designed by Lindquist et al. (2011). Primer Name Sequence Alu F - GAG ATC GAG ACC ATC CCG GCT AAA - Alu R - CTC A GC CTC CCA AGT AGC TG - IPC F - AAG CGT GAT ATT GCT CTT TCG TAT AG - IPC R - ACA TAG CGA CAG ATT ACA ACA TTA GTA TTG - Alu Probe - HEX - GGG CGT AGT GGC GGG - BHQ1 - IPC Probe - 6FAM - TAC CAT GGC - ZEN - AAT GCT - IABkFQ - IPC Template - AAG CGT GAT AT T GCT CTT TCG TAT AGT TAC CAT GGC AAT GCT TAG AAC AAT ACT AAT GTT GTA ATC TGT CGC TAT GT - Real - Supermix TM (Bio - Alu Alu Alu Syzygy Taq DNA polymerase (5 U/µL; Empirical Bioscien ce, Grand Rapid run in duplicate and a negative control was included in each assay. Reactions were set up in 0.2 mL optically clear flat - capped PCR strips (USA Scie ntific ® , Ocala, FL). PCR amplification was - Rad Laboratories). The cycling parameters were 3 min at 95 °C, followed by 50 cycles of 15 s at 95 °C and 1 min at 60 °C. Fluorescence olor Real - Time PCR Detection System (Bio - Rad 21 concentrations were multiplied by the DNA extract volumes to calculate DNA yield. STR Analysis of Spent Casing DNA PowerPl ex ® Fusion: STR Amplification STRs were amplified using a PowerPlex ® Fusion System (Promega, Madison, WI) and an Applied Biosystems 2720 Thermal Cycler (Life Technologies, Carlsbad, CA). Six microliters Mix in a PCR tube. DNA extracts from buccal swabs were diluted 1:300 with water , water conducted using an initial denaturatio n step of 96 °C for 1 min, 30 cycles of 94 °C for 10 s, 59 °C for 1 min, and 72 °C for 30s, and a final 10 min 60 °C extension. Amplified DNA was denatured at 95 °C for 3 min and placed on ice for 3 min. One - Lane Standard 500 (Promega). DNA w as electrophoresed on an AB3500 Genetic Analyzer (Life Technologies). Capillary electrophoresis was performed using the parameters: oven temperature 60 ° C; pre - run voltage 15 kV; p re - run time 180 s; injection voltage 1.2 kV; injection time 24 s; run voltage 15 kV; run time 1500 s; capillary length 50 cm. Allele calls were made using GeneMapper ® v4.1 software (Life Technologies) at a threshold value of 100 rela tive fluorescence unit s (RFUs) and were verified using OSIRIS v2.2 (Goor et al., 2011). Alleles were compared to the reference profiles and were classified as consistent or not consistent with the handler. Percent profiles were calculated by dividing the number of consistent al leles by the total number of possible alleles for that individual. 22 Forty of the DNA extracts from the fumed casings in Collection 1 with the highest DNA ® it (Life AmpFlSTR ® ® DNA. PCR cycling conditions were 11 min at 95 °C followed by 30 cycles of 20 s at 94 °C, 2 min at 59 °C, and 1 min at 72 °C, and a final extension step of 45 min at 60 °C. - ® Size Standard (Life Technologies). Capillary electrophoresis was performed using the parameters: oven temperature 60 ° C; pre - run voltage 15 kV; pre - run time 180 s; injection voltage 1.6 kV; injection time 8 s; run voltage 19.5 kV; run time 1330 s; capillary length 50 cm. Allele calls were made as above. MtDNA Sequenci ng of Spent Casings Mitochondrial DNA was analyzed from 96 extracts from Collection 2. Samples were divided into three groups based on DNA quantitation (high, medium, and low) , and eight of each type (individually swabbed 0.45 caliber casings, cumulativel y swabbed 0.45 caliber casings, individually swabbed 0.22 caliber casings, and cumulatively swabbed 0.22 caliber casings) were selected for mtDNA sequencing from each group. HV1 and HV2 were amplified and sequ enced, using the primers in Table 2. 23 Table 2 . Primers used to amplify and sequence mtDNA from casings and reference samples. All samples were amplified with F15989, R16410, F15, and R499. F16190 and R285 were used when sequences failed or were not suitable for analysis. Primer Name Region Sequence F15989 HV1 5 - CCC AAA GCT AAG ATT CTA AT - R16410 HV1 5 - GAG GAT GGT GGT CAA GGG AC - F16190 HV1 5 - CCC CAT GCT TAC AAG CAA GT - F15 HV2 5 - CAC CCT ATT AAC CAC TCA CG - R499 HV2 5 - CGG GGG TTG TAT TGA TGA GAT T - R285 HV2 5 - GTT ATG ATG TCT GTG TGG AA - 2 - water , 1 unit AmpliTaq Gold ® polymerase 94 °C, 38 cycles of 30 s at 94 °C, 30 s at 60 °C, and 30 s at 72 °C, and a final extension of 5 min at 72 °C. Five microliters of PCR product was electrophoresed on a 1% agarose gel. Post PCR clean - up was performed using Diffinity RapidTips ® (Diffinity Genomics, Inc., West Henrietta, NY) . PCR prod ucts were aspirated through a RapidTip approximately 15 times, and were transferred to a new tube. ® Terminator v3.1 Cycle Sequencing master mix (1.82 µL of BDX64 BigDye ® enhancing buffer [MCLAB, San Francisco, CA], 0.68 µL of BigDye ® Terminator v3.1 Ready Reaction Mix [Life Technologies]), forward or reverse primer, 1 water parameters were 3 min at 96 °C followed by 30 cycles of 10 s at 96 °C, 5 s at 50 °C, and 2 min at 60 °C. 24 of 3 M sodium acetate, 1 - five microliters cold 95% ethanol was added to each sequencing reaction, which was vortexed for 10 s and centrifuged at maximum speed for 10 min. The sup ernatant was removed, and the pellet maximum speed for 5 min, and the supernatant removed. Th e 70% wash step was repeated two more times, and DNAs were vacuum dried for 10 min. Ten microl iters of Hi - Di Formamide was added and was vortexed for 10 s. DNAs were electrophoresed on an AB3500 Genetic Analyzer using the parameters: oven temperature 60 ° C; injection time 8 s; injection voltage 1.6 kV; run time 1400 s; run voltage 19.5 kV; capill ary length 50 cm. Sequences were aligned and analyzed using BioEdit v7.2 software (Hall, 1999), and compared to the Cambridge Reference Sequence (Anderson et al., 1981). Polymorphisms were identified and compared to volunteer reference sequences, and profi les were classified as consistent, inconsistent, mixed - consistent, or mixed - inconsistent (Table 3). Mixtures were identified when two peaks were detected at the same position in both the forward and reverse sequence s . Table 3 . Desc ription of categories used in mtDNA analysis. Category Description Consistent Profile was consistent with the handler Inconsistent Profile was not consistent with the handler Mixed - Consistent Profile consisted of a mixture of individuals, including the handler Mixed - Inconsistent Profile consisted of a mixture of individuals, not including the handler 25 Amplification of M tDNA from Reagent Blanks HV2 was amplified from 21 reagent blanks, selected at random. PCR was conducted in - verse primer (R499), 3 water , 1 unit AmpliTaq Gold ® polymerase (Life 94 °C, 38 cycles of 30 s at 94 °C, 30 s at 60 °C, and 30 s at 72 °C, and a final extension of 5 min at 72 °C. Fi ve microliters of PCR product was electrophoresed on a 1% agarose gel. Statistical Analysis Statistical tests were performed using XLSTAT 2014.2.01 (Addinsoft, Paris, France) with a significance level of 0.05. A Shapiro - Wilk test was conducted to determin e normality for all nuclear DNA data (including quantification and STR results) , a Kruskal - Wallis test was used to make multiple comparisons, and pairwise comparisons were made using Mann - Whitney. A Bonferroni correction was applied to account for multiple pairwise comparisons within a set of conducted to determine whether quantitation level, swabbing strategy, and cartridge caliber had a statistically signi ficant effect on mtDNA profile classification . 26 RESULTS Loading, Collecting , Cyanoacrylate Fuming, and DNA Isolation of Cartridge Casings Volunteers had a broad range of experience with loading cartridges, and as a result the amount of time spent handl ing each cartridge varied from several seconds to a couple of minutes. The microscope cover used to capture casings during Collection 1 was relatively inefficient, as multiple casings fell out and made contact with the firing range floor. The pop - up hamper utilized in Collection 2 was simpler to use, as the mesh made it easier to find and grasp casings, and resulted in few or none of them being dropped. The casings fumed at MSP had a white residue coating the outer surface, exemplified in Figure 1. The fi rst set of casings fumed at MSU did not have a change in appearance, though a similar white residue was present on the second and third sets. Figure 1 . Example of a non - fumed (left) and fumed (right) casing covered in a white re sidue. White flecks were present in the interface between the organic and aqueous layers during the phenol and occasionally the chloroform extractions, which were not transferred to the Amicon ® column. The extraction of DNA from both fumed and non - fumed c asings resulted in 27 black residue at the bottom of the tube following centrifugation of the spin baskets and during the phenol extraction. Solutions were clear after the chloroform extraction and Amicon ® purification. Reagent Blanks: Quantitation and MtDNA Amplification Results , however mtDNA f rom it failed to amplify. DNA from o nly one reagent blank, which had the se cond highest concentration, amplified mtDNA, and the resulting band (visualized via gel electrophoresis) was faint. Collection 1: Effect of Fuming on DNA Recovery and Analysis from Spent Casings Comparison of DNA Yields from Fumed and Non - Fumed Casings T he median DNA yields of fumed and non - fumed casings and the distributions of the data are shown in Figures 2 and 3, respectively. The non - fumed casings resulted in a DNA yield of 25.86 pg, while 11.53 was recovered from the MSU - fumed, and 4.95 pg from the MSP - fumed casings. Descriptive statistics are in Table 4. DNA yields were not normally distributed (Shapiro - Wilk, p < 0.0001), and there was a significant difference among the non - fumed, MSU - fumed, and MSP - fumed casings (Kruskal - Wallis, p < 0.0001). Pairwi se comparisons ( Table 5 ) showed that all differences in DNA yield were significant. Appendix B contains the DNA concentration and yield from each casing. 28 Figure 2 . Median DNA (pg) yields among the fumed and non - fumed casings. D ata for the non - fumed casings taken from Mottar (2014). 0 5 10 15 20 25 30 MSU-Fumed MSP-Fumed Non-Fumed Median DNA Yield (pg) 29 Figure 3 . Box plots displaying the distribution of the DNA yields (pg) of fumed and non - fumed casings. The box encompasses the interquartile range (the distance between the lower and upper quartiles), with the line through the box symbolizing the median. The mean is represented by a red +, extreme outliers are represented by x, mild outliers are represented by °, and maximum/minimum values are represented by blue squares. The whiskers represent the maximum/minimum values that are not outliers. The MSU - fumed casings contained an extreme outlier at 1420.2 pg that is not shown. Data for the non - fumed casings taken from Mottar (2014). Table 4 . Descript ive statistics of quantitation results of fumed and non - fumed casings. Data for the non - fumed casings taken from Mottar (2014). MSU - Fumed MSP - Fumed Non - Fumed DNA Concentration (pg/uL) Median 0.57 0.18 0.99 Average 2.15 0.41 1.65 Standard Deviation 7. 46 0.58 2.37 DNA Yield (pg) Median 11.53 4.95 25.86 Average 53.94 11.47 42.33 Standard Deviation 199.97 16.88 57.95 n 51 51 51 MSU - Fumed MSP - Fumed Non - Fumed 0 50 100 150 200 250 300 350 400 DNA Yield (pg) 30 Table 5 . Pairwise comparisons of DNA yields (pg) for fumed and non - fumed casings (Mann - Whitney, - fumed casings taken from Mottar (2014). et of casings fumed at not normally distributed, and there was no significant difference among the groups (Kruskal - Wallis, p = 0.561). Comparison of Commercial S Table 6 displays the median number of handler alleles, non - handler alleles, and percent n of 2 alleles consistent with the handler from casings fumed both at MSU and at MSP and 11 for non - fumed casings, while Fusion resulted in medians of 10 (MSU - fumed), 15 (MSP - fumed), and 23 (non - median of a 13% profile for bot h MSU and MSP fumed casings, while Fusion produced 25% (MSU - fumed) and 36% (MSP - fumed) profiles. The non - fumed casings resulted in median profiles of 67% (MiniFiler) and 60.5% (Fusion). The MSU - fumed casings, MSP - fumed casings, and non - fumed casings produc ed median percent Fusion profiles of 31%, 42%, and 70%, respectively, when only the loci smaller than 300 bp were examined . Fusion also generated a higher number of non - handler alleles, resulting in medians of 10 (MSU - fumed), 4 (MSP - fumed) and 3 (non - fumed ), compared to medians of 2 (MSU - fumed), 0 (MSP - fumed), and 1 (non - Pair P - Value MSU - Fumed MSP - Fumed 0.0065 MSU - Fumed Non - Fumed 0.0024 MSP - Fumed Non - Fumed < 0.0001 31 fumed) amplified using ( Table 7 ) showed that all but three - fumed c asings, percent profiles from the non - fumed casings, and number of non - handler alleles from the non - fumed casings). Appendix C contains the STR profile for each DNA extract (from casings and buccal swabs) D summarizes the number of handler alleles, non - handler alleles, and percent profiles produced using each kit. Table 6 . Median number of handler (H) alleles, non - handler (NH) alleles, and percent profiles produced from fumed and n on - - fumed casings taken from Mottar (2014). MSU - Fumed MSP - Fumed Non - Fumed Fusion Fusion Fusion Median #H Alleles 2 10 2 15 11 23 Median #NH Alleles 2 10 0 4 1 3 Median % Profile 13 25 13 36 67 61 n 22 22 19 19 11 11 Table 7 . Mann - Whitney pairwise comparisons between the number of handler (H) alleles, non - (Bonferroni - fumed casings taken from Mottar (2014). Pair #H Alleles (p - value) #NH Alleles (p - value) % Profile (p - value) - Fumed) < 0.0001 < 0.0001 0.0606 - Fumed) < 0.0001 < 0.0001 0.0003 - Fumed) 0.0005 0.1912 0.6862 Comparison of Fusion STR Profiles from Fumed and Non - Fumed Casings Figure 4 displays the median number of alleles consistent and not consistent with the handler (amplified with Fusion) for the full set of casings (DNA from three casings was not 32 amplifie d due to low extract volumes), and d escriptive statistics are in Table 8. The non - fumed casings generated the greatest number of alleles consistent with the handler, with a me dian of 12, while the fumed casings generated medians of 5 (MSU) and 5.5 (MSP). Pairwise comparisons (Table 9) showed that t he number of handler alleles did not differ significantly between the MSU - fumed and the MSP - fumed casings, while significantly more were prod uced from the non - fumed casings . MSU and MSP - fumed casings both resulted in a median percent profile of 13.2%, while non - fumed casings produced 30.8% of a full profile. The percent profile from non - fumed casings was significantly higher than the M SU and MSP - fumed casings. Unlike the number of handler alleles, t he MSU - fumed casings resulted in the largest number of non - handler alleles with a median of 7, which was significantly greater than those produced from the MSP - fumed and non - fumed casings. Th e distribution s of the number of handler and non - handler alleles are shown in Figures 5 and 6 , respectively . The STR profile for each casing is in Appendix E, and a summary of the data is in Appendix F . Figure 4 . Median number o f handler (H) and non - handler (NH) alleles amplified from fumed and non - fumed casings using Fusion. Data for the non - fumed casings taken from Mottar (2014). 0 2 4 6 8 10 12 14 Fumed at MSU Fumed at MSP Non-Fumed Median Number of Alleles # H Alleles # NH Alleles 33 Table 8 . Descriptive statistics of the number of handler alleles (H), non - h andler alleles (NH), and percent profile from fumed and non - fumed casings. Data for the non - fumed casings taken from Mottar (2014). MSU - Fumed MSP - Fumed Non - Fumed #H Alleles Median 5.0 5.5 12.0 Average 7.9 7.6 13.0 Standard Deviation 8.5 7.4 9.7 #NH Alleles Median 7.0 2.0 3.0 Average 8.6 3.6 4.5 Standard Deviation 8.1 4.6 4.9 % Profile Median 13.2 13.2 30.8 Average 18.8 18.4 31.0 Standard Deviation 19.4 17.9 22.9 n 49 50 51 34 Figure 5 . Box plots displaying the distribution of the number of handler alleles from fumed and non - fumed casings. The box encompasses the interquartile range (the distance between the lower and upper quartiles), with the line through the box symbolizing the median. The mean is represented by a red +, extreme outliers are represented by x, mild outliers are represented by °, and maximum/minimum values are represented by blue squares. The whiskers represent the maximum/minimum values that are not outliers. Data for the non - fumed casings taken from Mottar (2014). MSU - Fumed MSP - Fumed Non - Fumed 0 5 10 15 20 25 30 35 40 45 Number of Handler Alleles 35 Figure 6 . Box plots displaying the distribution of the number of non - handler alleles from fumed and non - fumed casings. The box encompasses the interquartile range (the distance between the lower and upper qu artiles), with the line through the box symbolizing the median. The mean is represented by a red +, extreme outliers are represented by x, mild outliers are represented by °, and maximum/minimum values are represented by blue squares. The whiskers represen t the maximum/minimum values that are not outliers. Data for the non - fumed casings taken from Mottar (2014). Table 9 . Pairwise comparisons of the number of handler alleles (H) and non - handler alleles (NH) and the percent profiles u sing the Mann - Data for the non - fumed casings taken from Mottar (2014). Pair #H Alleles (p - value) #NH Alleles (p - value) % Profile (p - value) MSU - Fumed MSP - Fumed 0.8690 < 0.0001 0.8418 MSU - Fumed Non - Fumed 0.0 025 0.0027 0.0038 MSP - Fumed Non - Fumed 0.0034 0.2383 0.0056 MSU - Fumed MSP - Fumed Non - Fumed 0 5 10 15 20 25 30 35 40 Number of Non - Handler Alleles 36 Influence of Loading/ Firing Order on DNA Yields and STRs The median DNA yield and number of handler alleles of casings from each set of cartridges fired is shown in Table 10. Cartridges 10 1 2 were not consistently loaded into the same magazine, and were either the last fired cartridges from magazine 1 or the first fired cartridges from magazine 2. Consequently, casings 10 12 were not included in pairwise comparisons, the p - values for which are in Table 11. No differences were significant. Table 10 . Median DNA yields (pg) and number of handler (H) alleles from spent casings. Casing number 1 3 refers to casings from the 1 st , 2 nd , and 3 rd cartridges fired, 4 6 refer s the 4 th , 5 th , and 6 th , etc. Cartridges 10 12 were sometimes loaded into magazine 1 and fired. Data for the non - fumed casings taken from Mottar (2014). Magazine 1 Magazine 2 Casing # 1 3 4 6 7 9 10 12 13 15 16 18 19 21 MSU - Fumed Yield (pg) 16.54 2.54 15.89 11.53 3.65 77.99 3.96 #H Alleles 6.0 3.0 5.0 8.0 2.5 24.5 3.0 MSP - Fumed Yield (pg) 3.18 15.63 5.13 3.60 15.38 1.77 3.96 #H Alleles 3.0 3.0 6.0 4.0 17.0 2.0 8.5 Non - Fumed Yield (pg) 12.90 38.47 17.13 37.26 32.10 7.59 72.62 #H A lleles 3.5 17.0 11.0 18.0 15.0 2.5 26.0 Table 11 . Mann - Whitney pairwise comparisons between DNA yields and number of handler (H) for the non - fumed casings taken from Mottar (2014). Casings 1 3 vs. 7 9 Casings 13 15 vs. 19 21 MSU - Fumed DNA Yield (p - value) 0.953 0.158 #H Alleles (p - value) 0.991 0.613 MSP - Fumed DNA Yield (p - value) 1.000 0.316 #H Alleles (p - value) 0.414 0.866 Non - Fumed DNA Yield (p - value) 0.776 0.050 #H Alleles (p - value) 0.118 0.120 37 Correlation Between DNA Concentration and STR Profiles The correlation coefficient between DNA concentration and the number of handler alleles generated using Fusion for fumed and non - fumed casings is shown in Table 12. The MSP and non - fumed casin gs had similar, moderate correlations (0.7432 and 0.7051, respectively), while the MSU - fumed casings had a weaker correlation (0.5850). Table 12 . h andler alleles generated using Fusion for fumed and non - fumed casings. Data for the non - fumed casings taken from Mottar (2014). Correlation Coefficient (r) MSU - Fumed 0.5850 MSP - Fumed 0.7432 Non - Fumed 0.7051 Collection 2: Effect of Swabbing Strategy a nd Cartridge Caliber on DNA Recovery and Analysis Comparison of DNA Yields Based on Swabbing Strategy and Cartridge Caliber Figure 7 displays the median DNA yields based on swabbing s trategy and cartridge caliber, and t he distribution of the data is in Fi gure 8. More DNA was recovered from 0.45 caliber casings than from 0.22 caliber casings, and cumulative swabbing resulted in higher yields than individual swabbing. Cumulatively swabbed 0.45 casings resulted in the largest median DNA yield (46.41 pg), foll owed by indiv idually swabbed 0.45 casings ( 18.13 pg ) , cumulatively swabbed 0.22 casings ( 17.40 pg ) , and individually swabbed 0.22 casings ( 13.31 pg ) . The DNA concentration of approximately 37% of individually swabbed 0.22, 15% of individually swabbed 0.45, 13% of cumulatively swabbed 0.22, and 2% of cumulatively swabbed 0.45 caliber casings fell at or below the lowest quantitation standard . Descriptive statistics and pairwise comparisons are shown in Tables 13 and 14, respectively. A ll differences were sign ificant with the exception 38 of individually swabbed 0.45 and cumulatively swabbed 0.22 casings. The quantitation results for each casing are in Appendix G. Figure 7 . Median DNA yield (pg) based on swabbing strategy and cartridge caliber. Individual/cumulative refers to the swabbing strategy and 0.45/0.22 refers to the caliber. 0 5 10 15 20 25 30 35 40 45 50 Individual, 0.45 Individual, 0.22 Cumulative, 0.45 Cumulative, 0.22 Median DNA Yield (pg) 39 Figure 8 . Box plots displaying the distribution of the DNA yield (pg) based on swabbing strategy and cartridge caliber. T he box encompasses the interquartile range (the distance between the lower and upper quartiles), with the line through the box symbolizing the median. The mean is represented by a red +, extreme outliers are represented by x, mild outliers are represented by °, and maximum/minimum values are represented by blue squares. The whiskers represent the maximum/minimum values that are not outliers. Individual/cumulative refers to the swabbing strategy and 0.45/0.22 refers to the caliber . Table 13 . Descriptive statistics of quantitation results based on swabbing strategy and cartridge caliber. Individual/cumulative refers to the swabbing strategy and 0.45/0.22 refers to the caliber. Individual, 0.45 Individual, 0.22 Cumulative, 0.45 Cumulat ive, 0.22 Median DNA Yield (pg) 18.13 13.31 46.41 17.4 Average DNA Yield (pg) 26.52 13.94 70.22 28.33 Standard Deviation (yield) 26.21 11.25 80.00 33.02 0.57 0.42 1.60 0.57 0.89 0.45 2.25 0.92 Standard Deviation (concentration) 0.85 0.36 2.16 1.08 n 60 59 60 60 Individual, 0.45 Individual, 0.22 Cumulative, 0.45 Cumulative, 0.22 0 50 100 150 200 250 300 350 400 450 500 DNA Yield (pg) 40 Table 14 . Mann - Whitney pairwise comparisons for DNA yield ( 0.0083). Individual/cumulative refers to the swabbing strategy, and 0.45/0.22 refers to the caliber. Pair P - Value Individual, 0.45 Individual, 0.22 0.0023 Individual, 0.45 Cumulative, 0.45 < 0.0001 Individual, 0.45 Cumulati ve, 0.22 0.9728 Individual, 0.22 Cumulative, 0.22 0.0039 Individual, 0.22 Cumulative, 0.45 < 0.0001 Cumulative, 0.45 Cumulative, 0.22 < 0.0001 Influence of Handling 0.45 or 0.22 Caliber Cartridges First on DNA Yields from Spent Casings The median DNA yields from casings handled first and second are compared in Figure 9. More DNA was recovered from the 0.22 caliber casings (both individually and cumulatively swabbed) when they were handled first than when they were handled second. Similarly, more DNA w as recovered from cumulatively swabbed 0.45 casings when they were handled first than when they were handled second. In contrast, more DNA was recovered from the individually swabbed 0.45 casings that were handled second rather than first. Pairwise compari sons between DNA yields of casings handled first and second for each caliber and swabbing strategy ( Table 15 ) revealed that t he only significant difference was in the cumulatively swabbed 0.22 casings. 41 Figure 9 . Median DNA yield s (pg) of 0.45/0.22 caliber casings based on swabbing strategy. Individual/cumulative refers to the swabbing strategy, and 0.45/0.22 refers to the caliber. Table 15 . Mann - Whitney pairwise comparisons between the DNA yields (pg) fro m casings handled first and second based on swabbing strategy and cartridge caliber (Bonferroni corrected caliber. P - Value Individual, 0.45 0.947 Individual, 0.22 0.462 Cumulative, 0.45 0.109 Cumulative, 0.22 0.006 Co mparison of Fusion STR Profiles The median number of handler and non - handler alleles based on swabbing strategy and cartridge cal iber and descriptive statistics are displayed in Figure 10 and T able 16, respectively . The cumulatively swabbed 0.45 casings resulted in the largest median number of handler alleles (17.5), followed by cumulatively swabbed 0.22 casings (8.5), individually swabbed 0.45 casings (6.0), and individually swabbed 0.22 casing s (4.0). T he distribution of the handler alleles is in Figure 11. Pairwise comparisons ( Table 17 ) showed that the only non - significant difference was 0 10 20 30 40 50 60 70 Individual, 0.45 Individual, 0.22 Cumulative, 0.45 Cumulative, 0.22 Median DNA Yield (pg) Handled 1st Handled 2nd 42 between individually swabbed 0.45 and cumulatively swabbed 0.22 casings. The median number of non - handler alleles was 4.5 for cumulatively swabbed 0.45 caliber casings, 2.5 for individually swabbed 0.45 caliber casings, 2.0 for cumulatively swabbed 0.22 caliber casings, and 1.0 for individually swabbed 0.22 caliber casings. The only pairs that were not signifi cantly different the individually swabbed 0.45 and 0.22 caliber casings, individually swabbed 0.45 and cumulatively swabbed 0.22 caliber casings, and cumulatively swabbed 0.45 and 0.22 caliber casings. The distribution of the non - handler alleles is in Figu re 12. Median profiles of 41.5% (cumulative, 0.45), 20.0% (cumulative, 0.22), 15.0% (individual, 0.45), and 10.0% (individual, 0.22) were produced. The percent profile differed significantly between all groups except for individually swabbed 0.45 and cumul atively swabbed 0.22 casings. All STR profiles are in Appendix H, and are summarized in Appendix I. Figure 10 . Median number of handler (H) and non - handler (NH) alleles based on swabbing strategy and cartridge caliber. Individua l/cumulative refers to the swabbing strategy and 0.45/0.22 refers to the caliber. 0 2 4 6 8 10 12 14 16 18 20 Individual, 0.45 Individual, 0.22 Cumulative, 0.45 Cumulative, 0.22 Median Number of Alleles # H Alleles #NH Alleles 43 Table 16 . Descriptive statistics of the number of handler alleles (H), non - handler alleles (NH), and percent STR profile based on swabbing strategy a nd cartridge caliber. Individual/cumulative refers to the swabbing strategy and 0.45/0.22 refers to the caliber. Individual, 0.45 Individual, 0.22 Cumulative, 0.45 Cumulative, 0.22 #H Alleles Median 6.0 4.0 17.5 8.5 Average 6.8 5.2 18.8 11.1 Standard Deviation 6.9 4.7 10.5 9.1 #NH Alleles Median 2.0 1.0 4.5 2.5 Average 2.6 1.7 6.2 3.8 Standard Deviation 3.1 1.9 5.5 3.8 % Profile Median 15.6 10.0 41.5 20.0 Average 20.6 12.7 45.8 26.9 Standard Deviation 16.6 11.1 25.8 21.7 n 60 59 60 60 44 Figure 11 . Box plots displaying the distribution of the number of handler alleles based on swabbing strategy and cartridge caliber. The box encompasses the interquartile range (the distance between the lower and upper quar tiles), with the line through the box symbolizing the median. The mean is represented by a red +, mild outliers are represented by °, and maximum/minimum values are represented by blue squares. The whiskers represent the maximum/minimum values that are not outliers. Individual/cumulative refers to the swabbing strategy and 0.45/0.22 refers to the caliber. Individual, 0.45 Individual, 0.22 Cumulative, 0.45 Cumulative, 0.22 0 5 10 15 20 25 30 35 40 45 Number of Handler Alleles 45 Figure 12 . Box plots displaying the distribution of the number of non - handler alleles based on swabbing strategy and cartridge caliber. The box encompasses the interquartile range (the distance between the lower and upper quartiles), with the line through the box symbolizing the median. The mean is represented by a red +, extreme outliers are represented by x, mild outliers are r epresented by °, and maximum/minimum values are represented by blue squares. The whiskers represent the maximum/minimum values that are not outliers. Individual/cumulative refers to the swabbing strategy and 0.45/0.22 refers to the caliber. Table 17 . Pairwise comparisons of the number of handler alleles (H), non - handler alleles (NH), and percent profile (Mann - refers to the swabbing strategy and 0.45/0.22 refers to the caliber. Pair # H Alleles (p - value) #NH Alleles (p - value) % Profile (p - value) Individual, 0.45 Individual, 0.22 0.0062 0.0964 0.0070 Individual, 0.45 Cumulative, 0.45 < 0.0001 < 0.0001 < 0.0001 Individual, 0.45 Cumulative, 0.22 0.0860 0.0125 0.0930 Individual, 0.22 Cu mulative, 0.45 < 0.0001 < 0.0001 < 0.0001 Individual, 0.22 Cumulative, 0.22 < 0.0001 < 0.0001 < 0.0001 Cumulative, 0.45 Cumulative, 0.22 < 0.0001 0.0145 < 0.0001 Individual, 0.45 Individual, 0.22 Cumulative, 0.45 Cumulative, 0.22 0 5 10 15 20 25 30 Number of Non - Handler Alleles 46 Influence of Loading/ Firing Order on DNA Yields and STRs Table 18 displays the median DNA yield and number of handler alleles from casings from each magazine based on swabbing strategy and caliber . Pairwise comparisons of DNA yields and number of handler alleles between the first and last casings ( Table 19 ) showed that n o differences were sign ificant . Table 18 . Median DNA yields (pg) and number of handler (H) alleles from spent casings. Casing number 1 3 refers to the casings from the 1 st , 2 nd , and 3 rd cartridges fired, 4 6 refers to the 4 th , 5 th , and 6 th , etc. Indi vidual/cumulative refers to the swabbing strategy and 0.45/0.22 refers to the caliber. Magazine 1 Magazine 2 Casing # 1 3 4 6 7 9 10 12 Individual, 0.45 Yield (pg) 12.26 17.83 29.04 25.28 #H Alleles 3.0 6.0 9.0 10.0 Individual, 0.22 Yield (pg ) 4.75 9.32 14.24 24.77 #H Alleles 3.0 5.0 6.0 3.5 Cumulative, 0.45 Yield (pg) 43.55 64.78 31.82 44.71 #H Alleles 20.0 22.0 14.0 15.0 Cumulative, 0.22 Yield (pg) 18.14 16.63 18.71 14.88 #H Alleles 8.0 9.0 8.0 10.0 Table 19 . Mann - Whitney pairwise comparisons of DNA yields (pg) and number of handler (H) alleles between casings from the first and last fired cartridges from each magazine based on al/cumulative refers to the swabbing strategy and 0.45/0.22 refers to the caliber. Casings 1 3 vs. 4 6 Casings 7 9 vs. 10 12 Individual, 0.45 DNA Yield (p - value) 0.46 0.68 #H Alleles (p - value) 0.16 0.98 Individual, 0.22 DNA Yield (p - value) 0 .28 0.01 #H Alleles (p - value) 0.03 0.72 Cumulative, 0.45 DNA Yield (p - value) 0.56 0.74 #H Alleles (p - value) 0.75 0.90 Cumulative, 0.22 DNA Yield (p - value) 0.74 0.07 #H Alleles (p - value) 0.87 0.48 47 Correlation Between DNA Concentration and STR Profi ling The correlation coefficient between DNA concentration and the number of handler alleles generated using Fusion for each swabbing strategy and caliber is shown in Table 20. Correlations were highly variable, ranging from 0.3030 (individual, 0.22) to 0 .8061 (cumulative, 0.22). Table 20 . handler alleles generated using Fusion for each caliber and swabbing strategy. Individual/cumulative refers to the swabbing strategy, and 0.45/0.22 refers to the caliber. Correlation Coefficient (r) Individual, 0.45 0.5647 Individual, 0.22 0.3030 Cumulative, 0.45 0.5825 Cumulative, 0.22 0.8061 Comparison of MtDNA Profiles MtDNA profiles were successfully ge nerated from all 96 DNA extracts tested. Figure 13 displays the classification results from all profiles, of which 50 were consistent (52%), 25 were mixed - consistent (26%), 18 were inconsistent (19%), and 3 were mixed - inconsistent (3%). In total, 78% of th e generated profiles included the handler and 29% contained a mixture. All mtDNA profiles are in Appendix J. 48 Figure 13 . Classification of mtDNA profiles for all samples (n=96). A major contributor could be identified in some mixtures, exemplified in Figure 14, when one peak was higher than the other in both strands. A major contributor could not be identified in other mixtures, when the two peaks were of approximately equal height (e.g., Figure 15). Figure 14 . MtDNA containing a mixture in which a major contributor could be determined (major = ACTTACC, minor = ACTCACC). Similar peak heights were observed in both strands. 49 Figure 15 . MtDNA containing a mixture in which a m ajor contributor could not be determined. Similar peak heights were observed in both strands. Figure 16 displays the mtDNA profile classifications for each DNA quantitation level (high, medium, and low). The highest DNA yields resulted in 21 consistent, 4 mixed - consistent, 6 inconsistent, and 1 mixed - inconsistent profile. The medium DNA yields resulted in 18 consistent, 9 mixed - consistent, 4 inconsistent, and 1 mixed - inconsistent profile. The lowest DNA yields resulted in 11 consistent, 12 mixed - consisten t, 8 inconsistent, and 1 mixed - inconsistent profile. s exact test indicated that mtDNA profile classification was independent of quantitation level (p = 0.433). 50 Figure 16 . Classification of mtDNA profiles for the high (A) , medium (B), and low (C) DNA quantity samples (n = 32 for each chart). Cumulatively and individually swabbed casings produced similar numbers of inconsistent profiles, however individually swabbed casings had fewer mixtures (Figure 17). The cumulatively swabbed casings resulted in 29 consistent, 19 mixed - consistent, 10 inconsistent, and no mixed - inconsistent profiles. The individually swabbed casings resulted in 31 consistent, 6 mixed - consistent, 8 inconsistent, and 3 mixed - exact test produced a p - value of 0.035, indicating the two variables are dependent/linked. Individually swabbed samples produced significantly more consistent profiles and significantly fewer mixed - consistent 51 profiles, while the number of inconsistent and mixed - inconsistent profiles did not differ significantly. Figure 17 . Percentage of each mtDNA profile category for cumulatively swabbed casings (A) and individually swabbed casings (B) (n = 48 for each chart). Figure 18 disp lays the mtDNA profile classifications for 0.45 and 0.22 caliber casings. Forty - five caliber casings resulted in 26 consistent, 14 mixed - consistent, 6 inconsistent, and 2 mixed - inconsistent profiles. Twenty - two caliber casings resulted in 24 consistent, 11 mixed - consistent, 12 inconsistent, and 1 mixed - - value of 0.321, indicating that mtDNA profile classification was independent of cartridge caliber . 52 Figure 18 . Percentage of each mtDNA profile category for 0.45 caliber casings (A) and 0.22 caliber casings (B) (n = 48 for each chart). Comparison of STR and mtDNA Results Overall, the STR and mtDNA results corresponded well with one another. Figure 19 shows the median number o f handler and non - handler STR alleles for samples of each mtDNA profile classification. DNA extracts that produced an inconsistent mtDNA profile also had a relatively high number of non - loader alleles when compared to extracts that resulted in a mtDNA prof ile consistent with the handler. Consistent mtDNA profiles had a median of 11 handler and 2 non - handler alleles, mixed - consistent profiles had a median of 9 handler and 3 non - handler alleles, and inconsistent profiles had a median of 7.5 handler and 3 non - handler alleles. The classification of each mtDNA profile and the corresponding number of handler and non - handler alleles for each sample are in Appendix K. 53 Figure 19 . Medium number of handler (H) and non - handler (NH) STR allele s for each classification of mtDNA profile. Only three samples were classified as mixed - inconsistent, which are not included in this graph. 0 2 4 6 8 10 12 Consistent Mixed-Consistent Inconsistent Median Number of STR Alleles mtDNA Classification # H Alleles # NH Alleles 54 DISCUSSION DNA profiling from spent cartridge casings has been suggested as a method for identifying the individual who loaded a weapon, but thus far it has not been highly successful (e.g. Horsman - Hall et al., 2009; Branch, 2010). Optimization of DNA extraction can greatly improve its recovery (Mottar, 2014), however there are a variety of other factors that may also affect DNA isolation and profiling . The goals of this research were to evaluate several of these to determine what effect, if any, they have on DNA yields and analysis from spent casings, in an attempt to better establish the quantity and qua lity of genetic information that can be obtained from them. Touch DNA samples are inherently variable, which presents challenges in a research setting. Some authors have stated that individuals differ greatly in the quantity of DNA they transfer through co ntact (e.g. Lowe et al., 2002), while others have shown that variation exists within an indivi dual (Thomasma and Foran, 2013), and that the number of cells deposited through touch is dependent on many factors, including the surface being handled and the co ndition of skin (e.g. Phipps and Petricevic, 2007). Meakin and Jamieson (2013) proposed that any activity liable to remove cells from hands may influe nce the transfer of touch DNA; f or example , less DNA is generally transmitted through con tact immediately after hand washing (Lowe et al., 2002; Phipps and Petricevic, 2007). These same variables had the potential to affect the touch samples in this research, but there are other factors to consider as well. For instance, t he volunteers in this study were largely from the firearms and DNA units of the MSP Forensic Laboratory, and therefore many had likely been wearing disposable gloves prior to (but not while) handling ammunition. It is possible that the action of removing gloves strips off loos e cells, causing less DNA to be transferred through touch. 55 Alternatively, wearing gloves for an extended period could prevent cell loss, increasing the quantity of DNA deposited onto a surface. Consequently, the amount of DNA transmitted to the casings pro bably differed between individuals who had been wearing gloves and those who had not. Another factor that might have influenced DNA yields was cartridge handling time, which varied widely because volunteers were simply instructed to load the cartridges int o a magazine, rather than hold them for a specific amount of time as other researchers have done (e.g. Horsman - Hall et al., 2009; Branch, 2010). Some volunteers had a great deal of experience working with firearms and quickly and effortlessly loaded the ca rtridges, while others had never handled ammunition and expended more time and energy. For example, one volunteer from Collection 1 took up to two minutes to load each cartridge and was not capable of loading 12 into a single magazine, and instead loaded t hree or six cartridges at a time. Volunteers typically found it easier to load the 0.22 caliber cartridges than the 0.40 and 0.45 caliber cartridges, and required less time and force to load them, although experience seemed to have a bigger impact on handl ing time. Balogh et al. (2003a) recovered DNA from paper that was touched for varying lengths of time (from 1 to 60 s), and reported that handling time did not affect STR profiling (using a Profiler Plus ® kit), h owever in that study volunteers placed singl e fingerprints on paper using the same amount of pressure. The individuals in the current research who spent extra time handling the cartridges usually moved them around in their hand, likely resulting in more cells being deposited. Another variable that h ad the potential to influence the amount of DNA recovered from casings was the order in which the cartridges were placed into the magazine and subsequently fired. However, loading/firing order did not have a significant effect on DNA yield in the current r esearch, for which there are several potential explanations. One is that the order the cartridges 56 were loaded in simply did not influence DNA yield. However, Van Oorschot et al. (2003) stated that the amount of DNA transferred to items although their experiment involved only four volunteers placing their hand on 30 sheets of plastic. DNA was then extracted and amplified using Profiler Plus ® , and STR peak heights were used as a metric of the amount of DNA dep osited , which is not a generally accepted method for determining DNA yield . The quantity of DNA transmitted to a surface therefore may not systematically decrease as multiple surfaces are touched. Another factor related to loading order is the force needed to place the cartridges into the magazine; although the last cartridge required more force to load, this might not have resulted in increased cell deposition . Additionally, it is possible that firing order did not significantly affect DNA yields because t he temperature of the casings did not get high enough to have a degradative effect. The temperature of the barrel of a fired gun can reportedly reach 1,200 °C (Lawton, 2001), but the casing likely does not get that hot as it is quickly ejected from the fir earm. Or , it may be that both loading and firing order do have an effect on DNA yield, but one counteracts the other. For example, the first loaded cartridge might contain the most DNA prior to firing, but the temperature inside the gun degrades it to the point that the amount recovered is similar to the last loaded/first fired cartridge. An alternate explanation for why loading/firing order did not affect DNA yield is that aspects of the experimental design of this research masked its true effect. Cartrid ges were fired in sets of three, so it was not known which casing was the first or last fired in each set. Instead, the sets of three were compared; if the amount of DNA deposited dropped substantially after the first cartridge loaded, this could have affe cted the results. For example, if the first cartridge had a large quantity of DNA and the second and third had much less , the average of the three would be much smaller than that of the first alone. The manner in which the cartridges were fired might 57 also have mitigated the effect of heat, as the temperature inside the chamber of the gun when the last cartridges were fired was less than it would have been had they all been fired in quick succession . Finally, because casings from each volunteer were assigned different treatment methods (i.e. fuming methods or swabbing strategies), DNA yields from the f irst and last fired cartridges loaded by the same individual could not be directly compared. Whether any of these variables affected the results is unclear, but since loading/firing order did not have a measurable effect on DNA recovery in the current research, knowing the order of casings collected from a crime scene does not appear to be critical to their analysis. This is fortunate, as it is unlikely that the order in which cartridges were fired will be determined when casings are found at a crime scene. There are also several characteristics of a casing itself that might influence DNA recovery. Cartridge casings can be made of brass, nickel, aluminum, and stee l, and spent casings submitted to a crime laboratory as evidence are likely to vary in metal composition. All casings in the current research were brass, an alloy of copper and zinc. Copper has antimicrobial properties and causes membrane damage when it ma kes contact with cells, which can be followed by DNA degradation (Grass et al., 2011). It is possible that the composition of the cartridge casing caused damage to touch DNA prior to and/or post firing, lowering the amount that was recovered . Bille et al. (n.d.) reported that when controlled quantities of cells were placed on unfired cartridges, significantly more DNA resulted from those made of nickel, aluminum, and steel than from brass. Similarly, Wan et al. (2015) had volunteers handle clean brass and n ickel cartridges, and the latter produced significan tly higher DNA yields . Given the findings of these studies, DNA analysts may consider directing their efforts to casings made of metals other 58 than brass when a crime laboratory receives them as evidence. It also might be better to process brass casings for DNA as soon as possible, or store them at - 20 °C until processing can occur. Another characteristic of cartridges that had an effect on the recovery of DNA in this research was caliber. There are two pot ential reasons for why the 0.45 caliber casings yielded significantly more DNA than did the 0.22 caliber casings . First, the former have a greater surface area on which DNA can be deposited, and second, the increased force required to load the bigger cartr idges may have resulted in additional cells being transferred to the casing surface. Or, both may have had an effect. Spear et al . (2005) reported similar finding s when investigating the recovery of fingerprints from spent casings. The authors placed print s on cartridges ranging in size from 0.22 to 0.45 caliber, and half were fired. Only one identifiable print was obtained from the spent casings and five from the unfired cartridges , all six of which were 9 mm (approximately 0.35 in) or 0.45 caliber. Based on the result s of the current research, as well as previous studies, forensic examiners should consider cartridge caliber when deciding whether to attempt DNA recovery from a casing, and when determining what methods to use during processing (discussed bel ow). Since more DNA was recovered from 0.45 than from 0.22 caliber casings, it was expected that 0.40 caliber casings would generate an intermediate amount. Instead, the 0.40 caliber casings resulted in the highest DNA yields in this study (a median of 23 pg, compared to 18 pg from the 0.45 caliber casings). However, it is difficult to compare the 0.40 and 0.45 caliber casings directly, because they were from separate collections. The groups of volunteers who participated in each collection differed (altho ugh some individuals overlapped), and Collection 1 took place in December while Collection 2 was in June, so the environmental conditions were dissimilar , which could have influenced DNA deposition and recovery. Bright and Petricevic 59 (2004) reported that w hen the hands of different individuals were directly swabbed, volunteers who repeatedly resulted in higher DNA yields had comparatively drier skin (although how this was determined was not described). People tend to have drier skin during winter (Rudikoff, 1998), potentially increasing the amount of DNA transferred to the casings in Collection 1. Furthermore, the data for the non - fumed 0.40 caliber casing s were taken from Mottar (2014) so the casings were process ed by another individual, though the analyst followed the same procedures . Given these differences, the higher yields of the 0.40 caliber casings should likely not be used to refute the idea that more DNA can be recovered from larger casings. On the other hand , it is possible that more DNA actually can be recovered from 0.40 than from 0.45 caliber casings. The latter are much bigger than 0.22 caliber casings (having surface areas of approximately 9 cm 2 and 3 cm 2 , respe ctively), while 0.40 caliber casings are very close in size (approximately 8 cm 2 ), thus similar amounts of DNA may be deposited on the larger two. However, bigge r cartridges hold more gunpowder, and therefore likely reach a higher temperature when they are fired, which could have increased DNA degradation. Additionally, it is feasible th at more DNA was recovered from the earlier loaded 0.40 caliber casings because volunteers who participated in both collections improved at the loading process , and spent less time and effort placing the 0.45 cal iber cartridges in the magazine . Gunshot resi due is expelled when cartridges are fired, so while it is not a characteristic of the casing itself it is likely always present on spent casings, and has the potential to influence DNA processing. The components of gunshot residue include burnt and unburnt powder, metals from the cartridge or firearm, and elements from the primer (including lead, antimony, and barium), smoke, and lubricant (reviewed by Dalby et al., 2010), which produce a black layer of soot coating the casing and could interfere with the r ecovery and/or analysis of DNA . Horsman - 60 Hall et al. (2009) stated that gunshot residue might inhibit PCR, and when the authors added control DNA to DNA recovered from 28 shotgun shells, inhibition was ob served in three of them (11%), although t here was no evidence of PCR inhibition in the current research or that of Mottar (2014). Most or all of the gunshot residue appeared to have been removed from solution in this study , as many of the components are not water soluble and pelleted when DNA was extracted. Troy et al. (2015) added a gunshot residue suspension to purified DNA and extracted it using either a robotic silica - based manual organic procedure . Peak height suppression occurred in several of the sam ples extracted using the silica - based technique, while the organically extracted samples resulted in peaks that - not removed. Once a casing has been collected from a shooting scene, it is likely to be transported to a crime laboratory for analysis such as visualization of fingerprints, microscopic examination, and DNA profiling. How the casing is handled upon discovery and collection has the potential to affect the amount of DNA recovered from it. Fingerprints are typically enhanced using chemical or physical means that might remove touch DNA, trap it in place, introduce contaminant DNA, or introduce substances that interfere with DNA extraction or analysis. If t he casing is manipulated by a firearms examiner, any cells that were deposited onto the casing when the cartridge was loaded could be inadvertently lost, and may even be replaced by DNA from the examiner if precautions are not taken. Additionally, the mann er in which the casing is processed for DNA can also affect yields, as there are many extraction and analysis techniques employed by forensic scientists to analyze DNA . Several of these factors were examined in the current research. 61 Cyanoacrylate fuming is a common fingerprint enhancement technique that can be performed using a portable or stationary fuming chamb er. The purpose of testing two methods in this study was to determine not only if cyanoacrylate fuming had an effect on the recovery and analysis o f DNA from spent casings, but also whether fuming casings on site was more beneficial than transporting them prior to fuming . For example, fuming casings immediately after collection may glue cells to the surface and prevent loss during transportation . On the other hand, cyanoacrylate itself might interfere with DNA retrieval and/or analysis. Transportation did not affect DNA recovery from fumed casings in the current research, as the MSU - fumed casings (which were transported before fuming) resulted in sign ificantly higher DNA yields . However, the MSP - fumed casings were coated in a heavier layer of cyanoacrylate than those fumed at MSU, so it is possible that the amount of cyanoa crylate affected the DNA yields. Even more DNA was recovered from the non - fumed casings, thus it is likely that the cyanoacrylate residue hindered DNA extraction, potentially because the cells were trapped in the cyanoacrylate, which remained in the interface between the organic and aqueous layers . Another disadvantage of cyanoacrylat e fuming was that casings fumed at MSU produced a large number of non - handler alleles. This may have resulted from the extra handling that was DNA could not be i dentified, if present, due to the small number of alleles that were generated). It was difficult to position casings inside of the chamber, and they therefore spent a longer period of time exposed to the atmosphere of the laboratory, which might have contr ibuted to the increased number of non - handler alleles. In contrast, the casings fumed in the commercial chamber at the MSP laboratory were kept isolated from one another on paper bags and none fell over and made contact with the surface of the fuming chamb er, while the non - fumed casings 62 were not removed from their paper bags until just before swabbing. Consequently, if casings are to be fumed prior to DNA processing, precautions such as cleaning the chamber and fuming different evidence samples separately s hould be taken to avoid contamination. Several researchers have examined the influence of cyanoacrylate fuming on DNA recovery and also found it to be deleterious. Von Wurmb et al. (2001) placed blood and saliva on glass slides , half of which were fumed wi th cyanoacrylate , cells/ DNA was removed (details not given) and a Chelex extraction performed. STRs were amplified using Profiler Plus ® , and while profiles were generated from The authors also reported that when cyanoacrylate was directly added to controlled amounts of purified DNA, PCR was inhibited, although this probably occurred because Chelex does not separate it from substances like cyanoacrylate in the solution. Pitilertpanya and Palmback (2007) placed fingerprints on soda cans, fumed them with cyanoacrylate , extracted DNA using a QiaAmp extraction kit , and amplified STRs using an ® No quantitative data or statistics were presented, but the authors stated that more cyanoacrylate resulted in profiles and non - fumed prints produced , which is consistent with the findings of the current study . In contrast, other researchers have found that cyanoacrylate fuming did not have an effect on DNA analysis . Gicale (2011) examined the recovery of DNA from deflagrated pipe bombs fumed with cyanoacrylate by having volunteers assemble bombs, fuming half on site fo r 15 min following deflagration, and extracting DNA; t here was no statistical difference in DNA yields between the two . Bille et al. (2009) also examined cyanoacrylate fuming of pipe bombs, but used a cell suspension to deliver a constant am ount of DNA, and deflagrated bombs were exposed to cyanoacrylate for 10 min. Only six bombs were tested and no statistics were reported, although DNA yields from fumed and u n - 63 fumed bombs were similar. These conflicting results are likely due to variations in experimental design, sample type (blood, saliva, touch samples, etc.), fuming method, and DNA extraction technique . Cyanoacrylate fuming may therefore not have the same effect on all types of samples, so both the sample and processing methods must be ta ken into account by the DNA analyst, and extraction procedures that do not separate molecules such as cyanoacrylate from the DNA (e.g. Chelex) should be avoided. Based on the results of the current study, cyanoacrylate fuming spent casings is not recommen ded for several reasons. The purpose of this technique is to enhance latent fingerprints, which is rarely successful when working with casings (e.g. Bentsen et al., 1995). Furthermore, even when fingerprints are visible on spent casings they are often part ial or have poor ridge detail, and are not easily identifiable (Bentsen et al., 1995). Fingerprints were not observed on any casings in this study wh ile STR results were produced from most, indicating that DNA is far more likely to provide investigative in formation from spent casings. However, cyanoacrylate fuming casings prior to DNA processing was detrimental to the identification of the loader, as it resulted in lower DNA yields and more non - handler alleles. The isolation and extraction method used to r ecover DNA can also affect the yields obtained from spent casings. One goal of the current study was to further the work of Mottar (2014) , who optimized extraction methods and compared soaking and swabbing as means of i solating DNA from spent casings, by e xamining whether swabbing casings cumulatively or individually was advantageous. Cumulative swabbing resulted in higher DNA yields than did i ndividual swabbing, however they were not three times larger, so cumulative swabbing recovered more DNA per swab bu t not per casing. The most probable reason for this is that DNA retrieved from one casing was deposited onto subsequently swabbed casings. Hebda et al. (2014) 64 examined the effects of cumulative swabbing when studying the collection and analysis of DNA from fingernail evidence. Blood from a male volunteer was placed on two of four female fingernails, which were cumulatively swabbed with a single swab wetted with digestion buffer, alternating between bloody and clean. The clean nails were re - swabbed using the double swab technique and DNA was extracted, quantified, and Y - STRs were amplified to assay blood transfer ; enough blood was transmitted to the clean nails to produce full Y - STR profiles. It is likely that DNA was also transferred in the current research, as cells picked up from an earlier swabbed casing were deposited onto those that were subsequently swabbed , resulting in the reduction of important genetic evidence. An interesting finding regarding swabbing strategy in this research was that while cumula tive swabbing did not triple DNA yields for either caliber, the increase over swabbing a single casing was much larger for the 0.45 caliber casings. This probably stemmed from the difference in surface area between the two calibers. The surface area of a 0 .45 caliber casing is approximately three times bigger than that of a 0.22 caliber casing, so when multiple 0.45 caliber casings are swabbed there is a greater increase in the total surface area (the surface area increases by 18 cm 2 and 6 cm 2 for 0.45 and 0.22 caliber casings, respectively). This larger increase in surface area would logically result in a bigger difference in DNA yield. Another factor that may have caused this discrepancy is the accuracy of the DNA quantitation. If a sample falls outside th e range of a standard curve, the calculation of its DNA concentration is likely not as accurate as samples that fall within the curve. The lowest quantitation standard in this research was near the limit of detection of the assay, so adding another standar d dilution was not an option. More individually swabbed 0.22 caliber samples fell at or below the smallest quantitation standard than any other caliber/swabbing strategy, so the calculated DNA concentrations for these samples 65 were probably less accurate. T he median DNA yield for the individually swabbed 0.22 casings could thus be artificially high, making it appear as though there was not a large difference between individually and cumulatively swabbed 0.22 caliber casings. Once DNA has been extracted, two analysis methods are commonly used by forensic scientists: STR profiling and mtDNA sequencing. The Fusion STR amplification kit greatly research (consistent with the findings of Mottar [2014]) because the design of t he Fusion kit makes it better suited to overcome the challenges associated with analyzing DNA from spent casings, the first of which is the small amount that is present. Fusion has a lower limit of detection, and thus allows smaller quantities of DNA to be amplified . Promega recommends 0.25 0.50 ng of template DNA for Fusion reactions, although Oostdik et al. (2014) showed that full Fusion profiles could be produced from 100 pg of DNA, and the percentage of alleles recovered dropped only slightly to 97.2% when 50 pg of DNA was amplified. Mulero et al. (2008), in contrast, reported that 125 pg input DNA was required to 750 pg was determined to be optimal. The amount of DNA added to each STR reaction in the cur rent study was almost always below 50 pg, so using a more sensitive kit was highly beneficial. There are several other strategies that can be employed to improve the amount of genetic information produced when anal yzing small quantities of DNA. One way to increase the DNA yield is whole genome amplification (WGA), in which all of the DNA present in an extract is amplified prior to analysis. There are several methods for performing WGA (reviewed by Zheng et al., 2011) and multiple commercial WGA kits have be en developed, including the Qiagen REPLI - g Kit and the Illustra GenomiPhi V2 DNA Amplification Kit . Both these kits were tested by Schneider et al. (2004), who found that full profiles could be obtained from 500 pg of starting 66 DNA, while drop - out started t o occur at 50 pg ( drop - in was not observed ) . However, this research was accomplished using high quality DNA from human cell lines. Barber and Foran (2006) examined the feasibility of using WGA for the analysis of forensic samples, testing control DNA, arti ficially degraded DNA, and DNA extracted from fresh blood, aged blood, hair, and aged bones. WGA was performed using improved primer extension pre - amplification and multiple displacement amplification, and different primer sets were used to assay various l engths of nuclear and mtDNA both prior to and following WGA. STR analysis was conducted using an Identifiler ® kit. The results showed that while WGA was successful when working with high quality DNA, it did not perform well with degraded samples, and in so me instances allelic drop - out/amplification failure became worse following WGA. This likely occurred because both improved primer extension pre - amplification and multiple displacement amplification utilize random primers, which decrease the size of the DNA as amplification proceeds. If the DNA fragments are small initially , this reduction may prevent their subsequent amplification. Consequently, the authors stated that WGA was of limited use when working with degraded forensic samples. Another method for im proving analysis success of low quantity DNA is to concentrate it prior to amplification (e.g. Hudlow et al., 2011) , which allows for more DNA to be added to an STR reaction, and may result in additional alleles being detected. However, any PCR inhibitors present in solution may also be concentrated, causing amplification failure. Furthermore, increasing the concentration of the DNA decreases solution volume, potentially prevent ing replicate testing. Following amplification, the signal intensity of STR prod ucts from low copy number DNA can be strengthened using post - PCR clean - up. The purpose of this procedure is to remove remaining primers, dNTPs, and salts from solution, all of which are small molecules that 67 could be preferentially injected into a capillary , reducing the amount of DNA that is loaded. Smith and Ballantyne (2007) extracted DNA from blood, then serially diluted and amplified it using Identifiler ® . Four post - PCR purification methods were tested and full STR profiles were produced from 20 pg of D NA using a MinElute column, although it also caused increased stutter and drop - in. More DNA can likewise be loaded into a capillary by increasing the voltage and time of the injection. Westen et al. (2009) tested various capillary electrophoresis condition s, including injection voltages and times ranging from 3 to 15 kV and 10 to 300 s , respectively . The standard setting was 3 kV/10 s, and an increase to 9 kV/15 s improve d sensitivity while maintaining crimination from background elevated PCR cycle number, and the authors reported that though drop - out, peak imbalance, and drop - in occurred using both methods, eleva ted PCR cycle number res ulted in increased stutter whereas boosted injection did not. The other problem faced when analyzing DNA from the spent casings was that the DNA was highly degraded. The size of the targets affects the successful amplification of a lleles, and Mottar (2014) observed that, when using Fusion to analyze DNA from spent casings, alleles were more frequently detected at the smaller loci. The kit amplifies nine loci that are all less than 300 bp in size , while Fusion targets 24 l oci , of which 14 are below 300 bp. M the eight largest loci in the Identifiler ® kit, so that more genetic information could be obtained from degraded samples than through Identifiler ® alone. However, even when examining onl y the Fusion loci smaller than 300 bp, Fusion still produced 68 information, and it (and other similar mega - STR kits) may be better suited for use in forensic laboratorie s when working with DNA from spent casings. There are several methods for decreasing amplicon size to improve the success of STR analysis of highly degraded DNA. P rimers can be redesigned to anneal closer to the actual ST R (Wiegand and Kleiber, 2001), h owe ver there are limits to how far they may be moved, as primers cannot be placed inside the STR itself. It is also not feasible for every STR to be small; if loci overlap in size, it is impossible to distinguish them from one another. One way to overcome thi s is to alter the mobility of the DNA fragments in a capillary by attaching non - nucleotide linkers to the primers (Grossman et al., 1994); t his allows loci to be differentiated even though the actual amplicon length is the same. Alternatively, additional f luorescent dyes can be used to discriminate between similar sized fragments . Most modern commercial STR kits utilizes a five ® has six dyes , which permits the amplification of more sma than 300 bp in size, and 23 are smaller than 400 bp. If a sample has a relatively high quantity of DNA , it would logically generate a more complete profile than one with less . However, the corr el ation coefficients between DNA yield and number of handler alleles were moderate in the current research (ranging from 0.3 to 0.8) , meaning that while yield does affect STR analysis, it cannot always be used to accurately predict whether or not profiling w ill be successful. Samples that resulted in greater amounts of DNA (e.g. non - fumed casings, cumulatively swabbed 0.45 casings, etc.) typically produced more handler alleles, with one exception. The large number of non - handler alleles generated from the MSU - fumed casings lowered the correlation between yield and handler alleles, as DNA from sources other than the handler contributed to the total DNA . The relationship between DNA 69 quantity and STR alleles is important to consider when working with evidence, be cause samples with very little DNA can still produce genetic information. Not all quantitation assays are as sensitive as the Alu assay utilized in the current study , so crime laboratories may commonly encounter samples that appear to have no DNA. This res earch shows that such low yielding samples could actual ly contain enough DNA to generate a partial profile. The relationship between DNA yield and profiling results did not exist when mtDNA was sequenced, as profiles were generated for all samples tested, even those with very little DNA and/or produced few or no STR alleles. However, the DNA quantitation assay used in this research does not measure mtDNA, thus its level in each sample was un known. MtDNA sequencing is more sensitive than nuclear DNA analysis , as there are hundreds of copies of mtDNA per cell ( Robin and Wong, 1988) compared to the two copies of nuclear DNA, potentially making its amplification more successful when analyzing touch samples (e.g. Balogh et al., 2003b). Additionally, mtDNA is bett er protected from degradation than nuclear DNA (Foran, 2006), which is beneficial when working with degraded samples such as DNA from spent casings. Consequently, all casings had enough high quality mtDNA for HV1 and HV2 to successfully amplify . It should be noted, though, that samples with a high DNA yield generated more accurate sequences (i.e. consistent with the handler) than low yielding samples, but the difference was not significant. One of the major advantages of mtDNA analysis over STRs in the curr ent research was that the mtDNA sequences were simpler to analyze. HV1 and HV2 were each a nalyzed as a single fragment , thus they either amplified or did not , and there was no drop - out, like there was in the STR profiles. Furthermore, mtDNA sequences were easier to compare to the handler than STR profiles. STR analysis involves the examination of many fragments of DN A as opposed to a 70 continuous sequence, and individual alleles were classified as consisten t with or not consisten t with the handler rather than the whole profile. Whether a mixture was present could not easily be ascertained when partial profiles were produced , which was the case for nearly all samples. For instance, if an STR profile consisted of six alleles, and four of them were consistent wit h the handler, it was impossible to determine if the profile was a mixture of the handler and another individual or resulted from a single individual who had four alleles in common with the handler. In contrast, a mixture was recognized in mtDNA when more than one peak was present at a given position , and determining if it could include the handler was straightforward. For example, if a mixture contained the polymorphisms 16126Y and 16147Y (with Y representing a mixture was 16126C and 16147T, the mixture was classified as including the handler . The only mixtures that were particularly difficult to interpret were those that i nvolved different length polymorphisms in the first C - stretch of HV2 (302 310); i n such samples, the sequences become out of phase downstream of the C - stretch causing multiple bases to appear at every nucleotide position, which is irresolvable . Other researchers have also had problems sequencing this region, and Stewart et al. (2001) reported various lengths of th at C - stretch in separate hairs from the same individual, so they proposed that length polymorphisms not be used for exclusionary purposes. Several authors have stated that when a mtDNA mixture is present in a forensic sample, no attempt shoul d be made to interpret it at all (e.g. Andréasson et al., 2006; Butler 2011), thus complex mixtures involving C - stretch polymorphisms would simply be classified as inconclusive. Only ~ 30% of the spent casings in the current research produced mtDNA mixtur es, however it is possible that this number was underestimated as not all mixtures can be detected through Sanger sequencing. Mixtures of a 1:5 ratio may be readily visualized in a sequence, but 71 more extreme ratios ranging from 1:20 to 1:300 are typically indistinguishable from background noise (Holland et al., 2011). Additionally, mixtures can consist of DNA from two individuals who have the same mtDNA sequence, which would appear as a single source sample. These would probably have no bearing on forensic evidence, though, because if a minor contributor is not detected , it will not complicate interpretation. Most sequences produced in this research were clean and easy to read, although low quality sequences, comprised of irregularly shaped or spaced peaks , were sometimes generated . This problem was generally solved by re - sequencing, often using less amplified DNA, potentially because when too much was added to the reaction, non - specific binding of the sequencing primer occurred . Primer specificity is very im portant; if there are multiple template regions of DNA , it is impossible to obtain a single, clean sequence. The sequencing protocol used in the current study has an annealing time of only 5 s to help prevent primers from binding non - specifically to the DN A. Too much template likely creates a greater opportunity for non - specific primer binding, resulting in low quality sequences. Overall, mtDNA sequencing was more effective for ing a cartridge, and therefore might be the better option for examining DNA from evidentiary casings. However, there are drawbacks to analyzing mtDNA in a crime laboratory that do not exist for STR analysis. MtDNA profiles are not unique, and the most common Caucasian and African American haplotypes have frequencies of approximately 4% and 2.7%, respectively (Holland and Parsons, 1999). Consequently, mtDNA cannot be utilized to positively identify the loader of a firearm, although it can be used to include, and more importantly to completely exclude , a suspect. Sequencing is also much more time consuming than STR analysis. Further, few crime laboratories perform mtDNA analysis, and may instead have to send samples to 72 agencies such as the regional FBI laboratories for testing, which already have a back log of cases (U.S. Department of Justice, 2012) , or private laboratories . Another shortcoming of mtDNA sequencing is that an evidentiary profile is only informative when it can be compared to a suspect, as there is current ly no searchable database for for ensic mtDNA profiles (although the FBI has stated it plans to add mtDNA to CODIS [Federal Bureau of Investigation, 2010]). In contrast, the existing database can be searched for possible matches to an STR profile, giving STRs more investigative value when there is no suspect. CODIS includes an index of arrestees and convicted offenders, so if an individual whose DNA is already in the database commits another crime, searching their profile will identify them. Several studies have shown that most individuals involved in firearm violence are repeat offenders, therefore their DNA would be in the system. For example, 60% of all youth homicides in Boston involve chronic offenders, and in Indianapolis, homicide suspects from 1997 1998 averaged 11.5 prior arrests (reviewed by McGarrell et al., 2006). Additionally, the investigation of the Brightwood Gang in Indianapolis in 1999 resulted in the arrest of 16 individuals, who combined had more than 20 prior convictions for violent felonies and over 70 other conviction s (McGarrell et al., 2006). DNA from spent cartridge casings would provide valuable evidence in such instances, as the results of a database search would often include the individual who loaded the weapon. STRs thus have the potential to provide greater in vestigative leads when no information about a suspect is available, even though mtDNA analysis is more sensitive and better for helping to identify the individual who loaded a firearm when is available for comparison. The greatest impedimen t to accurately identifying the loader in this research was the presence of DNA not consistent with the handler, as 85% of STR profiles and 48% of mtDNA 73 sequences contained at least one non - handler allele/polymorphism. There were multiple potential sources of these non - handler DNAs. Cartridges were not cleaned prior to loading, and DNA might have already been present on them. Some volunteers placed the cartridges on the table top prior to loading them into a magazine, which was not a clean surface and could have held DNA from other individuals. The same magazines were loaded by each volunteer and all cartridges of the same caliber were fired by a single weapon, so DNA may have been transferred between the casings and the magazine/firearm. The shooter wore gl oves when firing the cartridges, but the be identified due to the small number of alleles produced and the anonymous method used to collect buccal swabs, and the non - handler alleles did not appear to be from one consistent individual. The casings within each collection were captured using a single apparatus (a microscope cover for Collection 1 or a pop - up hamper for Coll ection 2), and as a result DNA might have bee n transferred between the collection apparatus and the casings. Additionally, many casings fell onto the floor during collection. Although some of these sources of inconsistent alleles/polymorphisms may be a product of the research setting (e.g. it is unli kely that 20 different individuals would load the same gun over the course of a few hours), others are likely to be present in a forensic scenario. For instance, it is doubtful that a criminal will clean ammunition prior to loading it, and casings are goin g to fall to the ground when ejected from a weapon. Non - handler DNAs were present in all types of casings, however they were most numerous in the MSU - fumed casings (as discussed above) and the cumulatively swabbed casings. This was a severe disadvantage t o cumulative swa bbing; although it resulted in higher DNA yields, it also held a stronger risk of inaccurate information . Individual swabbing was thus 74 superior as a method of accurately identifying the individual who loaded a firearm, because it produced l ess non - handler alleles/polymorphisms. This was especially true when analyzing mtDNA, since both strategies yielded the same amount of information (i.e. both generated complete sequences), but individual swabbing gave rise to fewer mixed and inconsistent p rofiles. However, when STRs were amplified, the decrease in the number of non - handler alleles produced from the individual swabbed casings was accompanied by a decrease in the number of handler allele s and the loss of valuable evidence. If an STR profile c ontains only a few alleles , there might not be enough information to hold any real evidentiary value, even when no non - handler alleles are present. Searching a database for profiles consistent with a small amount of alleles will likely return too many hits to be useful if there is no other information to narrow down the list of potential suspects. Conversely, if casings are cumulatively swabbed and generate a profile containing 20 alleles, some number of which are inconsistent with the handler , the informat ion could still provide investigative leads. For example, a database may be searched using low stringency conditions (e.g. search for profiles that are 75% consistent) to compensate for the possibility of the profile containing non - handler alleles; this ca n still result in many consistent profiles, but the number might be small enough to determine if any of the individuals were potentially involved in the crime. Due to this trade - off between many handler and few non - handler alleles, cumulative swabbing coul d be more advantageous if STRs are to be analyzed from spent casings, particularly when working with smaller caliber (e.g. 0.22) casings, which did not frequently yield more than a few alleles when individually swabbed. Another trade - off of individual swab bing is the amount of time it takes. If a crime scene involves a large number of casings it may not be feasible to swab them all individually, even if it will generate less mixtures, as swabbing casings individually is time consuming . 75 Non - handler DNA was m uch more abundant in the STR profiles than in the mtDNA sequences , and many casings produced several non - handler alleles but no non - handler polymorphisms. It is t hus likely that some of the non - handler STR alleles were the product of artifacts such as drop - in and stutter, rather than contaminant DNA. A recommended method to overcome the challenges presented by artifacts is replicate analysis (not used in this study ), in which DNA is amplified multiple times from the same extract and only alleles that are ob served at least twice are reported (Taberlet et al., 1996; Budowle et al., 2009). This might remove drop - in from a profile, but it may also eliminate correct alleles. Stochastic sampling can result in different DNA being transferred to separate STR reactio ns, and therefore dissimilar alleles being amplified. Consequently, low copy number samples are often not reproducible (reviewed by Budowle et al., 2009), so distinguishing between correct alleles and artifacts is difficult. One STR artifact that could be reproducible is stutter. A general observation in t he current research was that many non - handler STR alleles were in a stutter position, however classification of these alleles as stutter was not attempted due to peak height imbalance , which is extremely p revalent in low copy number samples (e.g. Gill et al., 2000) , although the GeneMapper ® software does identify and filter out some stutter peaks . Additionally, it is typical for non - artifact alleles to be in a stutter position of one another, since the freq uencies of STR alleles for each locus are generally normally distributed (i.e. form a bell curve), not random (e.g. Díaz et al., 2008). Thus , the most common allele is at or near the center of the distribution, with the next most frequent alleles occurring in its stutter positions. For instance, 12 might be the most common allele at a particular locus, with 11 and 13 following in frequency, so many individuals in a population will have some combination of those alleles. Consequently, just because an allele in a low copy number sample is located in a stutter position of another allele does not mean that it actually is 76 stutter. It may therefore be advisable to perform replicate analysis when working with spent casings in a forensic setting in an attempt to eli minate STR artifacts from evidentiary profiles and identify all of the alleles that are present in a sample. Alleles could easily be classified as consistent or not consistent with the handler in this study, which is extremely valuable in a research setti ng, as variables (e.g. swabbing strategy and STR kit) can be manipulated and the amount of accurate genetic information that is produced can be measured. If cumulative and individual swabbing were compared in the current research without knowing the handle non - handler alleles/polymorphisms would not have been recognized. Another group of researchers, Dieltjes et al. (2011), developed an extraction procedure for cartridges, bullets, and casi ngs and reported that profiles were generated from 6.9% of the 4,085 items tests, but whether the alleles were consistent with the individual who loaded or shot the firearm could not be established because the items were from criminal investigations for wh ich the handler was not known, thus the true effectiveness of the method is unclear. Profiles from evidence are typically compared to that of a suspect, although they may not be the individual who loaded the weapon. If an examiner assumed the evidence and the suspect are consistent, they would be biased and could attribute any inconsistent alleles to drop - in, even if they are not. Conversely, if all alleles are presumed to be correct, the presence of even one non - handler allele might result in the exclusion of the individual who loaded the firearm. Guidelines regarding the conclusions that are made from DNA from spent casings must therefore be developed/adopted and strictly followed. As a hypothetical example, a laboratory policy could state that 20% of the alleles in a profile can be inconsistent with a suspect who is said to be included as a possible source of the DNA, and that 50% must be inconsistent to completely exclude the suspect, while anything in between is 77 inconclusive. Additionally, the laboratory may require that a profile includes a minimum number of alleles (e.g. 10) in order for any conclusions to be made. If these policies were applied to the this research, approximately 62% of the Collection 2 profiles would have been classified as inconclusi ve/not enough information, 30% as including the loader, and 8% as excluding the loader. However, due to the prevalence of non - handler alleles in the MSU - fumed casings, only 13% of Collection 2 profiles would have included the loader, while 18% would have e xcluded them and 69% were inconclusive/not enough information. The presence of alleles/polymorphisms that are inconsistent with a suspect could have committed the crime, so being aware of potential sources of contamination would allow them to classify a profile as being consistent with the suspect even when alleles in consistent with the defendant are present . For instance, if a partial STR profile is produced that includes 20 alleles consistent with the suspect and two that are not, it is possible that t he inconsistent alleles are f rom the surface the casing fell on, the manufacturing process, or any of the other contamination sources mentioned previously, so the pr osecution would likely argue that the DNA still came from the suspect. On the other hand, the defense could contend that even one inconsistent allele excludes the suspect as the source of the DNA. Additionally, they may ar gue that the entire profile was th e result of contamination, and does not mean that they fired the gun, or even that they loaded the cartridge. Instead, the defense cells whe n it fell onto the floor, or that the suspect had handled the gun prior to the commission of the crime. Consequently, DNA analysts need to be aware of the possibility of contamination/artifacts in a profile from a spent casing and be cautious when making c laims about the consistency of the profile. 78 There are many other variables that remain to be investigated regarding the recovery and analysis of DNA from spent cartridge casings. First, experiments could be conducted to further explore the effect of loadin g and firing order, which were not examined individually in the current study. Whether loading order alone actually affects the amount of DNA deposited on casings may be determined by having volunteers load cartridges into a magazine, then manually cycling the weapon (without firing it) to eject them. Cartridges from each volunteer would be collected individually and processed for DNA in order to directly compare them. Firing order might then be examined by repeating the experiment, this time firing the wea pon, and comparing the spent casings to the cartridges to see if the results differed. Second, though fuming certainly had a negative impact on DNA recovery and analysis from spent casings, whether transportation had an effect was not as clear due to the d ifferences in the fuming methods. Fuming casings on site immediately following firing and after a period of time during which the casings were transported using the same or a similar fuming chamber would reduce or eliminate variables such as the amount of cyanoacrylate deposited on the casings, making comparisons more straightforward. Next, additional techniques for analyzing low copy number DNA could be tested to improve the success of DNA profiling. For instance, post - PCR clean - up might increase the quant ity of genetic information produced utilizing commercial STR kits. It would also be beneficial to better understand the sources of non - handler DNA . If a portion of the non - handler alleles are due to STR artifacts and not contaminant DNA, it is possible tha t techniques such as replicate analysis will eliminate them. Additionally, using the same magazines, gun, and collection apparatus for all volunteers may have contributed to non - handler alleles/polymorphisms in the current research, and exaggerated the lik elihood of DNA contamination on casings collected from a crime scene , which would be valuable knowledge in 79 court . Examin ation of all these variables could greatly improve the accuracy of using DNA profiling from spent cartridge casings to identify the load er of a firearm. 80 CONCLUSIONS Overall, a variety of factors were examined in this research to determine what, if any, effect they have on the recovery and analysis of DNA from spent casings . Several important facts were determined over the course of this study. Among the m are: Significantly m ore DNA was recovered from 0.45 than from 0.22 caliber casings. Loading/firing order did not have a significant effect on the recovery and analysis of DNA from spent casings. Cyanoacrylate fuming spe nt casings prior to DNA processing was detrimental to DNA recovery/ analysis. Cumulative swabbing recovered more DNA than individual swabbing, but also resulted in more non - handler alleles/polymorphisms. The Fusion STR kit generated more genetic information than MiniFiler MtDNA was more sensitive than STR analysis, and resulted in fewer mixed/incorrect profiles. Knowledge of these facts can aid law enforcement in the accurate identification of the individual who loaded a firearm based on DNA from spent cas ings, providing valuable evidence that may be used during the investigation of a firearm offense. 81 APPENDICES 82 APPENDIX A. ASSIGNMENT OF FUMING METHODS AND SWABBING STRATEGIES FOR COLLECTIONS 1 AND 2 Table A1 . Assignment of fuming methods for Colle ction 1 . Bag # Buccal Letter Casing # Fired Fuming Method 2 - 1 U 1 thru 3 Fumed at MSP 2 - 2 U 4 thru 6 Fumed at MSU 2 - 3 U 7 thru 9 Not Fumed 2 - 4 U 10 thru 12 Used in a different study 2 - 5 U 13 thru 15 Used in a different study 2 - 6 U 16 thru 18 Used in a different study 2 - 7 U 19 thru 21 Used in a different study 3 - 1 MM 1 thru 3 Used in a different study 3 - 2 MM 4 thru 6 Fumed at MSP 3 - 3 MM 7 thru 9 Fumed at MSU 3 - 4 MM 10 thru 12 Not Fumed 3 - 5 MM 13 thru 15 Used in a different study 3 - 6 MM 16 thru 1 8 Used in a different study 3 - 7 MM 19 thru 21 Used in a different study 8 - 1 S 1 thru 3 Used in a different study 8 - 2 S 4 thru 6 Used in a different study 8 - 3 S 7 thru 9 Fumed at MSP 8 - 4 S 10 thru 12 Fumed at MSU 8 - 5 S 13 thru 15 Not Fumed 8 - 6 S 16 t hru 18 Used in a different study 8 - 7 S 19 thru 21 Used in a different study 10 - 1 VV 1 thru 3 Used in a different study 10 - 2 VV 4 thru 6 Used in a different study 10 - 3 VV 7 thru 9 Used in a different study 10 - 4 VV 10 thru 12 Fumed at MSP 10 - 5 VV 13 th ru 15 Fumed at MSU 10 - 6 VV 16 thru 18 Not Fumed 10 - 7 VV 19 thru 21 Used in a different study 13 - 1 V 1 thru 3 Used in a different study 13 - 2 V 4 thru 6 Used in a different study 83 13 - 3 V 7 thru 9 Used in a different study 13 - 4 V 10 th ru 12 Used in a different study 13 - 5 V 13 thru 15 Fumed at MSP 13 - 6 V 16 thru 18 Fumed at MSU 13 - 7 V 19 thru 21 Not Fumed 15 - 1 HH 1 thru 3 Not Fumed 15 - 2 HH 4 thru 6 Used in a different study 15 - 3 HH 7 thru 9 Used in a different study 15 - 4 HH 10 thr u 12 Used in a different study 15 - 5 HH 13 thru 15 Used in a different study 15 - 6 HH 16 thru 18 Fumed at MSP 15 - 7 HH 19 thru 21 Fumed at MSU 23 - 1 L 1 thru 3 Fumed at MSU 23 - 2 L 4 thru 6 Not Fumed 23 - 3 L 7 thru 9 Used in a different study 23 - 4 L 10 th ru 12 Used in a different study 23 - 5 L 13 thru 15 Used in a different study 23 - 6 L 16 thru 18 Used in a different study 23 - 7 L 19 thru 21 Fumed at MSP 25 - 1 T 1 thru 3 Fumed at MSP 25 - 2 T 4 thru 6 Fumed at MSU 25 - 3 T 7 thru 9 Not Fumed 25 - 4 T 10 thru 12 Used in a different study 25 - 5 T 13 thru 15 Used in a different study 25 - 6 T 16 thru 18 Used in a different study 25 - 7 T 19 thru 21 Used in a different study 26 - 1 XX 1 thru 3 Used in a different study 26 - 2 XX 4 thru 6 Fumed at MSP 26 - 3 XX 7 thru 9 Fumed at MSU 26 - 4 XX 10 thru 12 Not Fumed 26 - 5 XX 13 thru 15 Used in a different study 26 - 6 XX 16 thru 18 Used in a different study 26 - 7 XX 19 thru 21 Used in a different study 27 - 1 N 1 thru 3 Used in a different study 27 - 2 N 4 thru 6 Used in a dif ferent study 84 27 - 3 N 7 thru 9 Fumed at MSP 27 - 4 N 10 thru 12 Fumed at MSU 27 - 5 N 13 thru 15 Not Fumed 27 - 6 N 16 thru 18 Used in a different study 27 - 7 N 19 thru 21 Used in a different study 24 - 1 OO 1 thru 3 Used in a different study 24 - 2 OO 4 thru 6 Used in a different study 24 - 3 OO 7 thru 9 Used in a different study 24 - 4 OO 10 thru 12 Fumed at MSP 24 - 5 OO 13 thru 15 Fumed at MSU 24 - 6 OO 16 thru 18 Not Fumed 24 - 7 OO 19 thru 21 Used in a different study 33 - 1 B 1 thru 3 Used in a different study 33 - 2 B 4 thru 6 Used in a different study 33 - 3 B 7 thru 9 Used in a different study 33 - 4 B 10 thru 12 Used in a different study 33 - 5 B 13 thru 15 Fumed at MSP 33 - 6 B 16 thru 18 Fumed at MSU 33 - 7 B 19 thru 21 Not Fumed 36 - 1 D 1 thru 3 Not Fumed 36 - 2 D 4 thru 6 Used in a different study 36 - 3 D 7 thru 9 Used in a different study 36 - 4 D 10 thru 12 Used in a different study 36 - 5 D 13 thru 15 Used in a different study 36 - 6 D 16 thru 18 Fumed at MSP 36 - 7 D 19 thru 21 Fumed at MSU 38 - 1 WW 1 thru 3 Fumed at MSU 38 - 2 WW 4 thru 6 Not Fumed 38 - 3 WW 7 thru 9 Used in a different study 38 - 4 WW 10 thru 12 Used in a different study 38 - 5 WW 13 thru 15 Used in a different study 38 - 6 WW 16 thru 18 Used in a different study 38 - 7 WW 19 thru 21 Fumed at MSP 40 - 1 SS 1 thru 3 Fumed at MSP 40 - 2 SS 4 thru 6 Fumed at MSU 85 40 - 3 SS 7 thru 9 Not Fumed 40 - 4 SS 10 thru 12 Used in a different study 40 - 5 SS 13 thru 15 Used in a different study 40 - 6 SS 16 thru 18 Used in a different st udy 40 - 7 SS 19 thru 21 Used in a different study 41 - 1 Y 1 thru 3 Used in a different study 41 - 2 Y 4 thru 6 Fumed at MSP 41 - 3 Y 7 thru 9 Fumed at MSU 41 - 4 Y 10 thru 12 Not Fumed 41 - 5 Y 13 thru 15 Used in a different study 41 - 6 Y 16 thru 18 Used in a different study 41 - 7 Y 19 thru 21 Used in a different study 50 - 1 II 1 thru 3 Used in a different study 50 - 2 II 4 thru 6 Used in a different study 50 - 3 II 7 thru 9 Fumed at MSP 50 - 4 II 10 thru 12 Fumed at MSU 50 - 5 II 13 thru 15 Not Fumed 50 - 6 II 16 t hru 18 Used in a different study 50 - 7 II 19 thru 21 Used in a different study Table A2 . Assignment of swabbing strategies for Collection 2. Bag # Caliber Buccal Letter Casing # Fired Swabbing Strategy 70 1 0.45 and 0.22 OOO 1 3 Cumulative 70 2 0.45 and 0.22 OOO 4 6 Cumulative 70 3 0.45 and 0.22 OOO 7 9 Individual 70 4 0.45 and 0.22 OOO 10 12 Cumulative 53 1 0.45 and 0.22 B 1 3 Cumulative 53 2 0.45 and 0.22 B 4 6 Cumulative 53 3 0.45 and 0.22 B 7 9 Cumulative 53 4 0.45 and 0.22 B 10 12 Individual 67 1 0.45 and 0.22 VVV 1 3 Individual 67 2 0.45 and 0.22 VVV 4 6 Cumulative 67 3 0.45 and 0.22 VVV 7 9 Cumulative 86 67 4 0.45 and 0.22 VVV 10 12 Cumulative 51 1 0.45 and 0.22 NN 1 3 Cumulative 51 2 0.45 and 0.22 NN 4 6 Individual 51 3 0.45 and 0.22 NN 7 9 Cumulative 51 4 0.45 and 0.22 NN 10 12 Cumulative 58 1 0.45 and 0.22 J 1 3 Cumulative 58 2 0.45 and 0.22 J 4 6 Cumulative 58 3 0.45 and 0.22 J 7 9 Individual 58 4 0.45 and 0.22 J 10 12 C umulative 56 1 0.45 and 0.22 AA 1 3 Cumulative 56 2 0.45 and 0.22 AA 4 6 Cumulative 56 3 0.45 and 0.22 AA 7 9 Cumulative 56 4 0.45 and 0.22 AA 10 12 Individual 66 1 0.45 and 0.22 DDD 1 3 Individual 66 2 0.45 and 0.22 DDD 4 6 Cumulative 6 6 3 0.45 and 0.22 DDD 7 9 Cumulative 66 4 0.45 and 0.22 DDD 10 12 Cumulative 69 1 0.45 and 0.22 R 1 3 Cumulative 69 2 0.45 and 0.22 R 4 6 Individual 69 3 0.45 and 0.22 R 7 9 Cumulative 69 4 0.45 and 0.22 R 10 12 Cumulative 52 1 0.45 and 0 .22 ZZZ 1 3 Cumulative 52 2 0.45 and 0.22 ZZZ 4 6 Cumulative 52 3 0.45 and 0.22 ZZZ 7 9 Individual 52 4 0.45 and 0.22 ZZZ 10 12 Cumulative 61 1 0.45 and 0.22 I 1 3 Cumulative 61 2 0.45 and 0.22 I 4 6 Cumulative 61 3 0.45 and 0.22 I 7 9 Cumulative 61 4 0.45 and 0.22 I 10 12 Individual 68 1 0.45 and 0.22 XXX 1 3 Individual 68 2 0.45 and 0.22 XXX 4 6 Cumulative 68 3 0.45 and 0.22 XXX 7 9 Cumulative 68 4 0.45 and 0.22 XXX 10 12 Cumulative 59 1 0.45 and 0.22 KKK 1 3 Cumulati ve 59 2 0.45 and 0.22 KKK 4 6 Individual 87 59 3 0.45 and 0.22 KKK 7 9 Cumulative 59 4 0.45 and 0.22 KKK 10 12 Cumulative 62 1 0.45 and 0.22 YYY 1 3 Cumulative 62 2 0.45 and 0.22 YYY 4 6 Cumulative 62 3 0.45 and 0.22 YYY 7 9 Individual 62 4 0.45 and 0.22 YYY 10 12 Cumulative 60 1 0.45 and 0.22 JJJ 1 3 Cumulative 60 2 0.45 and 0.22 JJJ 4 6 Cumulative 60 3 0.45 and 0.22 JJJ 7 9 Cumulative 60 4 0.45 and 0.22 JJJ 10 12 Individual 57 1 0.45 and 0.22 A 1 3 Indivi dual 57 2 0.45 and 0.22 A 4 6 Cumulative 57 3 0.45 and 0.22 A 7 9 Cumulative 57 4 0.45 and 0.22 A 10 12 Cumulative 63 1 0.45 and 0.22 EE 1 3 Cumulative 63 2 0.45 and 0.22 EE 4 6 Individual 63 3 0.45 and 0.22 EE 7 9 Cumulative 63 4 0.45 a nd 0.22 EE 10 12 Cumulative 54 1 0.45 and 0.22 BBB 1 3 Cumulative 54 2 0.45 and 0.22 BBB 4 6 Cumulative 54 3 0.45 and 0.22 BBB 7 9 Individual 54 4 0.45 and 0.22 BBB 10 12 Cumulative 39 1 0.45 and 0.22 SSS 1 3 Cumulative 39 2 0.45 and 0.22 SSS 4 6 Cumulative 39 3 0.45 and 0.22 SSS 7 9 Cumulative 39 4 0.45 and 0.22 SSS 10 12 Individual 55 1 0.45 and 0.22 C 1 3 Individual 55 2 0.45 and 0.22 C 4 6 Cumulative 55 3 0.45 and 0.22 C 7 9 Cumulative 55 4 0.45 and 0.22 C 10 12 Cum ulative 65 1 0.45 and 0.22 JJ 1 3 Cumulative 65 2 0.45 and 0.22 JJ 4 6 Individual 65 3 0.45 and 0.22 JJ 7 9 Cumulative 65 4 0.45 and 0.22 JJ 10 12 Cumulative 88 APPENDIX B. DNA QUANTITIES RECOVERED FROM SPENT CARTRIDGE CASINGS FROM COLLECTION 1 Table B1 . Quantitation results of casings fumed at MSU from Collection 1. Sample DNA Yield (pg) 2 - 2a 27.00 1.01E+00 27.27 2 - 2b 24.20 1.05E+00 25.41 2 - 2c 22.50 1.12E+01 252.00 3 - 3a 23.50 6.76E - 01 15.89 3 - 3b 25.50 6.10E - 01 15.56 3 - 3c 27.00 5.79E - 01 15.63 8 - 4a 26.00 1.58E+00 41.08 8 - 4b 19.40 5.61E - 01 10.88 8 - 4c 21.00 7.21E - 01 15.14 10 - 5a 26.50 3.10E - 02 0.82 10 - 5b 23.80 1.42E - 01 3.38 10 - 5c 21.60 7.33E - 02 1.58 13 - 6a 24.80 4.18E+00 103.66 13 - 6b 22.50 4 .81E+00 108.23 13 - 6c 24.00 2.18E+00 52.32 15 - 7a 26.60 1.83E - 01 4.87 15 - 7b 26.40 3.14E - 01 8.29 15 - 7c 21.00 1.25E+00 26.25 23 - 1a 22.00 1.05E+00 23.10 23 - 1b 25.50 3.17E - 01 8.08 23 - 1c 27.00 1.04E+00 28.08 24 - 5a 24.00 6.20E - 01 14.88 24 - 5b 26.40 1.90E - 0 1 5.02 24 - 5c 27.00 1.45E - 01 3.92 25 - 2a 27.20 2.71E - 02 0.74 25 - 2b 22.50 1.06E - 01 2.39 25 - 2c 26.00 2.14E - 02 0.56 26 - 3a 27.20 1.31E+00 35.63 26 - 3b 27.50 8.69E - 01 23.90 26 - 3c 23.00 6.36E+00 146.28 27 - 4a 21.70 2.88E - 01 6.25 27 - 4b 20.50 3.83E+00 78.52 27 - 4c 23.00 2.05E+00 47.15 33 - 6a 26.20 1.61E+00 42.18 89 33 - 6b 27.00 5.26E+01 1420.20 33 - 6c 25.50 4.94E - 01 12.60 36 - 7a 30.00 4.85E - 02 1.46 36 - 7b 27.50 1.11E - 01 3.05 36 - 7c 26.70 1.48E - 02 0.40 38 - 1a 22.80 4.37E - 01 9.96 38 - 1b 24.20 4.12 E - 01 9.97 38 - 1c 22.80 1.49E+00 33.97 40 - 2a 27.80 7.25E - 02 2.02 40 - 2b 29.70 8.56E - 02 2.54 40 - 2c 25.00 3.57E - 01 8.93 41 - 3a 25.50 2.05E - 01 5.23 41 - 3b 24.00 2.34E - 01 5.62 41 - 3c 25.80 6.21E - 01 16.02 50 - 4a 7.00 8.61E - 01 6.03 50 - 4b 24.00 2.68E - 01 6.43 5 0 - 4c 20.40 5.65E - 01 11.53 Table B2. Quantitation results of casings fumed at MSP from Collection 1. Sample DNA Yield (pg) 2 - 1a 29.30 1.32E - 01 3.87 2 - 1b 30.00 7.02E - 03 0.21 2 - 1c 27.00 8.43E - 01 22.76 3 - 2a 20.00 2.61E - 07 0.00 3 - 2b 26.30 4.29E - 04 0.01 3 - 3c 28.60 5.18E - 03 0.15 8 - 3a 28.40 6.72E - 01 19.08 8 - 3b 27.80 7.99E - 01 22.21 8 - 3c 25.00 4.14E - 01 10.35 10 - 4a 26.70 2.85E - 03 0.08 10 - 4b 28.80 2.61E - 01 7.52 10 - 4c 26.00 2.46E - 01 6.40 13 - 5a 29.20 5.63E - 01 16.44 13 - 5b 30.30 1.31E+00 39.69 13 - 5c 26.00 1.89E+00 49.14 15 - 6a 26.00 3.60E - 02 0.94 15 - 6b 26.70 5.55E - 02 1.48 15 - 6c 24.40 4.52E - 04 0.01 23 - 7a 32.00 1.28E+00 40.96 90 23 - 7b 29.80 3.34E+00 99.53 23 - 7c 26.30 1.18E - 01 3.10 24 - 4a 25.70 8.07E - 03 0.21 24 - 4b 27.50 7.81E - 01 21.48 24 - 4c 27.40 2.92E - 02 0.80 25 - 1a 27.00 2.48E - 01 6.70 25 - 1b 28.40 1.00E - 01 2.84 25 - 1c 22.20 7.15E - 02 1.59 26 - 2a 26.50 7.83E - 01 20.75 26 - 2b 22.60 4.56E - 01 10.31 26 - 2c 22.10 1.07E+00 23.65 27 - 3a 28.20 1.18E - 01 3.33 27 - 3b 23.20 1.09E - 01 2.53 27 - 3c 27.30 1.45E - 01 3.96 33 - 5a 27.60 6.95E - 02 1.92 33 - 5b 23.00 2.15E - 01 4 .95 33 - 5c 27.80 5.15E - 01 14.32 36 - 6a 30.00 6.87E - 02 2.06 36 - 6b 27.50 4.01E - 01 11.03 36 - 6c 26.70 3.72E - 01 9.93 38 - 7a 25.80 1.72E - 01 4.44 38 - 7b 27.00 1.87E - 01 5.05 38 - 7c 25.00 6.57E - 02 1.64 40 - 1a 27.80 1.77E - 01 4.92 40 - 1b 29.70 7.88E - 02 2.34 40 - 1c 25.00 1.27E - 01 3.18 41 - 2a 27.80 4.95E - 01 13.76 41 - 2b 29.00 5.45E - 01 15.81 41 - 2c 26.50 5.83E - 01 15.45 50 - 3a 28.50 8.62E - 01 24.57 50 - 3b 29.00 1.77E - 01 5.13 50 - 3c 28.80 8.98E - 02 2.59 91 Table B3. Quantitation results of non - fumed casings from Collec tion 1. Sample DNA Yield (pg) 2 - 3a 28.80 2.21E+00 63.65 2 - 3b 33.00 5.19E - 01 17.13 2 - 3c 26.00 8.25E - 01 21.45 3 - 4a 29.00 2.42E+00 70.18 3 - 4b 26.00 5.35E - 01 13.91 3 - 4c 24.00 3.40E - 01 8.16 8 - 5a 27.00 9.57E - 01 25.84 8 - 5b 2 5.00 1.66E+00 41.50 8 - 5c 29.00 1.97E+00 57.13 10 - 6a 26.20 2.59E - 01 6.79 10 - 6b 27.80 3.14E - 01 8.73 10 - 6c 28.40 2.28E - 01 6.48 13 - 7a 25.20 3.69E+00 92.99 13 - 7b 24.00 1.61E+01 386.40 13 - 7c 27.50 2.93E+00 80.58 15 - 1a 25.20 1.74E - 01 4.38 15 - 1b 30.80 3.6 7E - 01 11.30 15 - 1c 27.60 3.81E - 01 10.52 23 - 2a 25.60 5.14E+00 131.58 23 - 2b 24.00 4.15E+00 99.60 23 - 2c 25.20 1.38E+00 34.78 24 - 6a 26.80 1.24E - 01 3.32 24 - 6b 27.00 4.73E - 01 12.77 24 - 6c 27.40 3.06E - 01 8.38 25 - 3a 26.80 2.95E - 01 7.91 25 - 3b 24.80 3.54E - 01 8.78 25 - 3c 24.00 4.79E - 01 11.50 26 - 4a 24.50 1.70E+00 41.65 26 - 4b 26.80 1.78E+00 47.70 26 - 4c 27.00 1.38E+00 37.26 27 - 5a 21.20 1.38E+00 29.26 27 - 5b 18.80 1.39E+00 26.13 27 - 5c 24.50 1.31E+00 32.10 33 - 7a 24.50 2.16E+00 52.92 33 - 7b 24.40 2.65E+00 64.66 33 - 7c 25.60 1.19E+00 30.46 36 - 1a 22.20 9.91E - 01 22.00 36 - 1b 25.00 5.80E - 01 14.50 36 - 1c 28.00 8.94E - 01 25.03 92 38 - 2a 26.20 9.87E - 01 25.86 38 - 2b 27.20 1.55E+00 42.16 38 - 2c 26.00 7.87E - 01 20.46 40 - 3a 27.00 6.83E - 01 18.44 40 - 3b 28.80 1.83E+00 52.70 40 - 3c 27.20 3.53E - 01 9.60 41 - 4a 24.00 5.42E - 01 13.01 41 - 4b 29.30 4.04E+00 118.37 41 - 4c 25.50 7.36E - 01 18.77 50 - 5a 25.70 1.94E+00 49.86 50 - 5b 25.50 3.68E+00 93.84 50 - 5c 27.40 9.58E - 01 26.25 93 APPENDIX C. COMPARISON OF FUSION AND MI NIFILER STR PROFILES Red font: non - loader allele *: allele was above the threshold using OSIRIS, but below the threshold using GeneMapper ® . - ladder allele Blank cell: no alleles were amplified Gray cell: locus not amplified N/A: not applicable Ta ble C1. individual U. Locus 2 - 1c (Fusion) 2 - 1c 2 - 2a (Fusion) 2 - 2a U Amel X X,X D3 15 15,15 D1 15.3 , 16.3 11,17.3 D2 10,15 D10 12, 14 D13 13 9 9,13 Penta E 12 12,15 D16 11,13 6 11,13 D18 14,15 14 14,15 D2 17 17 17,25 CSF 10,12 Penta D 11 10,11 THO1 6 9 , 9.3 6,7 vWA 14,20 18 14,20 D21 28,30 D7 11 11,11 D5 11 11,11 TPOX 8,11 DYS391 N/ A D8 12 , , 13 , 15 12,12 D12 17,23 17,23 D19 15 13,13 FGA 24,25 D22 16 16,16 Fuming Method MSP - Fumed MSP - Fumed MSU - Fumed MSU - Fumed Buccal 94 Locus 2 - 2c (Fusion) 2 - 2c 2 - 3a 2 - 3a (Fusion) U Amel X X X X X,X D3 14 ,15, 18 15 15,15 D1 14 ,17.3 11, 17.3 11,17.3 D2 10, 11 , 11.3 10,15 10,15 D10 12,14 12 12,14 D13 10 , 11 9, 10 , 11 9 9,13 Penta E 12 12,15 D16 10 , 12 11 ,13 11 11,13 D18 12 , 17 12 , 17 13 ,14,15 14,15 14,15 D2 17, 18 ,25 17, 18 25 17,25 17,25 CSF 10 9 ,10 12 12 10,12 Penta D 16 10 10,11 THO1 6,7, 9.3 6,7 6,7 vWA 16 14 14,20 D21 30, 33.2 28,30, 33.2 28,30, 31 28,30 28,30 D7 10 , 12 11 11,11 D5 12 11 11,11 TPOX 8,11 8,11 8,11 DYS391 N/A D8 12, 14 , 15 12 12,12 D12 20 23 17,23 D19 13, 15 13 13,13 FGA 20 20, 17.2 , 24 ,25 24 , , , 24,25 D22 11 ,16 16 16,16 Fuming Method MSU - Fumed MSU - Fumed Non - Fumed Non - Fumed Buccal 95 Table C2. cartridge casings loaded by individual MM. Locus 3 - 3b (Fusion) 3 - 3b 3 - 4a (Fusion) 3 - 4a MM Amel X Y X,X D3 18 14,16 D1 12 12,16 D2 14 10,11 D10 14,15 D13 8,12 Penta E 12 7,21 D16 11 ,12 12 12,12 D18 13.2 , 16 12 14,14.2 D2 17, 18 , 22 17,23 17,23 CSF 12 12,13 Penta D 11 13,13 THO1 9.3 9,9.3 vWA 17,17 D21 29,31.2 D7 9,11 D5 9,10 TPOX 12 8,8 DYS391 N/A D8 10 ,15, 15.1 , , 13,15 D12 22 18,22 D19 13 14,15.2 FGA 23.2 22, 25 ,26.2 17.2 47.2 , , , 22,26 D22 16 11,12 Fuming Method MSU - Fumed MSU - Fumed Non - Fumed Non - Fumed Buccal 96 Table C3. rom spent cartridge casings loaded by individual S. Locus 8 - 3a (Fusion) 8 - 3a 8 - 3b (Fusion) 8 - 3b S Amel Y X,X D3 18 18,18 D1 12,15 D2 16 11,11.3 D10 13,15 D13 13 13 14 12,13 Penta E 12,13 D16 11 11 1 1,11 D18 12, 17 12,16 12,16 D2 17,25 17,25 17 17,25 CSF 10,11 Penta D 12 10,13 THO1 6, 9.3 6,9 vWA 15 ,17 17,18 D21 28, 30 28 28 28,28 D7 10, 12 10,10 D5 10,12 TPOX 11 8,11 DYS391 N/A D8 12 , 15 13 13,16 D12 18,18.3 18 18,18.3 D19 14 ,15 13.2,15 FGA 23, 24 22* 22,23 D22 15,15 Fuming Method MSP - Fumed MSP - Fumed MSP - Fumed MSP - Fumed Buccal 97 Table C3 Locus 8 - 3c (Fusion) 8 - 3c 8 - 4a (Fusion) 8 - 4a S Amel X X Y X,X D3 1 6 18,18 D1 12 12,15 D2 11,11.3 11,11.3 D10 15 13 13,15 D13 12 10 ,13 13 12,13 Penta E 12,13 D16 11 9 ,11, 13 11,11 D18 12,16 D2 17 18 17,25 CSF 6 10,11 Penta D 10,13 THO1 6 6 , 7 6,9 vWA 17 17 17,18 D21 28 28 28, 28 D7 10 10,10 D5 10,12 TPOX 8 8,11 DYS391 N/A D8 13,16 10 ,13, 15 ,16 13,16 D12 17 ,18 18,18.3 D19 13.2 13.2,15 FGA 22,23 D22 15,15 Fuming Method MSP - Fumed MSP - Fumed MSU - Fumed MSU - Fumed Buccal 98 Table C4. Fusion an individual VV. Locus 10 - 4b (Fusion) 10 - 4b VV Amel X X,Y D3 15 , 18 14,17 D1 12 , 14 ,15, 16.3 15,17.3 D2 11,14 D10 14 12,13 D13 9 ,11 12 11,11 Penta E 7,8 D16 12, 13 12,12 D18 13 ,16 14 12,16 D2 17 17,18 CSF 10 , 12 11,11 Penta D 12 9,12 THO1 6 ,9.3 9.3,9.3 vWA 15 , 18 17,17 D21 29 , 30.2 ,32.2 28,32.2 D7 10,11 D5 11,13 TPOX 8 11,11 DYS391 11 D8 8, 10 8,12 D12 23 15,25 D19 14 14,15.2 FGA 24 22 ,23 D22 11,15 Fuming Method MSP - Fumed MSP - Fumed Buccal 99 Table C5. individual V. Locus 13 - 5a (Fusion) 13 - 5a 13 - 5b (Fusion) 13 - 5b V Amel XY XY X,Y D3 14 14 14,14 D1 16 ,16.3 15 , 16 ,16.3, 17.3 16.3,17.3 D2 11 11,11.3 11,11.3 D10 15 13 ,15 15,16 D13 12 10, 12 10,12 Penta E 14 5,14 D16 12 11,12, 13 11,12 D18 16 16 14 16,17 D2 20 20,22 CSF 11 10,11 Penta D 12 11,1 2 THO1 9,9.3 9,9.3 9,9.3 vWA 16 16,18 16,18 D21 28,32.2 D7 12 11,12 D5 12 12 12,12 TPOX 8 8 8,8 DYS391 11 11 11 D8 12 9,12 9,12 D12 23 21,23 21,23 D19 12 12, 16 12,14 FGA 21.2, 22.2 21.2 21.2,22 D22 11 11 11,16 Fuming Method MSP - Fumed MSP - Fumed MSP - Fumed MSP - Fumed Buccal 100 Table C5 Locus 13 - 5c (Fusion) 13 - 5c 13 - 6a (Fusion) 13 - 6a V Amel Y XY X,Y D3 14 14,14 D1 16.3,17.3 16.3,17.3 D2 11.3 11 11,11.3 D10 16 15,16 15,16 D13 10,12 10,12 10, 12 10,12 10,12 Penta E 5,14 D16 11,12 11,12 D18 16 16,17 16,17 16,17 D2 20 20,22 20,22 20,22 20,22 CSF 10 10,11 11 10 10,11 Penta D 11 11,12 THO1 9,9.3 9,9.3 9,9.3 vWA 16 16,18 16,18 D21 32.2 32.2 28, 33.2 28,32.2 D7 11,12 11,12 D5 12 12,12 TPOX 8 8,8 DYS391 11 11 D8 9,12 9,12 9,12 D12 23 21,23 21,23 D19 12 12,14 12,14 FGA 21.2,22 21.2 21.2,22, 22.2 16.2 ,21.2,22 21.2,22 D22 11 11,16 Fuming Method MSP - Fumed MSP - Fumed MSU - Fumed MSU - Fumed Buccal 101 Table C5 Locus 13 - 6b (Fusion) 13 - 6b 13 - 6c (Fusion) 13 - 6c V Amel XY XY X,Y D3 14 14,15 14,14 D1 15.3 ,16.3, 17.3 14,17.3 16.3,17.3 D2 11,11.3 11,11.3 D10 15 13 , 16 15,16 D13 10,12 10,12 12 10 10,12 Penta E 5,14 5,14 D16 11,12 11,12 11, 13 11,12 D18 16,17 16,17, 18 12 16,17 D2 20,22 20,22, 23 23.3 17 ,20,22 20,22 CSF 11 10,11 12 10, 12 10,11 Penta D 11,12 11,12 THO1 7 , 9, 9.3 9 9,9.3 vWA 17 ,18 17 16,18 D21 28,32.2 28, 33.2 28,32.2 D7 11,12 9 ,11 11,12 D5 12 12,12 TPOX 7 8,8 DYS391 11 D8 9, 10 , 11 ,12 12, 14 , 15 9,12 D12 17 ,21,23 21, 23 21,23 D19 12,14 14 12,14 FGA 22 , 2 3 21 ,22, 23 17 20 ,22 21.2,22 D22 11 11,16 Fuming Method MSU - Fumed MSU - Fumed MSU - Fumed MSU - Fumed Buccal 102 Table C5 Locus 13 - 7a (Fusion) 13 - 7a 13 - 7b (Fusion) 13 - 7b V Amel X ,Y X ,Y X ,Y X,Y D3 14, 18 14 14,14 D1 16.3,17.3 16.3,17.3 16.3,17.3 D2 11 , 11.3 11 , 11.3 11,11.3 D10 15 ,16 15,16 D13 10 12 10, 12 10, 12 10,12 Penta E 5,14 5,14 5,14 D16 11 ,12 11 ,12 11 ,12 11 ,12 11,12 D18 17 16 16 ,17 16 ,17 16,17 D2 20,22 17 ,20,22, 25 20,22 20,22 20,22 CSF 11 10,1 1 10,11 10,11 Penta D 12 11,12 11,12 THO1 9 ,9.3 9 ,9.3 9,9.3 vWA 16, 18 16, 18 16,18 D21 28 ,32.2 32.2 28 ,32.2 28 ,32.2 28,32.2 D7 12 11,12 11 11,12 D5 12 12 12,12 TPOX 8 8,8 DYS391 11 11 11 D8 9,12 9,12 9,12 D12 20 ,21,23 21,23 21,23 D19 12 12,14 FGA 22 21.2, 31.2 , , 21.2, 22 21.2, 22 , 21.2,22 D22 11,16 11,16 Fuming Method Non - Fumed Non - Fumed Non - Fumed Non - Fumed Buccal 103 Table C5 Locus 13 - 7c (Fusion) 13 - 7c V Amel X ,Y X,Y D3 14 14,14 D1 16.3,1 7.3 D2 11 , 11.3 11,11.3 D10 15 ,16 15,16 D13 12 10,12 Penta E 5,14 5,14 D16 12 11 ,12 11,12 D18 16 ,17 16 16,17 D2 20 20,22 20,22 CSF 10 10,11 10,11 Penta D 12 11,12 THO1 9 ,9.3 9,9.3 vWA 14 , 17 , 18 16,18 D21 28 28 28,32.2 D7 11,12 11 1 1,12 D5 12,12 TPOX 8,8 DYS391 11 11 D8 9,12 9,12 D12 23 21,23 D19 12 12,14 FGA 22 , 23 , 32.2 21.2, , 21.2,22 D22 11,16 11,16 Fuming Method Non - Fumed Non - Fumed Buccal 104 Table C6. spent cartridge casings loaded by individual HH. Locus 15 - 7c (Fusion) 15 - 7c HH Amel X,X D3 14 14,18 D1 15.3 16,17.3 D2 11,14 D10 13,15 D13 10,11 Penta E 10,14 D16 9,12 D18 12 , 16,16 D2 17, 18 ,19 17,19 CSF 11,13 Penta D 10,10 THO1 6 , 7 9,9 vWA 14,16 D21 30,31 D7 11 11,12 D5 9,12 TPOX 9,11 DYS391 N/A D8 10*, 15* 10,13 D12 18* 20,21 D19 14 13,14 FGA 20 22,25 D22 16,16 Fuming Method MSU - Fumed MSU - Fumed Buccal 105 Table C7. Fusion an individual L. Locus 23 - 1a (Fusion) 23 - 1a 23 - 7a (Fusion) 23 - 7a L Amel X X X X,X D3 17 16,16 D1 15.3 16,17.3 D2 11 11 11,11 D10 13 13,15 13,15 D1 3 12 ,13 13 13 13,13 Penta E 7 7,7 D16 11 11 11 11,11 D18 12 ,16 12 15,16 19 15,16 D2 16 ,17, 18 , 19 , 24 17 17 17,17 CSF 12 11 ,12 12,13 Penta D 10 , 14 9,11 THO1 9 , 9.3 8,9.3 8,9.3 vWA 17 , 19 14, 17 ,18 14,18 D21 29 31.2 24 ,30 27,30 D7 8 10 8,10 D5 11 11,12 TPOX 8,8 DYS391 10 N/A D8 13,14 13,14 D12 21 18* 18,20 D19 15 14,15 FGA 23, 25 21,23 23 21,23 D22 16 15,16 Fuming Method MSU - Fumed MSU - Fumed MSP - Fumed MSP - Fumed Buccal 106 Table C7 Locus 23 - 7b (Fus ion) 23 - 7b 23 - 7c (Fusion) 23 - 7c L Amel X X X,X D3 16 16 16,16 D1 16 16,17.3 D2 11 11,11 D10 13 13,15 D13 13 13 13,13 Penta E 7 7,7 D16 11 11 11,11 D18 16 15,16 D2 17, 23 17 17,17 CSF 12 12,13 Penta D 9,11 THO1 8,9.3 8,9.3 vWA 14,18 14 14,18 D21 30 30 27 27,30 D7 8,10 12 8,10 D5 12 11,12 11,12 TPOX 8,8 DYS391 N/A D8 11 ,13,14 13,14 13,14 D12 18,20 20 18,20 D19 15 14,15 FGA 23 21,23 D22 15,16 Fumin g Method MSP - Fumed MSP - Fumed MSP - Fumed MSP - Fumed Buccal 107 Table C7 Locus 23 - 2a (Fusion) 23 - 2a 23 - 2b (Fusion) 23 - 2b L Amel X X X X X,X D3 16 16, 17 16,16 D1 16, 17.3 16, 17.3 16,17.3 D2 11 11 11,11 D10 15 13,15 D13 13 13 13 13,13 Penta E 7 7 7,7 D16 11 11 11 11 11,11 D18 15,16 15,16 15 15,16 15,16 D2 17 17 17 17 , 19 17,17 CSF 13 12 ,13, , 12 ,13, , 12,13 Penta D 8.2 9,11 THO1 8 ,9.3 8 ,9.3 8,9.3 vWA 14 14,18 14,18 D21 30 2 7,30 30 27,30 D7 8 8,10 8,10 D5 11,12 TPOX 8,8 DYS391 N/A D8 13 ,14 13 ,14 13,14 D12 20 18,20 18,20 D19 14 , 15 14,15 FGA 21,23 23.2 , 30 , , , 21, , 21,23 21,23 D22 , 16 15,16 Fuming Method Non - Fumed Non - Fumed Non - Fumed Non - Fumed Buccal 108 Table C8. individual OO. Locus 24 - 4b (Fusion) 24 - 4b 24 - 5a (Fusion) 24 - 5a OO Amel X X,X D3 14 , 16 15 15,1 8 D1 15.3 14,18.3 D2 11.3 ,14 11,14 D10 14,15 D13 10 11 ,12 9,12 Penta E 12 10,13 D16 13 11 13 12,12 D18 12 12 11,14 D2 22 22 ,25 17,25 CSF 10, 12 10 10,11 Penta D 11 9,12 THO1 9.3 6 6,9 vWA 17 17 17,18 D21 28, 33.2 28,31.2 D7 10 10,10 D5 12 , 13 11,11 TPOX 12 8 8,8 DYS391 N/A D8 10 14* , 15 12,17 D12 18 , 21 18.3,20 D19 14, 15 14 14,16 FGA 21 , 23 21 , 23 20 , 18,24 D22 16 11,15 Fuming Method MSP - Fumed MSP - Fumed MSU - Fumed MSU - Fumed Buccals 109 Table C9. individual XX. Locus 26 - 2a (Fusion) 26 - 2a 26 - 2b (Fusion) 26 - 2b XX Amel X X,X D3 15 14,15, 16 , 18 14,15 D1 12 14,17.3 14,17.3 D2 12,14 D10 14,16 D13 10 13 13 12,13 Penta E 12 12,12 D16 13 13 11,13 D18 12 ,17 18 17,18 D2 16 17,17 CSF 10,12 10 10,12 10,12 Penta D 12,12 THO1 9,9.3 9.3 9,9.3 vWA 17 17,19 D21 29 29,32 D7 12 12 9,12 D5 10 10, 13 TPOX 8 8,12 DYS391 N/A D8 10,13 10 10,13 D12 18, 21 20 18,22 D19 14 13,14 FGA 23 21,23 D22 16,17 Fuming Method MSP - Fumed MSP - Fumed MSP - Fumed MSP - Fumed Buccal 110 Table C9 Locus 26 - 2c (Fusion) 26 - 2c 26 - 3 a (Fusion) 26 - 3a XX Amel X X X,X D3 14,15 , 16 14,15 D1 15.3 ,17.3 15 , 15.3 14,17.3 D2 12,14 11 12,14 D10 14,16 D13 12 12,13 Penta E 7 12,12 D16 OL5.1 ,11,13 11 11,13 D18 17 12 , 15 , 16 13 , 24 , 17,18 D2 17 17 20 , 24 17 ,17 CSF 10,12 10,12 Penta D 3.2 , 10 , 11 12,12 THO1 7 ,9.3 8 ,9.3 9,9.3 vWA 17,19 17 17,19 D21 30 30 29,32 D7 9,12 9 9,12 D5 9 ,13 10 10,13 TPOX 8 8 8,12 DYS391 N/A D8 10,13, 15 13, 14 10,13 D12 18,22 18,22 D19 15 13,14 F GA 23, 24 21 21,23 D22 16 15 16,17 Fuming Method MSP - Fumed MSP - Fumed MSU - Fumed MSU - Fumed Buccal 111 Table C9 Locus 26 - 3c (Fusion) 26 - 3c XX Amel X Y X X,X D3 15, 16 , 17 14,15 D1 11 14,17.3 D2 10 , 11 12,14 D10 13 , 15 14,16 D1 3 10 10 , 11 ,12,13 12,13 Penta E 5 , 14 12,12 D16 9 ,11, 11.3 9 , 11 11,13 D18 17, 21 15 ,17, 21 17,18 D2 20 , 24 17, 20 , 24 17,17 CSF 10,12, 13 10 10,12 Penta D 9 , 11 12,12 THO1 9 9,9.3 vWA 14 , 16 , 18 17,19 D21 31 , 32.2 31 , 33.2 29,32 D7 8 ,9, 11 9,12 D5 7 10,1 3 TPOX 11 ,12 8,12 DYS391 10 N/A D8 10, 12 ,13,1 4 , 16 10,13 D12 18, 21 18,22 D19 14.2 , 15 13,14 FGA 20 , 22 , 24 21, 22 ,23 21,23 D22 15 ,16 16,17 Fuming Method MSU - Fumed MSU - Fumed Buccal 112 Table C10. cartridge casings loaded by individual N. Locus 27 - 4b (Fusion) 27 - 4b 27 - 4c (Fusion) 27 - 4c N Amel X X X,X D3 15 ,17, 18 16,17 D1 12 ,17.3 15.3,17.3 D2 11, 11.3 11,11 D10 13, 15 13,13 D13 11 , 13 12,14 Penta E 13 13,15 D16 11 10 12,13 D18 12 , 16 13,14 D2 17 , 25 17 , 25 20,23 CSF 10 11 11,12 Penta D 10 10,12 THO1 6, 9 ,9.3 6,9.3 vWA 17,18 17,18 D21 28 28 30,32.2 D7 10 11,12 D5 10 ,12 12,12 TPOX 8 8,8 DYS391 N/A D8 12 ,13 13 1 3,13 D12 18 , 18.3 19,20 D19 13.2 , 15 13,14 FGA 22 , 29.1 21,25 D22 15 11,15 Fuming Method MSU - Fumed MSU - Fumed MSU - Fumed MSU - Fumed Buccal 113 Table C11. individual B. Locus 33 - 5c (Fusion) 33 - 5c 33 - 6a (Fusion) 33 - 6a B Amel X X,Y D3 14 ,16 18 16,18 D1 14 ,16.3 16.3,17.3 D2 11.3 ,14 14,15 D10 16 15 13,15 D13 10 10 10,12 Penta E 7,18 D16 9, 11 , 12 11 , 12 ,13 9,13 D18 12 ,13, 19.2 13,15 D2 17 22 20 , 22 20,25 CSF 10,12 12 12 10,12 Penta D 12,13 THO1 8 6 , 8 , 9.3 8,9.3 vWA 16 ,17,18 17,18 17,18 D21 28 ,29, 30 31 29,31 D7 10 9,12 D5 13 12 ,13 11,13 TPOX 12 8,12 8,8 DYS391 11 D8 8,13 8 , 13 8,13 D12 23 21 22,23 D19 14 ,15 13,15 FGA 21,23 D22 16 15,16 Fuming Method MSP - Fumed MSP - Fumed MSU - Fumed MSU - Fumed Buccal 114 Table C11 Locus 33 - 6b (Fusion) 33 - 6b 33 - 6c (Fusion) 33 - 6c B Amel X X,Y D3 14 ,16,1 8 18 16,18 D1 14 , 15.3 ,16.3,17.3 15.3 , 16.3 16.3,17.3 D2 11.3 ,14,15 14,15 D10 15 13,15 D13 10,12 10,12 10,12 Penta E 7, 12 7,18 D16 11 ,13 9,13 9,13 D18 12 12 16 , 18 13,15 D2 18 , 22 ,25 18 ,20, 22 ,25 22 ,25 20,25 CSF 10,12 12 12 10,12 P enta D 9 , 11 ,12,13 12,13 THO1 6 ,9.3 9.3 8,9.3 vWA 17 17 17,18 D21 28 ,29,31, 33.2 28 ,29, 32 , 33.2 32.2 29,31 D7 9, 10 ,12 12 9,12 D5 11, 12 , 13 11,13 TPOX 8, 12 8 8,8 DYS391 11 11 D8 8, 10 , 15 8,13 8,13 D12 18 ,21,23 22 22,23 D19 13, 14 , 1 5 13,15 FGA 21,23 21,23 19.2 21,23 D22 15,16 17 15,16 Fuming Method MSU - Fumed MSU - Fumed MSU - Fumed MSU - Fumed Buccal 115 Table C11 Locus 33 - 7a (Fusion) 33 - 7a 33 - 7b (Fusion) 33 - 7b B Amel X , Y X , Y X,Y D3 16, 17 ,18 16 16,18 D1 16.3, 17.3 16.3,17.3 D2 14 ,15 14,15 D10 13,15 D13 10 12 10,12 10,12 Penta E 7,18 D16 9,13 9, 12 ,13 9,13 9,13 D18 15 15 13,15 13,15 13,15 D2 25 20 20,25 CSF 12 10, 12 10,12, , 10,12 Penta D 12,13 THO1 8, 9 , 9.3 8, 9.3 8,9.3 vWA 18 17 ,18 17,18 D21 29 29 29 29,31 D7 9 9,12 D5 13 11,13 TPOX 8,8 DYS391 11 D8 8 ,13 8 ,13 8,13 D12 22,23 22,23 D19 13, 14 13,15 FGA 31 , 20.2 , 23 , , 21, 23 , 48.2 21,23 D22 15,16 Fuming Method Non - Fumed Non - Fumed Non - Fumed Non - Fumed Buccal 116 Table C12. individual WW. Locus 38 - 1b (Fusion) 38 - 1b 38 - 1c (Fusion) 38 - 1c WW Amel X X X,X D3 16 16,18 D1 15 11,12 D2 11,14 D10 15,16 15,16 D13 12 10 , 12 8,9 Penta E 11,12 D16 , 11 ,12, 13 12,12 D18 12 12,15 D2 18 18 18 17,21 CSF 11,12 11,12 Penta D 10,12 THO1 7 , 9.1 ,9.3 6 9.3,9.3 vWA 16 ,17 15,17 D21 28,30 D 7 10,11 D5 13,13 TPOX 8,12 DYS391 N/A D8 10 10,12 D12 18, 23 17* , 21 18,19.3 D19 15 14 13,14 FGA 23 21 20,24 D22 16 16,16 Fuming Method MSU - Fumed MSU - Fumed MSU - Fumed MSU - Fumed Buccal 117 Table C13. profiles generated from spent cartridge casings loaded by individual SS. Locus 40 - 1a (Fusion) 40 - 1a SS Amel X,Y D3 14,18 D1 12 ,17.3 14,17.3 D2 10 11,11 D10 14,14 D13 10,12 Penta E 7,19 D16 11,12 D18 10,12 D2 20 17,20 CSF 11,12 Penta D 9,12 THO1 8,8 vWA 15 ,17 17,17 D21 29,32.2 29,32.2 D7 12,12 D5 13,13 TPOX 8 9,9 DYS391 11 11 D8 13 12,13 D12 15 15,24 D19 14,15 FGA 22,24 D22 18 16,16 Fuming Method MSP - Fumed MSP - Fumed Buccal 118 Table C14. individual Y. Locus 41 - 2c (Fusion) 41 - 2c 41 - 3c (Fusion) 41 - 3c Y Amel X X X,Y D3 14 ,16,17 16,17 D1 14 14, 15 12,14 D2 14 14,15 D10 1 4 14,15 D13 11 13,14 Penta E 14, 18 5,14 D16 11 10 11 11,12 D18 17 12 , 20.2 17,17 D2 17 17 17,24 CSF 12,14 OL,12,14 Penta D 8,13 THO1 9.3 9,9.3 vWA 14 14, 18 14,16 D21 30.2 29,30.2 D7 8 8 8,10 D5 12,12 TPOX 8 8,8 DYS391 11 D8 ,10, 13 10,14 D12 21 20 ,21 17,21 D19 13 14 13,16.2 FGA 21 ,27 22 22,27 D22 11,16 Fuming Method MSP - Fumed MSP - Fumed MSU - Fumed MSU - Fumed Buccal 119 Table C14 Locus 41 - 4b (Fusion) 41 - 4b Y Amel X ,Y X,Y D3 14 16,17 D1 16.3 , 17.3 12,14 D2 11.3 14,15 D10 15 , 16 14,15 D13 12 13,14 Penta E 5,14 5,14 D16 11 ,12 11 ,12 11,12 D18 16 ,17 16 ,17 17,17 D2 20 20 , 22 17,24 CSF 10 7 , 10 , 11 OL,12,14 Penta D 12 8,13 THO1 9, 9.3 9,9.3 vWA 16, 18 14,16 D21 28 , 32.2 28 , 32.2 29,30.2 D7 11 , 12 11 , 12 8,10 D5 12 12,12 TPOX 8 8,8 DYS391 11 11 D8 9 , 12 10,14 D12 21, 23 17,21 D19 12 , 14 13,16.2 FGA 21.2 ,22 25.2 , , 22,27 D22 11, 16 11,16 Fuming Method Non - Fumed Non - Fumed Buccal 120 Table C15. Fusion individual II. Locus 50 - 3a (Fusion) 50 - 3a 50 - 4c (Fusion) 50 - 4c II Amel X X X,Y D3 15 16 17,17 D1 15,18.3 D2 14 11.3 10,11 D10 15 13,15 D13 12 12 11,12 Penta E 13,14 D16 11 12,12 D18 13 13 17 16,17 D2 23 19,21 CSF 10 11 12,12 Penta D 9,13 THO1 7 ,8,9.3 8,9.3 vWA 17 15 15,17 D21 31, 31.2 29,31 D7 10,12 D5 12 12 11,12 TPOX 11 8,8 DYS391 11 D8 14 11,13 D12 20 18,20 18,20 D19 14 14 14,15.2 FGA 50.2 21,23 D22 17* 12 15,16 Fuming Method MSP - Fumed MSP - Fumed MSU - Fumed MSU - Fumed Buccal 121 Table C15 Locus 50 - 5b (Fusion) 50 - 5b II Amel Y Y X,Y D3 17 17,17 D1 15,18.3 15,18.3 D2 10,11 10,11 D10 13 13,15 D13 11,12 12 11,12 Penta E 13,14 13,14 D16 11 ,12 12 12,12 D18 16 16,17 16,17 D2 19,21 19,21 CSF 12 12 12,12 Penta D 9,13 THO1 6 ,8, 9 ,9.3 8,9.3 vWA 15,17 15,17 D21 31 29 29,31 D7 12 10 10,12 D5 12 11,12 TPOX 8 8,8 DYS391 11 D8 11,13 11,13 D12 18,20, 18,20 D19 14 14,15.2 FGA 23, 20 ,21,23, 25.2 , , , , , 21,23 D22 15,16 15,16 Fuming Method Non - Fumed Non - Fumed Buccal 122 APPENDIX D. COMPARISON OF HANDLER AND NON - HANDLER ALLELES AMPLIFIED WITH MINIFILER AND FUSION Table D1. Comparison of the number of handler alleles (H) and percent profile produced by #H Alleles #NH Alleles % Profile Sample Fusion Fusion Fusion 33 - 6b 8 35 6 21 44.0% 76.1% 2 - 2c 6 18 8 25 38.0% 46.2% 26 - 3c 9 13 10 39 56.0% 31.0% 13 - 6b 12 37 5 7 67.0% 84.1% 13 - 6a 10 34 2 1 56.0% 77.3% 27 - 4b 1 15 3 24 6.0% 37.5% 13 - 6c 5 15 3 11 28.0% 34.1% 27 - 4c 0 2 0 1 0.0% 5.0% 33 - 6a 2 14 1 13 11.0% 30.4% 8 - 4a 3 10 2 10 21.0% 25.0% 38 - 1c 0 3 1 7 0.0% 7.3% 26 - 3a 0 10 4 18 0.0% 23.8% 15 - 7c 2 4 2 10 13.0% 9.8% 23 - 1a 4 9 9 11 29.0% 23.7% 2 - 2a 0 8 0 10 0.0% 20.5% 41 - 3c 2 9 0 9 12.0% 20.5% 24 - 5a 2 6 5 7 13.0% 14.6% 3 - 3b 4 6 2 16 25.0% 1 4.6% 50 - 4c 1 7 1 4 6.0% 16.3% 33 - 6c 1 12 1 5 6.0% 26.1% 38 - 1b 0 12 2 11 0.0% 29.3% 123 Table D2. Comparison of the number of handler alleles (H) and percent profile produced by #H Alleles #NH Allel es % Profile Sample Fusion Fusion Fusion 23 - 7b 5 23 0 2 36.0% 60.5% 13 - 5c 9 19 0 0 50.0% 43.2% 13 - 5b 0 28 0 6 0.0% 63.6% 23 - 7a 5 23 2 2 36.0% 60.5% 26 - 2c 1 25 0 7 6.0% 59.5% 50 - 3a 2 8 4 8 13.0% 18.6% 2 - 1c 2 12 0 1 1 3.0% 30.8% 8 - 3b 3 5 0 1 21.0% 12.5% 26 - 2a 2 12 0 4 13.0% 28.6% 24 - 4b 1 6 1 23 7.0% 14.6% 8 - 3a 4 15 0 12 29.0% 37.5% 41 - 2c 1 17 0 4 6.0% 40.5% 13 - 5a 3 21 1 3 17.0% 47.7% 33 - 5c 1 15 0 11 6.0% 32.6% 26 - 2b 4 15 0 4 25.0% 35.7% 8 - 3c 0 15 0 0 0.0% 37.5% 10 - 4b 1 10 2 20 7.0% 24.4% 40 - 1a 0 8 0 6 0.0% 20.5% 23 - 7c 0 10 0 1 0.0% 26.3% Table D3. Comparison of the number of handler alleles (H) and percent profile produced by - fumed casings. #H Alleles #NH Alleles % Profile S ample Fusion Fusion Fusion 13 - 7b 17 43 0 0 94.4 % 97.7 % 23 - 2a 12 22 2 0 85.7 % 57.9 % 23 - 2b 10 23 3 2 71.4 % 60.5 % 41 - 4b 3 18 11 21 17.6 % 40.9 % 13 - 7a 8 31 3 2 44.4 % 70.4 % 50 - 5b 12 30 2 4 75 .0% 69.8 % 13 - 7c 11 29 0 4 61.1 % 65.9 % 33 - 7b 12 16 1 1 66.7 % 34.8 % 3 - 4a 0 2 1 3 0 .0% 4.9 % 2 - 3a 12 28 0 3 75 .0% 71.8 % 33 - 7a 5 23 1 4 27.8 % 50 .0% 124 APPENDIX E. FUSION STR PROFILES FROM COLLECTION 1. Red font: non - loader allele *: allele was above the threshold using OSIRIS, but below the threshold using GeneMapper ® . - ladder allele Blank cell: no alleles were amplified N/A: not applicable 125 Table E1. Fusion profiles generated from spent cartridge casings loaded by individual U. Locus 2 - 2a 2 - 2b 2 - 2c 2 - 1a 2 - 1b 2 - 1c 2 - 3a 2 - 3b 2 - 3c U Amel X X X X X X, Y X X,X D3 15 14 ,15, 18 15 15 15 15,15 D1 15.3 , 16.3 14 ,17.3 11,17.3 11 17.3 11,17.3 D2 10, 11 , 11.3 10,15 15 10,15 D10 12,14 12 12 14 12,14 D13 9 11 10 , 11 12 13 9,13 Penta E 12 12 15 12,15 D16 6 10 , 12 13 11,13 11,13 11,13 11,13 D18 12 , 17 14 14,15 13 ,14,15 14,15 14,15 D2 17 17, 18 ,25 19 25 17,25 17,25 CSF 10 10 12 10 10,12 Penta D 11 16 10 10,11 THO1 9 , 9.3 6 6,7, 9.3 6,7 7 6 6,7 6,7 7 6,7 vWA 18 16 14 14,20 14 14 15 14,20 D21 30, 33.2 30 28,30, 31 28,30 D7 11 10 , 12 11 11,11 D5 12 11 11 11 11 11,11 TPOX 8,11 8,11 11 8,11 DYS391 N/A D8 13 , 15 , 12 12, 14 , 15 12 12 12 12 12, 13 , 15 12,12 D12 18 20 17 17,23 23 17 17,23 D19 13, 15 15 13 13 13, 13 FGA 20 17.2 ,24, 25 24,25 D22 16 11 ,16 16 16,16 Fuming Method MSU - Fumed MSU - Fumed MSU - Fumed MSP - Fumed MSP - Fumed MSP - Fumed Non - Fumed Non - Fumed Non - Fumed Buccal 126 Table E2. Fusion profiles generated from spent cartridge casings loaded by in dividual MM. Locus 3 - 3a 3 - 3b 3 - 3c 3 - 2a 3 - 2b 3 - 2c 3 - 4a 3 - 4b 3 - 4c MM Amel X X Y Y X X,X D3 18 14,16 D1 12 11 16 12,16 D2 14 11 10,11 D10 14,15 D13 14 8 8,12 Penta E 12 7,21 D16 11 ,12 12, 13 12 12 11 12,12 D18 13.2 , 16 16 14.2 15 , 17 14,14.2 D2 17, 18 , 22 17,23 CSF 12 12,13 Penta D 11 13 13,13 THO1 9.3 6 9.3 6 ,9,9.3 9,9.3 vWA 17 17,17 D21 29,31.2 D7 8 9,11 D5 11 9,10 TPOX 12 8 8,8 DYS391 N/A D8 10 ,15, 15.1 , 13 12 ,13,15 13,15 D12 22 22 18 18,22 D19 13 14,15.2 FGA 23.2 17.2 22,26 D22 16 16 11,12 Fuming Method MSU - Fumed MSU - Fumed MSU - Fumed MSP - Fumed MSP - Fumed MSP - Fumed Non - Fumed Non - Fumed Non - Fumed Buccal 127 Table E3. Fusion profiles generated from spent cart ridge casings loaded by individual S. Locus 8 - 4a 8 - 4c 8 - 3a 8 - 3b 8 - 3c 8 - 5a 8 - 5b 8 - 5c S Amel X Y X Y X X X X X,X D3 16 14 , 16 18 18 18 18 18,18 D1 14 , 15.3 12 11 ,15 12,15 12,15 D2 11.3 16 11,11.3 11 11,11.3 D10 13 13 15 15 15 13 ,15 D13 10 ,13 10 ,12 13 14 12 13 13 12,13 Penta E 12 13 12,13 D16 9 ,11, 13 11, 13 11 11 11 11 11 11 11,11 D18 12 12, 17 12 12 12 12,16 D2 18 20 ,25 17,25 17 17 17,25 CSF 10, 12 11 13 10,11 Penta D 11 12 10,1 3 THO1 6 , 7 6,9, 9.3 6, 9.3 6 7 ,9 6,9 6,9 vWA 17 17,18 15 ,17 17 18 17,18 D21 28, 33.2 28, 30 28 28 29 , 34 28,28 D7 9 ,10, 11 10, 12 10 12 10 10 10,10 D5 13 12 10 10,12 TPOX 8 12 11 8 11 8,11 DYS391 N/A D8 10 ,13, 15 , 16 10 , 13 , 15 12 , 15 13 13,16 10 ,13,16 13,16 6 ,13, 14 ,16 13,16 D12 17 ,18 17 18,18.3 18 18 18,18.3 18.3 18,18.3 D19 14 , 15, 17 14 ,15 13.2 13.2,15 7 ,15, 16 , 19.2 13.2,15 FGA 21 ,23, 23, 24 22* 21 23, 22,23 D22 16 , 15,15 Fuming Method MSU - Fumed MSU - Fumed MSP - Fumed MSP - Fumed MSP - Fumed Non - Fumed Non - Fumed Non - Fumed Buccal 128 Table E4. Fusion profiles generated from spent cartridge casings loaded by individual VV. Locus 10 - 5a 10 - 5b 10 - 5c 10 - 4a 10 - 4b 10 - 4c 10 - 6a 10 - 6b 10 - 6c VV Amel X X X X X,Y D3 12 15 , 18 16 14,17 D1 12 , 14 ,15, 16.3 15,17.3 D2 14 11,14 D10 14 12,13 D13 12 9 ,11 11,11 Penta E 13 7,8 D16 11 12 12, 13 12,12 D18 10 , 17 13 ,16 12,16 D2 17 ,18 CSF 10 , 12 11,11 Penta D 10 12 9,12 THO1 8 6 9,9.3 6 ,9.3 9.3,9.3 vWA 15 , 18 15 , 18 17,17 D21 28 29 , 30.2 , 32.2 28,32.2 D7 9 10,11 D5 12 13 11,13 TPOX 8 11,11 DYS391 11 D8 14 10 11 8, 10 8, 13 8,12 D12 18 , 22 24 23 15,25 D19 14 14 14,15.2 FGA 24 32.2 , 22,23 D22 11,15 Fuming Method MSU - Fumed MSU - Fumed MSU - Fumed MSP - Fumed MSP - Fumed MSP - Fumed Non - Fumed Non - Fumed Non - Fumed Buccal 12 9 Table E5. Fusion pro files generated from spent cartridge casings loaded by individual V. Locus 13 - 6a 13 - 6b 13 - 6c 13 - 5a 13 - 5b 13 - 5c 13 - 7a 13 - 7b 13 - 7c V Amel XY XY XY XY XY Y X,Y X,Y X,Y X,Y D3 14 14 14,15 14 14 14, 18 14 14 14,14 D1 16.3,17.3 15.3 ,16.3, 17.3 14,17.3 16 ,16.3 15 , 16 , 16.3,17.3 16.3,17.3 16.3,17.3 16.3,17.3 D2 11 11,11.3 11 11,11.3 11.3 11,11.3 11,11.3 11,11.3 11,11.3 D10 15,16 15 13 , 16 15 13 ,15 16 15,16 15,16 15,16 D13 10, 12 10,12 12 12 10, 12 10,12 10 10,12 10,12 Penta E 5,14 14 5,14 5,14 5,14 5,14 D16 11,12 11,12 11, 13 12 11,12, 13 11,12 11,12 12 11,12 D18 16,17 16,17 12 16 14 17 16,17 16,17 16,17 D2 20,22 20,22 23.3 20 20,22 20,22 20 20,22 CSF 11 11 12 11 10 11 10,11 10 10,11 Penta D 11,12 12 11 12 11,12 12 11,12 THO1 9,9.3 7 , 9, 9.3 9 9,9.3 9,9.3 9,9.3 9,9.3 9,9.3 9,9.3 9,9.3 vWA 16,18 17 ,18 17 16 16,18 16 16,18 16,18 14 , 17 ,18 16,18 D21 32.2 28,32.2 28,32.2 28,32.2 28 28,32.2 D7 11,12 11,12 9 ,11 12 12 11,12 11,12 11,12 D5 12 12 12 12 12 12 12,12 TPOX 8 7 8 8 8 8,8 DY S391 11 11 11 11 11 11 11 D8 9,12 9, 10 , 11 , 12 12, 14 , 15 12 9,12 9,12 9,12 9,12 9,12 9,12 D12 21,23 17 ,21,23 21, 23 23 21,23 23 20 ,21,23 21,23 23 21,23 D19 12,14 12,14 14 12 12, 16 12 12 12 12,14 FGA 21.2,22, 22.2 22 , 23 17 21.2, 22.2 21.2,22 22 21.2,2 2 22, 23 , 32.2 21.2,22 D22 11 11 11 11 11,16 11,16 11,16 Fuming Method MSU - Fumed MSU - Fumed MSU - Fumed MSP - Fumed MSP - Fumed MSP - Fumed Non - Fumed Non - Fumed Non - Fumed Buccal 130 Table E6. Fusion profiles generated from spent cartridge casings loaded by individu al HH. Locus 15 - 7a 15 - 7b 15 - 7c 15 - 6a 15 - 6b 15 - 6c 15 - 1a 15 - 1b 15 - 1c HH Amel X X X X,X D3 16 14 15 14,18 D1 11 , 14 15.3 16,17.3 D2 11.3 11, 11.3 , 14 14 11,14 D10 15 13 13,15 D13 10, 12 10,11 Penta E 12 10, 14 D16 9 , 13 11 9,12 D18 12 , 17 12 , 13 12 16,16 D2 17,19 CSF 11,13 Penta D 11 , 13 10,10 THO1 6 , 7 , 9.3 6 , 7 8 9.3 9 9,9 vWA 17 14,16 D21 30,31 D7 11 10 11,12 D5 12 9,12 TPOX 8 12 9,11 DYS391 N/A D8 10,13 10*, 15* 12 ,13 13 10, 11 14.1 10,13 D12 18 ,20 18* 22 , 23 20 20,21 D19 15 15 14 13,14 FGA 24 21 23 22,25 D22 16 16,16 Fuming Method MSU - Fumed MSU - Fumed MSU - Fumed MSP - Fumed MSP - Fu med MSP - Fumed Non - Fumed Non - Fumed Non - Fumed Buccal 131 Table E7. Fusion profiles generated from spent cartridge casings loaded by individual L. Locus 23 - 1a 23 - 1b 23 - 1c 23 - 7a 23 - 7b 23 - 7c 23 - 2a 23 - 2b 23 - 2c L Amel X X X X X X X X X,X D3 17 14 , 16 16 16 1 6 16 16, 17 16,16 D1 15.3 12 , 14 14 16 16,17.3 16,17.3 16,17.3 D2 11 11 11 11 11 11,11 D10 13 16 13,15 13 15 14 13,15 D13 8 , 10 13 13 13 13,13 Penta E 7 7 7 7 7,7 D16 11 12 11 11 11 11 11 11, 12 11,11 D18 12 ,1 6 12 15,16 16 15,16 15 15 15,16 D2 23 17 17, 23 17 17 17,17 CSF 12 10 ,12 13 12 12,13 Penta D 10 , 14 12 , 13 8.2 9 9,11 THO1 9 , 9.3 6 , 7 6 ,9.3 8,9.3 8,9.3 8,9.3 8,9.3 3 ,8,9.3 8,9.3 vWA 17 , 19 17 16 14, 17 ,18 14,18 14 14 14,18 14 14,18 D21 29 24 ,30 30 27 30 30 27,30 D7 8 10 8,10 12 8 8,10 D5 11 12 11,12 11,12 TPOX 8,8 DYS391 10 10 N/A D8 10 ,13, 15 13 13,14 11 ,13,14 13,14 13,14 13,14 11 ,13,14 13,14 D12 21 21 18* 18,20 20 20 18,20 18 18,20 D19 14 15 15 15 14,15 15 14,15 FGA 21,23 21,23 21, , 21,23 D22 16 ,16 15,16 Fuming Method MSU - Fumed MSU - Fumed MSU - Fumed MSP - Fumed MSP - Fumed MSP - Fumed Non - Fumed Non - Fumed Non - Fumed Buccal 132 Table E8. Fusion profiles generated from spent cartridge casings loaded by individual OO. Locus 24 - 5a 24 - 5b 24 - 5c 24 - 4a 24 - 4b 24 - 4c 24 - 6a 24 - 6b 24 - 6c OO Amel X X Y X,X D3 15 16 14 , 16 15 15,18 D1 14 15.3 14,18.3 D2 15 11.3 ,14 11,14 D10 14,15 D13 12 12 10 9,12 Penta E 12 12 12 10,13 D16 11 12 8 , 13 13 9 , 11 11 12,12 D18 12 12 12 11,14 11,14 D2 22 20 20 17,25 CSF 10 10, 12 10,11 Penta D 11 10 9,12 THO1 6 7 9.3 6 6,9 6, 7 ,9, 9 .3 6, 9.3 6,9 vWA 17 15 17 17 16 17,18 D21 28, 33.2 28, 33.2 28,31.2 D7 9 ,10 10 8 10 10,10 D5 12 , 13 11,11 TPOX 8 8 12 12 8,8 DYS391 8 N/A D8 14* , 15 9 , 10 10 10 13 , 16 12,17 D12 18 , 21 18 19 ,20 18.3,20 D19 14 14, 15 14,16 FGA 21 , 23 21 , 23 22 18,24 D22 16 16 11,15 Fuming Method MSU - Fumed MSU - Fumed MSU - Fumed MSP - Fumed MSP - Fumed MSP - Fumed Non - Fumed Non - Fumed Non - Fumed Buccal 133 Table E9. Fusion profiles generated from spent cartridge casi ngs loaded by individual T. Locus 25 - 2a 25 - 2b 25 - 2c 25 - 1a 25 - 1b 25 - 1c 25 - 3a 25 - 3b 25 - 3c T Amel X X X,X D3 15 16, 18 16,17 D1 14 11 16,17.3 D2 14 11,14 D10 14 15 14,17 D13 12 11 9 11,11 Penta E 11,12 D16 9 ,11 12 11,12 D18 12 13 13 15 13,17 D2 20,24 CSF 12 10,11 Penta D 12 8,10 THO1 8 , 9 6 6 6,7, 9.3 6, 8 9.3 6,7 vWA 18 18 15 15 , 17 19,20 D21 32.2 29 29,29 D7 8,10 D5 12 13 12,12 TPOX 8 12 8,11 DYS391 N/A D8 10 13,14 D12 19,23 22 19,23 D19 13,16.2 FGA 21 24 24 24 24,24 D22 11 11,18 Fuming Method MSU - Fumed MSU - Fumed MSU - Fumed MSP - Fumed MSP - Fumed MSP - Fumed Non - Fumed Non - Fume d Non - Fumed Buccal 134 Table E10. Fusion profiles generated from spent cartridge casings loaded by individual XX. Locus 26 - 3a 26 - 3b 26 - 3c 26 - 2a 26 - 2b 26 - 2c 26 - 4b 26 - 4b 26 - 4c XX Amel X X Y X X X X X X,X D3 16 , 15, 16 , 17 15 14,15, 16 , 18 14,15 14,15 14,15 14,15 14,15 D1 15 , 15.3 11 12 14,17.3 15.3 ,17.3 14,17.3 14,17.3 14,17.3 D2 11 10 10 , 11 12,14 14 14 12 12,14 D10 15 13 , 15 14,16 13 ,14 14 14,16 D13 12 10 10 13 11 ,12 12,13 Penta E 7 5 , 14 12 12,12 D16 11 10 ,11, 9 ,11, 11.3 13 13 11,13, 11,13 13 11, 12 ,13 11,13 D18 12 , 15 , 16 16 17, 21 12 ,17 17 17 17, 17.2 15 ,17,18 17,18 D2 20 , 24 20 , 24 16 17 17 17 17,17 CSF 10,12, 13 10 10,12 10 10,12 Penta D 3.2 , 10 , 11 9 , 11 12,12 THO1 8 ,9.3 8 ,9,9.3 9 9,9.3 9.3 7 ,9.3 9,9.3 7 ,9,9.3 9.3 9,9. 3 vWA 17 14 , 15 14 , 16 , 18 17 17,19 17,19 17,19 17,19 17,19 D21 31 , 32.2 29 30 32 29,32 D7 9 8 8 ,9, 11 12 12 9,12 9 9,12 D5 10 7 10 9 ,13 10 10 10,13 TPOX 8 11 ,12 8 8 12 8,12 DYS391 10 N/A D8 13, 14 10, 12 ,13, 1 4 , 16 10,13 10 10,13, 15 10,13 10,13 10,13, 17 10,13 D12 17 18, 21 18, 21 20 18,22 18,22 19 ,22 18,22 18,22 D19 15 14 14.2 , 15 14 13 14 14 13,14 FGA 21 20 , 22 , 24 23 23, 24 21,23 21.2 ,23 23 21,23 D22 15 11 15 ,16 16 6 16, 18 16,17 Fuming Method MSU - Fumed MSU - Fumed MSU - Fumed MSP - Fumed MSP - Fumed MSP - Fumed Non - Fumed Non - Fumed Non - Fumed Buccal 135 Table E11. Fusion profiles generated from spent cartridge casings loaded by individual N. Locus 27 - 4a 27 - 4b 27 - 4c 27 - 3a 27 - 3b 27 - 3c 27 - 5a 27 - 5b 27 - 5c N Amel X X X X X X, Y X, Y X,X D3 15 ,17, 18 16 17 14 ,17, 18 16,17 D1 12 ,17.3 15.3 14 15.3,17.3 15.3,17.3 D2 11, 11.3 11 11,11 D10 13, 15 13 13,13 D13 11 , 13 14 12,14 Penta E 13 18 13,15 D16 14 11 10 13 11 ,12 12 11 ,12 12,13 D18 16 , 19.2 12 , 16 17 14 18 13, 18 14, 16 13,14 D2 17 , 25 20 20,23 CSF 10 11,12 Penta D 10 8 12 10,12 THO1 6, 6, 9 ,9.3 6,9.3 4 6,9.3 6,9.3 9.3 6,9.3 vWA 17,18 16 16 ,17,18 17 18 17,18 D21 30.1 , 34.1 28 28 32.2 30,32.2 D7 10 11,12 D5 10 ,12 10 11 , 13 12,12 TPOX 8 8,8 DYS391 N/A D8 13 12 ,13 13 13 11 ,13 13, 14 13, 15 13,13 D12 20 18 , 18.3 25 17 ,19 20 19,20 D19 13.2 , 15 15.2 15.2 13,14 FGA 22 , 29.1 24 21, 46.2 21,25 D22 19 15 17 11 11,15 Fuming Meth od MSU - Fumed MSU - Fumed MSU - Fumed MSP - Fumed MSP - Fumed MSP - Fumed Non - Fumed Non - Fumed Non - Fumed Buccal 136 Table E12. Fusion profiles generated from spent cartridge casings loaded by individual B. Locus 33 - 6a 33 - 6b 33 - 6c 33 - 5a 33 - 5b 33 - 5c 33 - 7a 33 - 7b 33 - 7c B Amel X X X X X,Y X,Y X,Y X,Y D3 18 14 ,16,18 18 16 14 ,16 16, 17 ,18 16 18 16,18 D1 14 , 15.3 , 16.3,17.3 15.3 , 16.3 14 ,16.3 16.3,17.3 16.3,17.3 D2 11.3 ,14 11.3 ,14,15 11 14,15 14 14,15 D10 15 15 16 13,15 D13 10 10,12 10 10 10,12 Pent a E 7, 12 7 7,18 D16 11 , 12 ,13 11 ,13 9,13 9 9, 11 , 12 9,13 9, 12 ,13 9,13 D18 12 ,13, 19.2 12 16 , 18 17 15 13,15 13 13,15 D2 22 18 , 22 ,25 17 17 25 20,25 CSF 12 10,12 12 10,12 12 12 10,12 Penta D 9 , 11 ,12,13 12,13 THO1 6 , 8 , 9.3 6 ,9.3 9. 3 8, 9 ,9.3, 8 8, 9 ,9.3 8,9.3 8 8,9.3 vWA 17,18 17 17 16 ,17,18 18 17,18 18 17,18 D21 31 28 ,29,31, 33.2 32.2 31 28 ,29, 30 29 29,31 D7 9, 10 ,12 12 10 9 12 9,12 D5 12 ,13 11, 12 , 13 13 13 11,13 TPOX 8,12 8, 12 8 12 8,8 DYS391 11 11 D8 8 , 13 8, 10 , 15 8,13 8 13, , 8,13 8,13 8,13 8,13 8,13 D12 21 18 ,21,23 22 23 22,23 22,23 D19 13, 14 , 15 15 14 ,15 13, 14 13,15 FGA 21,23 31 , 21,23 D22 15,16 17 11 16 15,16 Fuming Method MSU - Fumed MSU - Fumed MSU - Fumed MSP - Fumed MSP - Fum ed MSP - Fumed Non - Fumed Non - Fumed Non - Fumed Buccal 137 Table E13. Fusion profiles generated from spent cartridge casings loaded by individual D. Locus 36 - 7a 36 - 7b 36 - 7c 36 - 6a 36 - 6b 36 - 6c 36 - 1a 36 - 1b 36 - 1c D Amel X X X X,Y Y Y X,Y D3 15 16 16 17 15 17 ,18 D1 17.3 15,15 D2 11,14 D10 13 13,14 D13 11 11,11 Penta E 7,13 D16 5 , 8 ,13 13 10 , 12 ,13 12 ,13 13,13 D18 12 14 14 12,14 D2 17 16 20 17,20 CSF 11,12 Penta D 9,11 THO1 9 9 8 8,9.3 8,9.3 9 ,9.3 9.3 8,9.3 vWA 17 16 15 15 15,17 D21 28,30 D7 9, 10 12 9,12 D5 9 11,12 TPOX 9 9,11 DYS391 10 D8 15 8, 15 13 8, 16 , 18 8,13 13 9 ,13 8,13 D12 24 19 18 15,19 D19 18 15 14 ,15 FGA 22.1 , 21,26 D22 12,17 Fuming Method MSU - Fumed MSU - Fumed MSU - Fumed MSP - Fumed MSP - Fumed MSP - Fumed Non - Fumed Non - Fumed Non - Fumed Buccal 138 Table E14. Fusion profiles generated from spent cartridge casings loaded by individual WW. Locus 38 - 1a 38 - 1b 38 - 1c 38 - 7a 38 - 7b 38 - 7c 38 - 2a 38 - 2b 38 - 2c WW Amel Y X X X X, Y X X,X D3 14 ,16 16 16,18 16, 17 15 , 17 16,18 D1 11, 14 , 17.3 15 11 11 15 11,12 D2 11,14 14 11,14 11,14 D10 15,16 13 ,16 15 15,16 D13 10 , 12 8 11 9 8,9 Penta E 12 10 7 , 8 11,12 D16 12 11 ,12, 13 , 12 12 12 12 12,12 D18 12 15 15 12 12, 16 , 17 14 ,15 12,15 D2 18 18 18 17,21 CSF 11,12 11,12 Penta D 9 ,12 12 10,12 THO1 6 ,9.3 7 , 9.1 ,9.3 6 6 ,9.3 8 , 9.3 9.3 9.3 8,9.3 9.3, 9.3 vWA 16 ,17 17*, 18* 15,17 15,17 15,17 D21 28 28, 32.2 28,30 D7 11 10 10,11 D5 13 11 13,13 TPOX 8 11 8,12 DYS391 11 N/A D8 17 10 10,12, 14 10 10 10,12 10,12 D12 21 18, 23 17* , 21 18 23 18,19.3 19.3, 25 18,1 9.3 D19 15 14 14 14 14, 15.2 13 13,14 FGA 23 21 23 20,24 D22 16 16,16 Fuming Method MSU - Fumed MSU - Fumed MSU - Fumed MSP - Fumed MSP - Fumed MSP - Fumed Non - Fumed Non - Fumed Non - Fumed Buccal 139 Table E15. Fusion profiles generated from spent cart ridge casings loaded by individual SS. Locus 40 - 2a 40 - 2b 40 - 1a 40 - 1b 40 - 1c 40 - 3a 40 - 3b 40 - 3c SS Amel Y X,Y X,Y D3 16 14, 17 14, 17 14,18 D1 17.3 12 ,17.3 15 , 18.3 14,17.3 D2 11 10 10 11 11,11 D10 13 ,14 13 , 15 14,14 D13 11 10,12 Penta E 7 14 7,19 D16 12 10 ,12 11,12 D18 10 16 10,12 D2 17 20 22 19 17,20 CSF 11,12 12 11,12 Penta D 9 9 9,12 THO1 8 7 ,8 8,8 vWA 17 15 ,17 18 15 17,17 D21 29,32.2 29 29 29, 31 29, 32.2 D7 12,12 D5 13 11 13,13 TPOX 8 8 8 9,9 DYS391 11 11 11 D8 12 13 , 10 , 11 , 13 11 ,13 12,13 D12 15 15, 23 15 15, 20 18 15,24 D19 14 13 ,14 14,15 FGA 23.1 22,24 D22 16 18 13 16,16 Fuming Me thod MSU - Fumed MSU - Fumed MSU - Fumed MSP - Fumed MSP - Fumed MSP - Fumed Non - Fumed Non - Fumed Non - Fumed Buccal 140 Table E16. Fusion profiles generated from spent cartridge casings loaded by individual Y. Locus 41 - 3a 41 - 3b 41 - 3c 41 - 2a 41 - 2b 41 - 2c 41 - 4a 41 - 4b 41 - 4c Y Amel XY X X Y X,Y X,Y X,Y D3 15 ,17 14 ,16,17 14 17 16,17 D1 14, 15 14 16.3 , 17.3 12,14 D2 11 14 11.3 14 14,15 D10 15 14 14 15, 16 14,15 D13 11 12 9 ,13 13,14 Penta E 7 14, 18 5,14 5,14 D16 10 11 11 11,12 13 11,12 D18 12 , 20.2 17 16 16 ,17 17 17,17 D2 17 17 20 17,24 17,24 CSF 12,14 12 10 OL,12,14 Penta D 12 8 8,13 THO1 9.3 9.3 9.3 6 9,9.3 6 ,9.3 9,9.3 vWA 14 17 14, 18 17 14 16, 18 14 14,16 D21 33.3* 30.2 28 , 32.2 30.2 29,30.2 D7 8 10 10 8 11 , 12 10, 12 8,10 D5 12 12 12,12 TPOX 8 8 8 11 8,8 DYS391 11 11 D8 12 12 , 13 , 15 , 19 10, 13 , 13 10, 15 9 , 12 10,14 10,14 D12 24 20 ,21 21 21, 23 17, 20 17,21 D19 14 13 12 , 14 13, 15.2 13,16.2 FGA 23 , 24 21 ,27 21.2 ,22 20 ,27 22,27 D22 11,16 11,16 Fuming Method MSU - Fumed MSU - Fumed MSU - Fumed MSP - Fumed MSP - Fumed MSP - Fumed Non - Fumed Non - Fumed Non - Fumed Buccal 141 Table E17. Fusion profiles generated from spent cartridge casings loaded by individual II. Locus 50 - 4a 50 - 4b 50 - 4c 50 - 3a 50 - 3b 50 - 3c 50 - 5a 50 - 5b 50 - 5c II Amel X X X XY XY X,Y Y X,Y X,Y D3 16 15 15 17 17 17 17,17 D1 15,18.3 12 ,15 15,18.3 15,18.3 D2 11.3 14 11, 11.3 10,11 10,11 10,11 D10 15 15 13 15 13,15 D13 12 12 11,12 11 11 ,12 Penta E 13,14 13,14 D16 12 11 12 11 ,12 11 ,12 11 ,12 12,12 D18 13 16 16 16,17 D2 23 25 19 19,21 CSF 10 12 12,12 Penta D 9,13 THO1 6 , 7 ,8 7 ,8,9.3 7 ,8,9.3 6 ,8, 9 ,9.3 8 8,9.3 vWA 15 15 17 17 17, 18 15,17 15, 17 15,17 D21 29 31 29 31 29,31 D7 12 10,12 D5 12 12 11 12 12 11,12 TPOX 11 11 8 8,8 DYS391 11 11 D8 11 14 13 11,13 11,13, 14 , 16 11,13 11,13, 15 , 16 11,13 D12 18,20 20 23 18 18,20, 18, 19 18,20 D19 14 14 13 , 14 16. 2 14 15.2 14,15.2 FGA 23 25 22 ,23 23, 21, 21,23 D22 12 17* 15 15,16 15,16 Fuming Method MSU - Fumed MSU - Fumed MSU - Fumed MSP - Fumed MSP - Fumed MSP - Fumed Non - Fumed Non - Fumed Non - Fumed Buccal 142 APPENDIX F. HANDLER AND NON - HANDLER ALLELES AMPLIFIED WITH FUSION FROM COLLECTION 1 Table F1. Summary of the number of handler (H) alleles, non - handler (NH) alleles, and percent profile produced using Fusion for all samples in Collection 1. Sample Treatment Fusion % Profile # H alleles (Fusion) # NH alleles (Fusion) 2 - 1a Fumed at MSP 17.9% 7 0 2 - 1b Fumed at MSP 15.4% 6 2 2 - 1c Fumed at MSP 30.8% 12 1 2 - 2a Fumed at MSU 20.5% 8 10 2 - 2b Fumed at MSU 7.7% 3 2 2 - 2c Fumed at MSU 46.2% 18 25 2 - 3a Non - Fumed 71.8% 28 3 2 - 3b Non - Fumed 41.0% 16 1 2 - 3c Non - Fumed 28.2% 11 3 3 - 2a Fumed at MSP 2.4% 1 2 3 - 2b Fumed at MSP 7.3% 3 0 3 - 2c Fumed at MSP 4.9% 2 0 3 - 3a Fumed at MSU 0.0% 0 0 3 - 3a Fumed at MSU 9.8% 4 5 3 - 3b Fumed at MSU 14.6% 6 16 3 - 4a Non - Fumed 4.9% 2 3 3 - 4b Non - Fumed 2.4% 1 3 3 - 4c Non - Fumed 21.9% 9 4 8 - 3a Fumed at MSP 37.5% 15 12 8 - 3b Fumed at MSP 12.5% 5 1 8 - 3c Fumed at MSP 37.5% 15 0 8 - 4a Fumed at MSU 25.0% 10 10 8 - 4b Fumed at MSU - - - 8 - 4c Fumed at MSU 45.0% 18 26 8 - 5a Non - Fumed 31.7% 13 7 8 - 5b Non - Fumed 48.8% 20 1 8 - 5c Non - Fumed 34.1% 14 5 10 - 4a Fumed at MSP 9.8% 4 6 10 - 4b Fumed at MSP 24.4% 10 20 10 - 4c Fumed at MSP - - - 10 - 5a Fumed at MSU 0.0% 0 0 10 - 5b Fumed at MSU 2.4% 1 6 10 - 5c Fumed at MSU 4.9% 2 7 143 10 - 6a Non - Fumed 0.0% 0 1 10 - 6b Non - Fumed 9.7% 4 2 10 - 6c Non - Fumed 0.0% 0 0 13 - 5a Fumed at MSP 47.7% 21 3 13 - 5b Fumed at MSP 63.6% 28 6 13 - 5c Fumed at MSP 43.2% 19 0 13 - 6a Fumed at MSU 77.3% 34 1 13 - 6b Fumed at MSU 84.1% 37 7 13 - 6c Fumed at MSU 34.1% 15 11 13 - 7a Non - Fumed 70.4% 31 2 13 - 7b Non - Fumed 97.7% 43 0 13 - 7c Non - Fumed 65.9% 29 4 15 - 1a Non - Fumed 9.7% 4 3 15 - 1b Non - Fumed 4.9% 2 2 15 - 1c Non - Fumed 2.4% 1 1 15 - 6a Fumed at MSP 4.9% 2 2 15 - 6b Fumed at MSP 2.4% 1 7 15 - 6c Fumed at MSP 0.0% 0 0 15 - 7a Fumed at MSU 2.4% 1 3 15 - 7 b Fumed at MSU 22.0% 9 18 15 - 7c Fumed at MSU 9.8% 4 10 23 - 1a Fumed at MSU 23.7% 9 11 23 - 1a Fumed at MSU 10.5% 4 8 23 - 1b Fumed at MSU 13.2% 5 14 23 - 2a Non - Fumed 57.9% 22 0 23 - 2b Non - Fumed 60.5% 23 2 23 - 2c Non - Fumed 31.6% 12 4 23 - 7a Fumed at MSP 60.5 % 23 2 23 - 7b Fumed at MSP 60.5% 23 2 23 - 7c Fumed at MSP 26.3% 10 1 24 - 4a Fumed at MSP 0.0% 0 1 24 - 4b Fumed at MSP 14.6% 6 23 24 - 4c Fumed at MSP 4.9% 2 3 24 - 5a Fumed at MSU 14.6% 6 7 24 - 5b Fumed at MSU 7.3% 3 3 24 - 5c Fumed at MSU 12.2% 5 8 24 - 6a No n - Fumed 2.4% 1 2 24 - 6b Non - Fumed 1220.0% 5 14 24 - 6c Non - Fumed 12.2% 5 2 25 - 1a Fumed at MSP 0.0% 0 1 144 25 - 1b Fumed at MSP 7.3% 3 0 25 - 1c Fumed at MSP 7.3% 3 1 25 - 2a Fumed at MSU 0.0% 0 2 25 - 2b Fumed at MSU 7 .3% 3 6 25 - 2c Fumed at MSU 0.0% 0 2 25 - 3a Non - Fumed 12.2% 5 3 25 - 3b Non - Fumed 17.1% 7 1 25 - 3c Non - Fumed 7.3% 3 12 26 - 2a Fumed at MSP 28.6% 12 4 26 - 2b Fumed at MSP 35.7% 15 4 26 - 2c Fumed at MSP 59.5% 25 7 26 - 3a Fumed at MSU 23.8% 10 18 26 - 3b Fumed at MSU 9.5% 4 12 26 - 3c Fumed at MSU 31.0% 13 39 26 - 4a Non - Fumed 52.4% 22 1 26 - 4b Non - Fumed 52.4% 22 7 26 - 4c Non - Fumed 57.1% 24 3 27 - 3a Fumed at MSP 2.5% 1 4 27 - 3b Fumed at MSP 15.0% 6 3 27 - 3c Fumed at MSP 0.0% 0 1 27 - 4a Fumed at MSU 7.5% 3 8 27 - 4b Fumed at MSU 37.5% 15 24 27 - 4c Fumed at MSU 5.0% 2 1 27 - 5a Non - Fumed 22.5% 9 7 27 - 5b Non - Fumed 30.0% 12 6 27 - 5c Non - Fumed 37.5% 15 9 33 - 5a Fumed at MSP 10.9% 5 3 33 - 5b Fumed at MSP 10.9% 5 6 33 - 5c Fumed at MSP 32.6% 15 11 33 - 6a Fumed at MSU 30.4% 14 13 33 - 6b Fumed at MSU 76.1% 35 21 33 - 6c Fumed at MSU 26.1% 12 5 33 - 7a Non - Fumed 50.0% 23 4 33 - 7b Non - Fumed 34.8% 16 1 33 - 7c Non - Fumed 26.1% 12 0 36 - 1a Non - Fumed 27.3% 12 1 36 - 1b Non - Fumed 6.8% 3 1 36 - 1c Non - Fumed 11.4% 5 5 36 - 6a Fumed at MSP 4. 5% 2 2 36 - 6b Fumed at MSP 6.8% 3 3 145 36 - 6c Fumed at MSP 11.4% 5 3 36 - 7a Fumed at MSU 2.3% 1 3 36 - 7b Fumed at MSU 18.2% 8 5 36 - 7c Fumed at MSU 4.5% 2 3 38 - 1a Fumed at MSU 17.1% 7 7 38 - 1b Fumed at MSU 29.3% 1 2 11 38 - 1c Fumed at MSU 7.3% 3 7 38 - 2a Non - Fumed 41.5% 17 0 38 - 2b Non - Fumed 41.5% 17 18 38 - 2c Non - Fumed 21.9% 9 4 38 - 7a Fumed at MSP 17.1% 7 2 38 - 7b Fumed at MSP 14.6% 6 3 38 - 7c Fumed at MSP 2.4% 1 1 40 - 1a Fumed at MSP 20.5% 8 6 40 - 1b Fumed at MSP 0.0% 0 0 40 - 1c Fumed at MSP 5.1% 2 1 40 - 2a Fumed at MSU 7.7% 3 0 40 - 2b Fumed at MSU 17.9% 7 1 40 - 2c Fumed at MSU - - - 40 - 3a Non - Fumed 33.3% 13 10 40 - 3b Non - Fumed 30.8% 12 18 40 - 3c Non - Fumed 0.0% 0 1 41 - 2a Fumed at MSP 4.5% 2 0 41 - 2b Fumed at MSP 7.1% 3 4 41 - 2c Fumed at MSP 40.5% 17 4 41 - 3a Fumed at MSU 11.4% 5 1 41 - 3b Fumed at MSU 6.8% 3 10 41 - 3c Fumed at MSU 20.5% 9 9 41 - 4a Non - Fumed 9.1% 4 3 41 - 4b Non - Fumed 40.9% 18 21 41 - 4c Non - Fumed 40.9% 18 8 50 - 3a Fumed at MSP 18.6% 8 8 50 - 3b Fumed at MSP 14.0% 6 4 50 - 3c Fumed at MSP 14.0% 6 1 50 - 4a Fumed at MSU 0.0% 0 0 50 - 4b Fumed at MSU 20.9% 9 2 50 - 4c Fumed at MSU 16.3% 7 4 50 - 5a Non - Fumed 39.5% 17 11 50 - 5b Non - Fumed 69.8% 30 4 50 - 5c Non - Fumed 46.5% 20 4 146 APPEN DIX G. DNA QUANTITIES RECOVERED FROM SPENT CARTRIDGE CASINGS FROM COLLECTION 2 Table G1. Quantitation results for individually swabbed 0.45 casings from Collection 2. Sample Yield (pg) 39 - 4.45a 27.50 4.57E - 01 12.57 39 - 4 .45b 34.00 8.16E - 01 27.74 39 - 4.45c 31.80 1.49E+00 47.38 51 - 2.45a 34.00 2.42E - 01 8.23 51 - 2.45b 26.70 4.08E - 01 10.89 51 - 2.45c 22.00 9.96E - 01 21.91 52 - 3.45a 27.40 1.06E+00 29.04 52 - 3.45b 30.70 1.71E+00 52.50 52 - 3.45c 28.60 7.27E - 01 20.79 53 - 4.45a 33.2 0 2.56E+00 84.99 53 - 4.45b 25.30 1.67E+00 42.25 53 - 4.45c 28.50 1.17E+00 33.35 54 - 3.45a 34.20 2.91E - 01 9.95 54 - 3.45b 31.10 1.57E - 01 4.88 54 - 3.45c 35.00 2.40E - 01 8.40 55 - 1.45a 30.30 2.76E - 01 8.36 55 - 1.45b 30.20 2.79E - 01 8.43 55 - 1.45c 29.20 4.20E - 01 12 .26 56 - 4.45a 29.40 2.95E - 01 8.67 56 - 4.45b 29.00 5.00E - 02 1.45 56 - 4.45c 33.80 4.61E - 02 1.56 57 - 1.45a 36.00 4.35E - 01 15.66 57 - 1.45b 31.70 3.48E - 01 11.03 57 - 1.45c 33.00 1.34E - 01 4.42 58 - 3.45a 21.00 1.51E+00 31.71 58 - 3.45b 28.40 2.05E+00 58.22 58 - 3.45 c 27.20 1.30E+00 35.36 59 - 2.45a 30.00 8.70E - 01 26.10 59 - 2.45b 30.90 5.77E - 01 17.83 59 - 2.45c 33.30 3.83E - 01 12.75 60 - 4.45a 30.20 8.37E - 01 25.28 60 - 4.45b 31.20 1.07E+00 33.38 60 - 4.45c 34.80 3.74E+00 130.15 61 - 4.45a 28.90 1.78E - 01 5.14 61 - 4.45b 29.50 5.13E - 01 15.13 147 61 - 4.45c 25.50 8.66E - 01 22.08 62 - 3.45a 28.80 3.93E - 01 11.32 62 - 3.45b 27.50 4.24E+00 116.60 62 - 3.45c 30.50 2.76E+00 84.18 63 - 2.45a 24.60 1.22E+00 30.01 63 - 2.45b 34.00 1.12E+00 38.08 63 - 2.45c 28.40 1.17E+00 33.23 65 - 2.45a 29.60 4.94E - 01 14.62 65 - 2.45b 26.50 1.37E+00 36.31 65 - 2.45c 32.50 7.95E - 01 25.84 66 - 1.45a 28.80 1.11E+00 31.97 66 - 1.45b 31.70 1.02E+00 32.33 66 - 1.45c 30.70 2.42E - 01 7.43 67 - 1.45a 29.50 1.09E+00 32.16 67 - 1.45b 34.50 2.09E+00 72.11 67 - 1.45c 34. 20 4.41E - 01 15.08 68 - 1.45a 30.50 9.75E - 02 2.97 68 - 1.45b 36.00 3.60E - 01 12.96 68 - 1.45c 30.00 1.79E - 01 5.37 69 - 2.45a 28.90 3.02E - 01 8.73 69 - 2.45b 30.00 1.43E - 01 4.29 69 - 2.45c 29.80 6.39E - 02 1.90 70 - 3.45a 33.40 4.46E - 01 14.90 70 - 3.45b 33.10 5.57E - 01 1 8.44 70 - 3.45c 30.30 1.33E+00 40.30 Table G2. Quantitation results of individually swabbed 0.22 casings from Collection 2. Sample Yield (pg) 39 - 4.22a 32.80 7.74E - 01 25.39 39 - 4.22b 32.00 4.16E - 01 13.31 51 - 2.22a 32.60 1.46E - 01 4.76 51 - 2.22b 33.60 1.12E - 01 3.76 51 - 2.22c 36.30 1.54E - 01 5.59 52 - 3.22a 27.40 6.34E - 02 1.74 52 - 3.22b 31.50 4.69E - 01 14.7 7 52 - 3.22c 31.30 2.01E - 01 6.29 53 - 4.22a 33.00 7.32E - 01 24.16 53 - 4.22b 35.50 5.04E - 01 17.89 53 - 4.22c 29.70 2.17E - 01 6.44 148 54 - 3.22a 31.30 4.69E - 01 14.68 54 - 3.22b 29.60 4.81E - 01 14.24 54 - 3.22c 32.50 2.91E - 01 9.46 55 - 1.22a 33.70 7.07E - 01 23.83 55 - 1.22b 28.30 9.86E - 01 27.90 55 - 1.22c 31.20 1.90E - 01 5.93 56 - 4.22a 32.50 5.92E - 01 19.24 56 - 4.22b 30.10 5.07E - 01 15.26 56 - 4.22c 36.00 6.06E - 01 21.82 57 - 1.22a 29.80 6.60E - 02 1.97 57 - 1.22b 27.70 9.23E - 02 2.56 57 - 1.22c 33.00 4.99E - 02 1.65 58 - 3.22a 30.00 1.24E - 01 3.72 58 - 3.22b 31.80 6.50E - 02 2.07 58 - 3.22c 35.50 3.51E - 01 12.46 59 - 2.22a 26.00 6.27E - 01 16.30 59 - 2.22b 29.50 5.80E - 01 17.11 59 - 2.22c 30.00 4.50E - 01 13.50 60 - 4.22a 29.30 5.20E - 01 15.24 60 - 4.22b 26.50 1.32E+00 34.98 60 - 4.22c 33. 50 1.35E+00 45.23 61 - 4.22a 29.30 6.95E - 02 2.04 61 - 4.22b 29.00 3.18E - 02 0.92 61 - 4.22c 28.50 4.89E - 02 1.39 62 - 3.22a 31.50 9.92E - 01 31.25 62 - 3.22b 34.00 1.14E+00 38.76 62 - 3.22c 31.60 3.55E - 01 11.22 63 - 2.22a 30.40 7.72E - 01 23.47 63 - 2.22b 33.20 1.08E+00 35.86 63 - 2.22c 31.00 1.00E+00 31.00 65 - 2.22a 28.10 5.23E - 01 14.70 65 - 2.22b 28.70 2.78E - 01 7.98 65 - 2.22c 29.30 3.18E - 01 9.32 66 - 1.22a 34.00 1.70E - 01 5.78 66 - 1.22b 28.40 8.60E - 02 2.44 66 - 1.22c 27.70 5.88E - 02 1.63 67 - 1.22a 30.30 8.63E - 01 26.15 67 - 1. 22b 35.80 6.03E - 01 21.59 67 - 1.22c 37.50 5.60E - 01 21.00 68 - 1.22a 28.30 1.48E - 01 4.19 149 68 - 1.22b 28.40 3.47E - 02 0.99 68 - 1.22c 27.30 1.74E - 01 4.75 69 - 2.22a 31.10 7.13E - 02 2.22 69 - 2.22b 29.20 1.56E - 01 4.56 69 - 2.22c 25.70 8.81E - 02 2.26 7 0 - 3.22a 28.20 9.74E - 01 27.47 70 - 3.22b 31.80 6.33E - 01 20.13 70 - 3.22c 31.80 8.16E - 01 25.95 Table G3. Quantitation results for cumulatively swabbed 0.45 casings from Collection 2. Sample Yield (pg) 39 - 1.45 29.70 1.45E+00 43.07 39 - 2.45 28.60 2.46E+00 70.36 39 - 3.45 31.70 2.05E+00 64.99 51 - 1.45 32.20 1.76E - 01 5.67 51 - 3.45 32.50 5.62E - 01 18.27 51 - 4.45 29.60 6.59E - 01 19.51 52 - 1.45 35.40 1.28E+01 453.12 52 - 2.45 33.10 2.41E+00 79.77 52 - 4.45 35.30 2.90E+00 102.37 53 - 1.45 27.80 5.30E+00 147.34 53 - 2.45 27.70 2.55E+00 70.64 53 - 3.45 27.60 7.64E+00 210.86 54 - 1.45 31.30 7.70E+00 241.01 54 - 2.45 24.80 3.20E+00 79.36 54 - 4.45 26.60 2.50E+00 66.50 55 - 2.45 32.80 7.42E - 01 24.34 55 - 3.45 35.20 1.85E+00 65.12 55 - 4.45 29.80 1.39E+ 00 41.42 56 - 1.45 31.50 1.08E+00 34.02 56 - 2.45 35.00 1.26E+00 44.10 56 - 3.45 34.10 2.25E+00 76.73 57 - 2.45 31.70 4.69E+00 148.67 57 - 3.45 33.50 2.88E - 01 9.65 57 - 4.45 31.00 6.26E - 01 19.41 58 - 1.45 28.20 1.57E+00 44.27 58 - 2.45 28.70 1.70E+00 48.79 58 - 4.4 5 28.30 1.58E+00 44.71 59 - 1.45 34.80 7.45E - 01 25.93 150 59 - 3.45 32.30 3.05E - 01 9.85 59 - 4.45 29.00 5.62E - 01 16.30 60 - 1.45 32.80 1.28E+00 41.98 60 - 2.45 34.70 1.25E+00 43.38 60 - 3.45 35.70 2.78E+00 99.25 61 - 1.45 32.50 1.34E+00 43.55 61 - 2. 45 32.50 1.48E+00 48.10 61 - 3.45 31.80 1.00E+00 31.80 62 - 1.45 32.00 2.77E+00 88.64 62 - 2.45 35.00 3.58E+00 125.30 62 - 4.45 34.00 3.15E+00 107.10 63 - 1.45 30.50 8.61E - 01 26.26 63 - 3.45 30.30 1.05E+00 31.82 63 - 4.45 29.00 1.23E+00 35.67 65 - 1.45 32.40 3.57E +00 115.67 65 - 3.45 27.80 3.93E+00 109.25 65 - 4.45 28.50 3.78E+00 107.73 66 - 2.45 18.20 2.29E+00 41.68 66 - 3.45 33.50 5.22E+00 174.87 66 - 4.45 30.30 1.98E+00 59.99 67 - 2.45 27.50 4.95E - 01 13.61 67 - 3.45 29.70 8.86E - 01 26.31 67 - 4.45 28.50 5.11E+00 145.64 68 - 2.45 30.70 2.11E+00 64.78 68 - 3.45 32.00 4.79E - 01 15.33 68 - 4.45 31.50 3.71E - 01 11.69 69 - 1.45 29.40 4.08E - 01 12.00 69 - 3.45 34.10 5.76E - 01 19.64 69 - 4.45 32.20 9.05E - 01 29.14 70 - 1.45 36.20 1.62E+00 58.64 70 - 2.45 34.50 1.94E+00 66.93 70 - 4.45 35.10 2. 61E+00 91.61 151 Table G4. Quantitation results for cumulatively swabbed 0.22 casings from Collection 2. Sample Yield (pg) 39 - 1.22 30.30 1.51E+00 45.75 39 - 2.22 28.20 1.37E+00 38.63 39 - 3.22 33.30 1.42E+00 47.29 51 - 1.22 28.10 3.11E - 01 8.74 51 - 3.22 26.50 6.58E - 01 17.44 51 - 4.22 32.10 5.16E - 01 16.56 52 - 1.22 32.30 6.61E - 01 21.35 5 2 - 2.22 30.80 7.13E - 01 21.96 52 - 4.22 29.20 2.59E - 01 7.56 53 - 1.22 30.00 5.70E - 01 17.10 53 - 2.22 32.00 5.15E - 01 16.48 53 - 3.22 32.10 5.83E - 01 18.71 54 - 1.22 29.80 1.86E+00 55.43 54 - 2.22 28.10 1.49E+00 41.87 54 - 4.22 29.30 1.16E+00 33.99 55 - 2.22 31.40 4.68 E - 01 14.70 55 - 3.22 33.50 2.99E - 01 10.02 55 - 4.22 28.10 8.02E - 02 2.25 56 - 1.22 31.80 1.20E+00 38.16 56 - 2.22 27.70 9.37E - 01 25.95 56 - 3.22 31.50 5.69E - 01 17.92 57 - 2.22 30.50 2.11E - 02 0.64 57 - 3.22 30.10 9.34E - 02 2.81 57 - 4.22 29.00 1.59E - 01 4.61 58 - 1.22 33.40 5.43E - 01 18.14 58 - 2.22 32.80 2.54E - 01 8.33 58 - 4.22 33.30 1.92E - 01 6.39 59 - 1.22 30.10 2.61E - 02 0.79 59 - 3.22 32.20 5.33E+00 171.63 59 - 4.22 28.50 2.96E+00 84.36 60 - 1.22 29.80 4.92E+00 146.62 60 - 2.22 28.30 3.91E+00 110.65 60 - 3.22 34.00 2.76E+00 9 3.84 61 - 1.22 34.20 3.78E - 01 12.93 61 - 2.22 33.00 5.04E - 01 16.63 61 - 3.22 32.30 5.69E - 01 18.38 62 - 1.22 26.30 1.64E+00 43.13 62 - 2.22 30.30 5.73E - 01 17.36 62 - 4.22 34.10 5.39E - 01 18.38 152 63 - 1.22 31.50 8.06E - 01 25.39 63 - 3.22 33.50 7.29E - 01 24.42 63 - 4.22 27.90 9.01E - 01 25.14 65 - 1.22 31.40 2.97E - 01 9.33 65 - 3.22 31.40 1.77E+00 55.58 65 - 4.22 32.60 1.25E+00 40.75 66 - 2.22 27.20 5.62E - 01 15.29 66 - 3.22 32.10 6.28E - 01 20.16 66 - 4.22 31.00 8.11E - 02 2.51 67 - 2.22 33.00 7.89E - 01 26.04 67 - 3.22 35 .30 4.08E - 01 14.40 67 - 4.22 35.20 3.11E - 01 10.95 68 - 2.22 26.80 1.53E - 01 4.10 68 - 3.22 34.30 1.65E+00 56.60 68 - 4.22 34.60 4.77E - 01 16.50 69 - 1.22 34.50 2.52E - 01 8.69 69 - 3.22 34.50 2.92E - 01 10.07 69 - 4.22 29.70 5.01E - 01 14.88 70 - 1.22 32.60 2.16E - 01 7.04 70 - 2.22 28.90 3.74E - 01 10.81 70 - 4.22 20.00 3.69E - 01 7.38 153 APPENDIX H. FUSION STR PROFILES FROM COLLECTION 2 Red font: non - loader allele *: allele was above the threshold using OSIRIS, but below the threshold using GeneMapper ® . - ladder allele Blank cell: no alleles were amplified N/A: not applicable 154 Table H1. Fusion profiles generated from spent cartridge casings loaded by individual SSS. Locus 39 - 4.45a 39 - 4.45b 39 - 4.45c 39 - 4.22a 39 - 4.22b SSS Amel X X X X ,X D3 15 15,16 16 15 15,16 D1 18.3 12 12 16 ,18.3 12,18.3 D2 14 11 11 11,14 11,14 D10 15 14 14,15 D13 11 11 11,12 Penta E 13,17 D16 10 ,14 12,14 ,12,14 9 ,14 12,14 D18 12, 15 12, 21 12 12 ,12 D2 19,24 CSF 10 10 10,12 Penta D 9,14 THO1 9.3 9.3 9.3 9,9.3 6 ,9,9.3 9,9.3 vWA 19 19 18 18 18,19 D21 31.2 31.2 31.2 D7 9,10 D5 11 ,11 TPOX 8 8 ,8 DYS391 N/A D8 13,15 13 10 ,13,15 13,15 D12 17,19 17,19 D19 13 13 13,14 13, 14 FGA 25 25 21,25 D22 16 16 ,16 155 Table H1 Locus 39 - 1.45 39 - 2.45 39 - 3.45 39 - 1.22 39 - 2.22 39 - 3.22 SSS Amel X X , Y* X , Y X , Y X X X ,X D3 15 15,16 14 ,15,16 15,16, 18 14 ,15,16 14 ,15,16 15,16 D1 12 11 ,12 16.3,17.3 17.3*, 18.3 17.3 11 12,18.3 D2 11,14 11,14 11,14 14 11 11,14 D10 14,15 14,15 ,16 15 14 14,15 D13 11,12 10 ,12 11,12 11,12 Penta E 14 13 13 13,17 D16 11 ,12,14 11 ,14 11 ,12,14 ,12,14 12 12,14 12,14 D18 12 12, 15,16,17 15 12 ,16,17 - 12 12 ,12 D2 24 19*,24 22 19,24 2 4 19,24 CSF 10 10 10, 11 ,12 12 10 10 10,12 Penta D 9 14 14 9,14 14 9,14 THO1 7,8 ,9,9.3 6 ,9,9.3 9,9.3 9,9.3 6, 9,9.3 6, 9,9.3 9,9.3 vWA 18,19 18,19 18 17* 17 18,19 D21 31.2 30 ,31.2 28 ,31.2 31.2, 32 31.2 31.2 ,31.2 D7 8 10, 12 9,10 9,10 D5 11 11 12 11 11 ,11 TPOX 8 8 8 8 ,8 DYS391 N/A D8 13,15 9,10,12 ,13,15 9 ,13,15 11 ,13 13,15, 16 10 ,13,15 13,15 D12 17,19 17,19, 20 17,19, 25 17,19 17, 18.3 17,19 D19 13, 15.2 12 ,13 12 ,13,14 13,14 14 13 13,14 FGA 21 21, 22 21.2,22 ,25 21 21 21,25 D22 16 11 ,16 16 16 16 16 ,16 156 Table H2. Fusion profiles generated from spent cartridge casings loaded by individual NN. Locus 51 - 2.45a 51 - 2.45b 51 - 2.45c 51 - 2.22a 51 - 2.22b 51 - 2.22c NN Amel X X X , Y X X X , X D3 16 14 14,16 14,16 D1 16 12 12,16 16 12,16 D2 10, 1 1.3 11 11 10 10,11 D10 14,15 15 14 14 14,15 D13 8 8,12 Penta E 7,21 D16 12 12 12 12 12 ,12 D18 14.2 12 ,14 14,14.2 14,14.2 D2 17,23 17,23 17,23 CSF 12,13 Penta D 13 13 ,13 THO1 9,9.3 6 9.3 6 ,9 7 ,9,9.3 9,9.3 vWA 17 17 17 17 17 17 ,17 D21 29 31.2, 33.2 29 29,31.2 D7 11 9 9,11 D5 9 9 9,10 TPOX 8 ,8 DYS391 N/A D8 13, 14 ,15 13, 14 ,15 13,15 13 13,15 13,15 D12 18.3 22 18 22 18,22 D19 15.2 14,15.2 14,15.2 FGA 22,26 D22 11 ,11 157 Table H2 Locus 51 - 1.45 51 - 3.45 51 - 4.45 51 - 1.22 51 - 3.22 51 - 4.22 NN Amel X X , Y X X Y X , X D3 14, 15 ,16 14,16 18 14 18 14,16 D1 16, 17.3 12 12 12,16 D2 11, 14 11.3 10,11 D10 14,15 14,15 D13 12 8,12 Penta E 7,21 7,21 D1 6 11 ,12 12 12 12 12 12 12 ,12 D18 14 14,14.2 12 14 18 14,14.2 D2 23 23 17 17,23 17 17,23 CSF 13 12 12,13 Penta D 13 9 13 ,13 THO1 6 ,9.3 9 9,9.3 9.3 9 6,9 9,9.3 vWA 16 ,17 16 ,17 15 17 18 15 ,17 17 ,17 D21 29,31.2 31.2 29 29,31.2 D7 9 9 9 ,11 D5 9 9,10 TPOX 8 8 ,8 DYS391 N/A D8 13,15 9,12 ,13,15 13 13 13,15 D12 17 ,18,22 18 18,22 18,22 D19 14 15.2 14 14,15.2 FGA 21.2 ,22 26 22,26 D22 15 11 ,11 158 Table H3. Fusion profiles generated from spent cartridge c asings loaded by individual ZZZ. Locus 52 - 3.45a 52 - 3.45b 52 - 3.45c 52 - 3.22a 52 - 3.22b 52 - 3.22c ZZZ Amel X X X X X X ,X D3 14 18 17 18 14,18 D1 15,17.3 15,17.3 D2 10,11 11 10,11 D10 12,14 12,14 D13 12 9,12 Penta E 13 12 9,13 D 16 12, 16 9,12 9,12 12 9,12 D18 16 10.2,14 16 16 16 16,20 D2 19 19,21 21 20 ,21 19,21 CSF 12 11,12 Penta D 13 12,13 THO1 6,9.3 9.3 6,9.3 6 6 6,9.3 vWA 17 17 17 17 17 17 ,17 D21 28.2 28,29 28,29 D7 15 12 12 10,12 D5 11 13 13 11,13 TPOX 8 8,11 DYS391 N/A D8 10 10,12 10,12 10,12 10,12 D12 24 24,25 24 25 24,25 D19 14 14 13,14 FGA 19 19,26 D22 16 15,16 159 Table H3 Locus 52 - 1.45 52 - 2.45 52 - 4.45 52 - 1.22 52 - 2.22 52 - 4.22 ZZZ Amel X , Y X X X X X X ,X D3 17 14, 17 14,18 14,18 14,18 18 14,18 D1 15, 18.3 15 15,17.3 17.3 15 15,17.3 D2 10,11 11 11 10,11 11 10,11 D10 15 14 14 12,14 D13 11 ,12 9 9,12 9 12 9,12 Penta E 14 13 9,13 D16 12 9,12 9 ,12 9 9,12 9,12 D18 16, 17 16,20 16,20 16 16,20 16 16,20 D2 19,21, 25 19 19 21 21 19,21 CSF 12 12 11 11,12 Penta D 9 ,13 12 13 12,13 THO1 6, 8 ,9.3 6,9.3 6,9.3 6,9.3 6 6,9.3 6,9.3 vWA 15 ,17 17 17 17 17 ,17 D21 29 28,29 28,29 28,29 28 28,29 D7 10,12 12 10 10 12 10,12 10,12 D5 11, 12 13 11 13 11,13 TPOX 8 8 11 11 8,11 DYS391 11 N/A D8 11,12, 13 10,12 10,12 10,12 10,12 10,12 D12 18,18.3,20 24 24 24 15 ,24,25 24 24,25 D19 14, 15.2 14 13,14 13,14 13 13 13,14 FGA 21,23 19,26 19,26 26 26 19, 25 ,26 19,26 D22 15,16 16 15,16 160 Table H4. Fusion profiles generated from spent cartridge casings loaded by individual B. Locus 53 - 4.45a 53 - 4.45b 53 - 4.45c 53 - 4.22a 53 - 4.22b 53 - 4.22c B Amel X X X X ,X D3 18 18* 18 ,18 D1 11 ,12 12 12,15 D2 11.3 11,11.3 D10 15 14 13, 15 D13 13 13 12,13 Penta E 12,13 12,13 D16 11 11 11 11 ,11 D18 16 12,16 12,16 D2 17 25 17,25 CSF 10 10,11 Penta D 13 10,13 THO1 6,9 6 6,9 6,9 vWA 17,18 17,18 17 17,18 D21 28 28 ,28 D7 10 10 ,10 D5 12 10,12 TPOX 11 8,11 DYS391 N/A D8 13,16 13,16 16 9, 13 13,16 D12 17 ,18,18.3 18 18,18.3 18,18.3 18,18.3 D19 15 15 13.2,15 FGA 22 23 22,23 D22 15 ,15 161 Table H4 Locus 53 - 1.45 53 - 2.45 53 - 3.45 53 - 1.22 53 - 2.22 53 - 3.22 B Amel X X X X X X ,X D3 18 18 18 14 16 ,18 18 ,18 D1 12,15, 17 12,15 12,15 12 11 12,15 D2 11.3, 14 11,11.3 11,11.3 11.3 11,11.3 D10 15 13 13,15 13,15 D13 10 ,12 12,13 Penta E 12,13, 18 12,13 12,13 D16 11 11 11 11 11, 12 11 ,11 D18 1 2,16 12,16, 18 12,16 16, 17 12 12,16 D2 23 ,25 17,25 17,25 17,25 CSF 10, 12 10 10,11 10,11 Penta D 10 10,13 12 10 10,13 THO1 6,9 6,9 6,9 6,9 7 ,9 6,9 vWA 17,18 17,18 17,18 17 17,18 D21 28 28 28 32.2 28 28 ,28 D7 10 10 10 10 10 ,10 D5 10,12 10 10 14 10,12 TPOX 8 11 8,11 DYS391 N/A D8 9,10 ,13, 14 ,16 13,16 13,16 13 11,15 ,16 13,16 D12 17 ,18.3 18,18,3 18,18.3 18,18.3 D19 13.2,15 13.2,15 13.2,15 13.2,15 FGA 22 23 22,23 22 22 22,23 D22 15 15 15 ,15 162 Table H5. Fusion p rofiles generated from spent cartridge casings loaded by individual BBB. Locus 54 - 3.45a 54 - 3.45b 54 - 3.45c 54 - 3.22a 54 - 3.22b 54 - 3.22c BBB Amel X X X X X X ,X D3 16 16 16, 17 15 15,16 D1 12 12 12 11,12 D2 11 14 11,14 D10 13 14 ,14 D13 12 11 11,12 Penta E 13,16 D16 11 ,12,13 ,13 12 12 ,12 12,13 D18 14,16 14 14 14,16 D2 24 24 24 24 ,24 CSF 10 10 10 ,10 Penta D 9 9 9 ,9 THO1 8,9 8 9 9 8,9 vWA 17 17 17 18 17 ,17 D21 28 28 29 29,30 D7 7,8 D5 9 9, 11 TPOX 8 ,8 DYS391 N/A D8 14 14 8,9 14 14 14 ,14 D12 21 20 21 20 19 20,21 D19 12,13 FGA 22 20,22 D22 11,16 163 Table H5 Locus 54 - 1.45 54 - 2.45 54 - 4.45 54 - 1.22 54 - 2.22 54 - 4.22 BBB = 54 Amel X X X X X X X ,X D3 1 5,16 15,16 15,16 15 15,16 15 15,16 D1 11,12 11 11,12 12, 15.3 11,12 12 11,12 D2 11,14 11 11.3 11, 11.3 11 11,14 D10 14 14 14 14 14 ,14 D13 11,12 11 10 12 11,12 Penta E 13 16 13 ,14 13,16 D16 12,13 12,13 11 ,12 12,13* 12,13 12,13 12,13 D18 14,16 14,16 12 ,14,16 ,17 16 16 14,16 D2 19,21 ,24 24 24 18 24 22 24 ,24 CSF 10 10* 12 10 10 ,10 Penta D 9 9 9 ,9 THO1 8,9 8 8,9, 9.3 6 ,9, 9.3 8,9, 9.3 7 ,8 8,9 vWA 17 17 17 17 17 17 17 ,17 D21 29 30 29,30 28,33.2 29,30 D7 7,8 6.3 ,7, 9 7,8 10 7,8 8 7,8 D5 9,11 11 11 12 8 ,11 11, 12 9,11 TPOX 8 8 8 12 8 8 ,8 DYS391 N/A D8 14 14 14 13 ,14 13 ,14 14 14 ,14 D12 20,21 20, 23 17 ,20 20 19.3 ,20,21 20,21 D19 12,13 13 12 12 13 12 12,13 FGA 20,22 22 21 20,22 20,22 D22 11 11 ,16 164 Table H6. Fusion profiles generated from spent cartridge casings loaded by individual C. Locus 55 - 1.45a 55 - 1.45b 55 - 1.45c 55 - 1.22a 55 - 1.22b 55 - 1.22c C Amel X X ,X D3 18 15,18 15,18 D1 14,16 D2 10 11 ,11 D10 14 14,15 D1 3 12 11,12 Penta E 11,13 D16 11 11,12 D18 16 ,17 11,17 D2 17,25 CSF 10 10,11 Penta D 9,10 THO1 6 8 6 ,6 vWA 11,14 D21 28,29 D7 9,13 D5 11,12 TPOX 8 12 ,12 DYS391 N/A D 8 13,14 13,14 D12 17,19 D19 13,14 FGA 20,24 D22 15,17 165 Table H6 Locus 55 - 2.45 55 - 3.45 55 - 4.45 55 - 2.22 55 - 3.22 55 - 4.22 C Amel X X X X X , Y X , Y X ,X D3 15, 16 15, 17 ,18 14 ,18 15 15,18 D1 11,12 14, 15,15.3 11 ,14, 1 5.3 ,16 14 14,16 D2 11 11 11 ,11 D10 15 15 15 14,15 D13 11,12 12 11,12 Penta E 7 ,11 5,14 11,13 D16 12, 13 11,12 11,12 8.3* 12, 14 11 11,12 D18 11, 14 ,17 11 11 12,18 11,17 D2 17 17,25 CSF 10 10 10,11 Penta D 10 12 9,10 TH O1 6 6 6 3,9,9.3 6 9.3 6 ,6 vWA 14, 18 14, 17 14, 17 18 11,14 D21 28,29 29 28,29 D7 9 13 10 9,13 D5 11,12 TPOX 8 ,12 12 12 ,12 DYS391 11 N/A D8 13,14 10 ,13,14 13,14 13, 15 13,14 13,14 13,14 D12 17, 19.3 17, 18 ,19, 19.3, 20 17,19, 21 15, 20 20 19, 21 17,19 D19 8.2 ,14 13,14 12 ,13 13 13,14 FGA 18, 24 20 20,24 D22 15,17 15,17 166 Table H7. Fusion profiles generated from spent cartridge casings loaded by individual AA. Locus 56 - 4.45a 56 - 4.45b 56 - 4.45c 56 - 4.22a 56 - 4.22b 56 - 4.22c AA Amel Y X X X , Y D3 9,17 15 ,15 D1 19.3 18.3,19.3 D2 11,11.3 D10 13,14 D13 9,12 Penta E 5 ,5 D16 9,12 D18 13 13,14 D2 19,21 CSF 10,11 Penta D 11,14 THO1 9.3 6,9.3 vWA 15, 17 D21 28 28,30.2 D7 9 9 ,9 D5 12,13 TPOX 8,11 DYS391 10 D8 15 12,15 D12 20 19,22 D19 15 15 13,15 FGA 18,21 D22 11,17 167 Table H7 Locus 56 - 1.45 56 - 2.45 56 - 3.45 56 - 1.22 56 - 2.22 56 - 3.22 AA Amel X X , Y X X , Y X , Y D3 15 14 ,15 16 14 15 15 ,15 D1 18.3,19.3 14 15 18.3,19.3 D2 11.3 14 11 11,11.3 D10 13, 16 14 13,14 D13 9 12 9,12 Penta E 15 5 ,5 D16 9 9 9, 11 11 ,12 13 9 9,12 D18 14 11,17 14, 16 16 1 4, 15 13 13,14 D2 17 20 20 19,21 CSF 11 10,11 Penta D 11,14 THO1 6 7,9 7 ,9.3 8 6 6,9.3 vWA 15,17 14 18 15,17 D21 28 28* 28,30.2 D7 9 ,9 D5 11 ,13 13 11 12,13 TPOX 11 11 8 8,11 DYS39 1 10 D8 12 10 10,13 9 ,12 12 12 12,15 D12 22 21,23 17 ,22 21,23 18 19,22 D19 16.2 13 13.2 13,15 FGA 21 20 ,21 20 18,21 D22 11,17 168 Table H8. Fusion profiles generated from spent cartridge casings loaded by individua l A. Locus 57 - 1.45a 57 - 1.45b 57 - 1.45c 57 - 1.22a 57 - 1.22b 57 - 1.22c A Amel X X X ,X D3 15 15,16 D1 14 11,14 D2 14,16 D10 15,16 D13 12 13,14 Penta E 7,12 D16 11, 12 9 11 ,11 D18 11 12,17 D2 18 17,25 CS F 10 10,12 Penta D 12 ,12 THO1 7 6 7,9.3 vWA 16 15 17 17,18 D21 32.2 28,30 D7 10 10,12 D5 11 9,12 TPOX 8,11 DYS391 N/A D8 9 ,15 13 12,15 D12 18.3 20 18,24 D19 19 13,15.2 FGA 23,24 D 22 16 15,16 169 Table H8 Locus 57 - 2.45 57 - 3.45 57 - 4.45 57 - 2.22 57 - 3.22 57 - 4.22 A Amel X X X X Y X ,X D3 15,16 16 18 16 18 15,16 D1 11,14 14 14 12,17.3 11,14 D2 14,16 14,16 D10 15,16 15,16 D13 13,14 14 13,14 Penta E 7, 12 7,12 D16 11 11 11 11 11 ,11 D18 12,17 17 12 17 12,17 D2 17,25 17,25 17,25 CSF 10,12 12 10 10,12 Penta D 12 12 ,12 THO1 7,9.3 7,9.3 7 6 6 8 7,9.3 vWA 17,18 16 18 14 18 17,18 D21 28,30, 31 30 28 28,30 D7 10,12 10 10,12 D5 9,12 12 9,12 TPOX 8,11 8,11 DYS391 N/A D8 12,15 13 12 13,14 12,15 D12 18,24 15 18, 19.3 ,24 20 18,24 D19 13,15.2 13,15.2 FGA 23,24 23,24 D22 15,16 15,16 170 Table H9. Fusion profiles generated from spent cartridge casings loaded by individual J. Locus 58 - 3.45a 58 - 3.45b 58 - 3.45c 58 - 3.22a 58 - 3.22b 58 - 3.22c J Amel Y X X X , Y D3 15,16 16, 18 16 15,16 D1 17.3 13 ,13 D2 13 11.3,13 D10 13, 14 D13 11 ,11 Penta E 7 16 11,16 D16 12 9 13 11 ,12 12,13 D18 10 16 10,18 D2 18 17,18 CSF 12 Penta D 8,9 THO1 6,8 9 10 6 ,7 6 6,8 vWA 18 15,17 18 ,18 D21 31 28 30.2,31.2 D7 12 ,12 D5 10,12 TPOX 10 8,10 DYS391 10 D8 12, 13 10,13,14 11,12, 13,16 11,12 D12 17,23 17 ,18 18 ,18 D19 14,15 14,15 FGA 20,22 D22 16 ,16 171 Table H9 Locus 58 - 1.45 58 - 2.45 58 - 4.45 58 - 1.22 58 - 2.22 58 - 4.22 J Amel X , Y X , Y X , Y X X , Y X X , Y D3 16, 18 14 ,15,16 16 15,16, 17 15,16 D1 12 ,13, 1 5 11 11 14,17.3 12 13 ,13 D2 11.3 11.3,13 D10 13, 15 13 14 14 14 13, 14 D13 10 11 12 11 11 ,11 Penta E 12 16 11,16 D16 12,13 9 ,12,13 11 ,12,13 11 11 ,12,13, 14 12,13 D18 16 ,18 10, 12,14 ,18 10 17 10 10,18 D2 18 17 19 17,18 CSF 10 10 11 Penta D 13 8 8,9 THO1 6, 9,9.3 6, 7 ,8 6,8, 9.3 6, 9.3 8 6, 9,9.3 6,8 vWA 15,17 ,18 18 18, 19 15 17 ,18 18 ,18 D21 28 30.2,31.2 D7 12 12 ,12 D5 10 12 12 10,12 TPOX 10, 11 8,10 DYS391 10 D8 15 11,12, 13 11, 13 14 11,12 8,9 ,11,12, 13, 15 11,12 D12 18, 18.3,21 18, 18.3 18, 20 18 17 ,18, 18.3,21 18 ,18 D19 13,13.2 ,14 15 14 13 14,15 FGA 21 20 23 20, 24 20,22 D22 14,15 15 16 ,16 172 Table H10. Fusion profiles generated from spent cartridge casings loaded by individual KKK. Locus 59 - 2.4 5a 59 - 2.45b 59 - 2.45c 59 - 2.22a 59 - 2.22b 59 - 2.22c KKK Amel Y X , Y Y X , Y X , Y D3 15 15 15 15,16 D1 17 11 15 ,16, 18.3 16 11,16 D2 10 14 14 14 10 10,14 D10 15 14 14,18 D13 10 ,10 Penta E 5,7 D16 12 11, 12 9,11 ,9,11 9 9 9,11 D18 18 14 ,18 16,18 D2 18,19 CSF 10 10,14 Penta D 9,14 THO1 6 8,9.3 8,9.3 8,9.3 8 8,9.3 8,9.3 vWA 15 17 14,17 14 14,17 D21 29 30 30 30 ,30 D7 10 8 8,10 D5 10 10 10,13 TPOX 8 ,8 DYS391 9 D8 13 ,14 11 ,14 11,12,1 3 ,14, 16 14 14,16 14,16 D12 15 15,20 20 15 15*,20 15,20 15,20 D19 13, 15.2 13 13,14 FGA 23 18 ,21, 22 24 21,24 D22 14,15 173 Table H10 Locus 59 - 1.45 59 - 3.45 59 - 4.45 59 - 1.22 59 - 3.22 59 - 4.22 KKK Amel Y X , Y X , Y X , Y X , Y X , Y D3 15, 18 16 15 16 15,16 15,16 15,16 D1 11, 12,15 16 11, 15 ,16 11, 17.3 11,16 11 11,16 D2 11 14 14 14 10 10,14 D10 14,18 14,18 18 14,18 D13 12 10 10 10 10 ,10 Penta E 13 7 7, 12 5,7 D16 9,11 9, 13 9,11 11 9,11 9,11 9,11 D18 18 18 16,18 16,18 16,18 D2 18 , 20 18 19 18 18,19 18,19 CSF 10 14 10 10,14 14 10,14 Penta D 9,14 14 9,14 8 9,14 THO1 8 9.3 9.3 8,9.3 8,9.3 6 ,8, 9 ,9.3 8,9.3 vWA 14,17 14 14 14,17 14,17 14,17 D21 28,29 ,30 28,29 30 30, 31.2 30 ,30 D7 10 8,10 8 8,10 D5 10 13 10,13 10,13 10,1 3 TPOX 8 8 8 ,8 DYS391 9 9 9 D8 13 ,14,16 16 13 ,16 13 ,14,16 14,16 14,16 14,16 D12 15, 17,19 ,20 20 15, 19 ,20 20 15, 18 ,20 15 15,20 D19 13,14 13 13,14 FGA 24 21,24 21 21,24 D22 14 14,15 14,15 174 Table H11. Fusion profiles generated from spent cartridge casings loaded by individual JJJ. Locus 60 - 4.45a 60 - 4.45b 60 - 4.45c 60 - 4.22a 60 - 4.22b 60 - 4.22c JJJ Amel X X X X , Y X X , Y D3 16,18 18 18 16 16,18 D1 16.3 16.3* 12, 14 ,16.3 12 12,16.3 12 12,16.3 D2 11 11 11 11, 11.3 11 ,11 D10 13, 16 13 13 ,13 D13 12 12 ,12 Penta E 17 7 7,17 D16 12 12 12 12 12 12 12 ,12 D18 17 17 17 17 17 ,17 D2 19 20 20,22 CSF 10 12 10,12 Penta D 10 10,11 THO1 9,9.3 9 9,9.3 6,7 ,9,9.3 6 ,9,9.3 9,9.3 9,9.3 vWA 18 14,18 14,18 14,18 14,18 14, 18 D21 28 ,30 29 ,30 30 30,30.2 30,30.2 D7 9,11 11 9 9,11 D5 12,13 TPOX 8 8 ,8 DYS391 10 10 D8 10,14 14 10,14 10,14 10 14 10,14 D12 20 19.3,20 20 19.3,20 19.3,20 D19 15.2 12 15.2 12,15.2 FGA 20 18 18 18,20 18,20 D22 11 11,15 175 Table H11 Locus 60 - 1.45 60 - 2.45 60 - 3.45 60 - 1.22 60 - 2.22 60 - 3.22 JJJ Amel X , Y X X , Y X , Y X , Y X , Y X , Y D3 16, 17 15 ,18 16,18 16,18 16, 17 ,18 16,18 D1 12,16.3 16.3 12,16.3 12,16.3 12 12, 14 ,16.3 12,16.3 D2 11 11 11 11 11 11 11 ,11 D10 13 13 13 13 ,13 D13 12 12 12 12 12 12 12 ,12 Penta E 17 5 ,7,17 7,17 7 17 7,17 D16 ,12, 13 12 12 12 12 12 12 ,12 D18 17 15 ,17 17 17 17 17 ,17 D2 20,22 20 19 ,20, 21 ,22 20,22 22 20,22 20,22 CSF 10 10 10,12 10 10,12 Penta D 11 9,11 10 10,11 10 10,11 T HO1 7 ,9,9.3 7,8 ,9,9.3 9,9.3 9,9.3 9,9.3 6,7 ,9,9.3 9,9.3 vWA 14,18 14, 17 ,18 14, 17 ,18 14,18 14,18 14,18 14,18 D21 30.2 28 ,30.2 30,30.2 30,30.2 30.2 29.2 ,30.2 30,30.2 D7 9,11 9,11 9,11 9,11 D5 12 12,13 12,13 13 12,13 TPOX 8 8 8 8 ,8 DYS391 10 10 10 10 10 ,10 D8 10, 12 ,14 10, 13 ,14 10, 12 ,14, 15 10,14 10,14 10,14 10,14 D12 19.3,20 19.3, 21 19.3,20, 22 19.3,20 20, 21 19.3,20 19.3,20 D19 12,15.2 12,15.2 12,15.2 12,15.2 12 12,15.2 FGA 18,20 18 20, 21,22 18,20 20 18,20 D22 16 15 11,15 11,15 11,15 176 Table H12. Fusion profiles generated from spent cartridge casings loaded by individual I. Locus 61 - 4.45a 61 - 4.45b 61 - 4.45c 61 - 4.22a 61 - 4.22b 61 - 4.2c I Amel X X X X ,X D3 15 14* 16 16 ,16 D1 16,17.3 D2 11 11 11 ,11 D10 15 13,15 D13 12 13 ,13 Penta E 7 ,7 D16 4 9 ,11, 13 11 11 ,11 D18 12 ,15 13 15 15,16 D2 17 17 ,17 CSF 11 12,13 Penta D 9,11 THO1 5,11 6 ,9.3 6 ,8 9.3 8,9.3 vWA 14, 15 ,18, 19 16 14,18 D21 27 27,30 D7 8,10 D5 13 11, 12 TPOX 10 8 ,8 DYS391 N/A D8 13 12 ,13 10 13,14, 15 13,14 D12 22 18,20 D19 13 ,15, 15.2 15 15 14 14,15 FGA 21,23 D22 15,16 177 Table H12 Locus 61 - 1.45 61 - 2.45 61 - 3.45 61 - 1.22 61 - 2.22 61 - 3.22 I Amel X X X X X X ,X D3 16 16 16 ,16 D1 15 ,17.3 15 17.3 16,17.3 D2 11 11 11 ,11 D10 13 13,15 D13 13 ,13 Penta E 9 7 ,7 D16 9 11, 13 11 11 ,11 D18 15 15 15,16 D2 17 17 17 17 17 ,17 CSF 13 12,13 Penta D 9,11 THO1 6 ,9.3 8,9.3 8,9.3 8,9.3 vWA 14,18 16 17 18 14,18 D21 29 29 ,30 30 30 27,30 D7 12 8 9 8,10 D5 11 11,12 TPOX 8 8 8 ,8 DYS391 N/A D8 11 ,13 13,14 13,14 13 10 13,14 D12 24,25 18 18 18,20 D19 13.2 ,14,15 13.2 14,15 FGA 21,23 21, 23 D22 15,16 178 Table H13. Fusion profiles generated from spent cartridge casings loaded by individual YYY. Locus 62 - 3.45a 62 - 3.45b 62 - 3.45c 62 - 3.22a 62 - 3.22b 62 - 3.22c YYY Amel X X X X X X ,X D3 17,18 16 ,17 17,18 D1 15, 15.3 15, 19.3 15.3 14 14,15 D2 10 10 10 10 10 ,10 D10 16 16 16 ,16 D13 9 13 12 9,13 Penta E 5 5,10 D16 13 13 12 ,14 13 13,14 D18 13,15 13,15 13 15 13, 18 13,15 D2 20 21 18 18,20 CSF 11,12 Penta D 9,11 THO1 6,7 6,7 7, 9.3 9,9.3 6,7, 9.3 6,7 6 ,7 vWA 18 14 18 14* 14 14 14,18 D21 27 30 27 30 27,30 D7 10,12 12 9 10,12 D5 11 12 11,12 TPOX 8,11 DYS391 10 N/A D8 13 13 13 13 13 ,13 D12 18 18,21 18,21 19 18,21 18,21 D19 15.2 13 14,15.2 FGA 24 21 24, 26 20,24 D22 11 11 ,11 179 Table H13 Locus 62 - 1.45 62 - 2.45 62 - 4.45 62 - 1.22 62 - 2.22 62 - 4.22 YYY Amel X X X X X X ,X D3 17 15 ,17,18 17,18 17,18 17,18 D1 14,15 12 ,14,15 14,15, 16 14 14,15 D2 10 10 10 ,10 D10 16 16 13,15 16 ,16 D13 9 ,13 9,13 Penta E 5,10 5,10 5 5,10 D16 13,14 13,14 12 ,13,14 13 13 ,14 13,14 D18 13,15 13,15 13 ,14 ,15 13 13,15 D2 18 18 18 18,20 CSF 11 12 11 11,12 Penta D 11 9,11 THO1 6,7 6,7 6,7, 9.3 6 6 6,7 6,7 vWA 14,18 14 ,18 14,18 18 14,18 D21 27 27 27,30 27 27,30 D7 10,12 10 10 12 10,12 D5 12 12 10 11,12 TPOX 11 8,11 8 8,11 DYS391 N/A D8 13 13 13 13 12 ,13 13 13 ,13 D12 18,21 17 ,18,21 18,21 18 18,21 D19 14,15.2 13 ,14,15.2 14,1 5.2 15.2 14 14,15.2 FGA 20, 23* 20,24 20,24 D22 11 11 ,11 180 Table H14. Fusion profiles generated from spent cartridge casings loaded by individual EE. Locus 63 - 2.45a 63 - 2.45b 63 - 2.45c 63 - 2.22a 63 - 2.22b 63 - 2.22c EE Amel X X X,Y D 3 16 16,18 D1 16.3,17.3 D2 11 14,15 D10 13 13,15 D13 10,12 Penta E 7,18 D16 11 ,13 9 9,13 D18 16 14 13,15 D2 20,25 CSF 10,12 Penta D 12,13 THO1 9.3 8 9 8,9.3 vWA 18 17,18 D21 30 .2 31.2 32.2 29,31 D7 9,12 D5 11,13 TPOX 8 ,8 DYS391 11 ,11 D8 8 8 8 16 8 8,13 D12 22,23 D19 15 13,15 FGA 21,23 D22 11 15,16 181 Table H14 Locus 63 - 1.45 63 - 3.45 63 - 4.45 63 - 1.22 63 - 3.22 63 - 4 .22 EE Amel X X , Y X , Y X X , Y X,Y D3 14 ,16 16, 17 16, 17 16,18 D1 11 16.3 17.3 16.3,17.3 D2 11.3 ,15 11 ,14,15 15 14,15 D10 13,15 D13 12 13 10,12 Penta E 7,18 D16 12 ,13 11,14 12 9 9,13 D18 12 ,13,15, 17 13 13,15 D2 17, 2 0 18 20,25 CSF 11 10 10,12 Penta D 12,13 THO1 7 ,8 9 ,9.3 6 ,8,9.3 8 8,9.3 vWA 18 17,18 D21 28 29 29,31 D7 10 9,12 D5 11,13 TPOX 8 ,8 DYS391 11 ,11 D8 8, 9 ,13 10 ,13, 14 8, 9,15,16 8, 10 ,13 ,16 13 8,13 D12 19.3 20,21 15 22 22,23 D19 13 13,15 13,15 13,15 FGA 21.2 21 21 21,23 D22 15,16 182 Table H15. Fusion profiles generated from spent cartridge casings loaded by individual JJ. Locus 65 - 2.45a 65 - 2.45b 65 - 2.45c 65 - 2.22a 65 - 2.22b 65 - 2.22c JJ Amel X X X X X D3 14,17 17 16 14,17 D1 12,16 16 12,16 D2 11 14 11,14 D10 13 14 ,14 D13 11,12 Penta E 18 11 10,11 D16 10 ,11 11 12 11 11, 12,13 11 ,11 D18 14,19 14 14,19 D2 18,19 18,19 CSF 11 11 11,13 Penta D 11,14 THO1 5,11 8 ,9.3 9 9 9.3 9,9.3 vWA 16 11,14 13,14 18 11,15 D21 31 29 30.2 30.2,31.2 D7 9 10 9,10 D5 11 12,13 TPOX 8 8 ,8 DYS391 10 N/A D8 13 13 13 13 13 13 ,13 D12 20 16,21 15,20 ,21 19 15 15,20 D19 11,13.2 F GA 20 22,24 D22 14 11,14 183 Table H15 Locus 65 - 1.45 65 - 3.45 65 - 4.45 65 - 1.22 65 - 3.22 65 - 4.22 JJ Amel X X X X X X ,X D3 14,17, 18 14,17 14, 16 ,17, 18 18 16 ,17 17 14,17 D1 12,16 12 12 16 12,16 D2 11,14 11,14 11,14 14 14 11,14 D10 14 14 14 14 14 14 ,14 D13 11,12 11,12 11,12 Penta E 10,11 10, 12 10,11 D16 11 11 11 11 12 11 11 ,11 D18 14, 17 14,19 16 ,19 14 19 14,19 D2 19 18,19 18,19 18 18,19 CSF 11,13 13 10 ,11,13 11,13 Penta D 11,14 11,14 THO1 9,9.3 9,9.3 6 ,9,9.3 6 ,9 9,9.3 9,9.3 9,9.3 vWA 14,18 14,18 14 ,15, 17,18 18 14,18 14 11,15 D21 30.2,31.2 30.2,31.2 28 ,30.2,31.2 30.2 30.2,31.2 D7 10 9,10 9,10 D5 11 ,12 11,12 11 11 12,13 TPOX 8 8 ,8 DYS391 N/A D8 13 13 12 ,13, 15 13 13 13 13 ,13 D12 15,20 1 5,20 15, 18 ,20 20 21 15,20 D19 11,13.2 11,13.2 14,15 11,13.2 FGA 22,24 22,24 D22 11,14 11,14 184 Table H16. Fusion profiles generated from spent cartridge casings loaded by individual DDD. Locus 66 - 1.45a 66 - 1.45b 66 - 1.45c 66 - 1.22a 66 - 1.22b 66 - 1.22c DDD Amel X , Y X , Y X X X X X , Y D3 14 14,18 18 18 14,18 D1 14,17.3 14 14,17.3 D2 11 11 11 ,11 D10 14 14 ,14 D13 10,12 Penta E 19 7,19 D16 ,11,12 7 12 11,12 D18 12 17 10 10,12 D2 20 17,20 CSF 11,12 Penta D 9 9,12 THO1 8 8 8 8 ,8 vWA 17 17 16 ,17 17, 18 17 17 ,17 D21 29,32.2 D7 12 ,12 D5 10 13 ,13 TPOX 9 ,9 DYS391 11 D8 12,13 12,13 13 16 12, 14 12,13 D12 15, 23 ,24 15 24 24 15,24 D19 13* ,14 14 14,15 14,15 FGA 22 22 24 22,24 D22 16 ,16 185 Table H16 Locus 66 - 2.45 66 - 3.45 66 - 4.45 66 - 2.22 66 - 3.22 66 - 4.22 DDD Amel X , Y X , Y X Y X , Y D3 14, 16,17 ,18 14,18 14 14,18 14 14 14,18 D1 14 14,17.3 14 17.3 14,17.3 D2 11 11, 11.3 11 11 11 ,11 D10 1 4 14 14 ,14 D13 10 10,12 10,12 Penta E 7, 13 7,19 7 7,19 D16 11,12 11,12 11,12 11,12 11,12 D18 12 10,12 10 10 10 10,12 D2 20 17,20 20 17,20 CSF 11 11,12 12 11,12 Penta D 12 9,12 THO1 8, 9 8 6,7 ,8, 9.3 6 ,8, 9.3 7 8 ,8 vWA 14 ,17 1 7 16 ,17 17 14 17 ,17 D21 28 ,32.2 29,32.2 29 29,32.2 D7 12 12 12 ,12 D5 13 13 13 13 ,13 TPOX 9 9 9 9 ,9 DYS391 11 11 11 D8 12,13 12,13 11 ,12,13, 14, 15,16 13 12,13 D12 15,24 15, 18,23 15, 21 15 15,24 D19 13 ,15 14,15 14,15 13.2 14,15 FGA 24 22,24 22,24 D22 16 16 16 16 ,16 186 Table H17. Fusion profiles generated from spent cartridge casings loaded by individual VVV. Locus 67 - 1.45a 67 - 1.45b 67 - 1.45c 67 - 1.22a 67 - 1.22b 67 - 1.22c VVV Amel X X X , Y X , Y D3 14 14, 15 15 14,17 D1 17.3 15,17.3 D2 11 11,14 D10 12,13 D13 12 11 11 ,11 Penta E 7,8 D16 12 12 ,12 D18 12 17 12,16 D2 17,18 CSF 11 ,11 Penta D 9,12 THO1 9.3 9.3 ,9.3 vWA 17 ,17 D21 28,32.2 D7 10,11 D5 11,13 TPOX 11 ,11 DYS391 11 D8 8 16 8,12 D12 18.3 15 15 15, 18.3 15,25 D19 15 14,15.2 FGA 21.2 22,23 D22 11,15 187 Table H17 Locus 67 - 2.45 67 - 3.45 67 - 4.45 67 - 2.22 67 - 3.22 67 - 4.22 VVV Amel Y X X X , Y D3 17 15 ,17 16 15 14,17 D1 17.3 16,16.3 ,17.3 15,17.3 D2 11 11,14 D10 13 14 12,13 D13 9,12 11 ,11 Penta E 12,21 7,8 D16 12 11,13 11 ,12 12 ,12 D18 17 14 ,16 12,16 D2 17, 24 19 17,18 CSF 10 11 ,11 Penta D 9, 11 9 9,12 THO1 9.3 7 ,9.3 9 9,9.3 9.3 ,9.3 vWA 17 14,16 17 ,17 D21 30,30.2 28 28,32.2 D7 12 10 10,11 D5 13 11,13 TPOX 8 ,11 11 ,11 DYS391 11 D8 11,16 14 10 9,11 ,12 8,12 D12 23 17,22 15, 18,18.3 18 15,25 D19 13,15 ,15.2 14 14,15.2 FGA 22,23 23 21 22,23 D22 11,15 188 Table H18. Fusion profiles generated from spent cartridge casings loaded by individual XXX. Locus 68 - 1.45a 68 - 1.45b 68 - 1.45c 68 - 1.22a 68 - 1.22b 68 - 1.22c XXX Amel X X X Y X , Y D3 14,16 D1 15.3 17.3 12,16 D2 11 11.3,14 D10 11 14 14,16 D13 9,12 Penta E 14 7 ,7 D16 13 ,13 13 12,13 D18 25 18 14,16 D2 20 18,25 CSF 10,12 Penta D 10 9,11 THO1 13.3 7,9.3 vWA 23 18 , 18 D21 28.2 30 28,30 D7 5 10 ,10 D5 11, 12 11 ,11 TPOX 8 8 ,8 DYS391 10 D8 14 15 9 ,15 10,16 14,15 D12 21 19,21 D19 14 15 14,15 FGA 23 23.2,25 D22 16 14,16 189 Table H18 Locus 68 - 2.45 68 - 3.45 68 - 4 .45 68 - 2.22 68 - 3.22 68 - 4.22 XXX Amel X X X X , Y D3 14, 18* 18 15 18 14,16 D1 16 12,16, 17.3 17 12, 17 12,16 D2 11* 11 11 11.3,14 D10 14 16 14,16 D13 12 9,12 Penta E 7 ,7 D16 9 ,13 12 13 9 ,13 12,13 D18 16, 20 14 12 14,16 D2 18 17 25 25 18,25 CSF 10 10,12 Penta D 13 11 9,11 THO1 6 ,7, 9 7 9.3 6 7,9.3 7,9.3 vWA 18 18 15,16 18 ,18 D21 30.2 32.2 28,30 D7 10 10 ,10 D5 12 11 ,11 TPOX 8 8 ,8 DYS391 10 D8 11,12,13 13 ,14 13 ,14 10, 16 14,15 D12 17,23 19,21 D19 15 14 14,15 FGA 22 25 23.2,25 D22 16 14,16 190 Table H19. Fusion profiles generated from spent cartridge casings loaded by individual R. Locus 69 - 2.45a 69 - 2.45b 69 - 2.45c 69 - 2.22a 69 - 2.22b 69 - 2.22c R Amel X X X X X ,X D3 16 16,17 D1 15 15 12 12,18.3 D2 11 ,11 D10 13,14 D13 12 ,12 Penta E 10,14 D16 9,11,16 12,14 D18 18 17,18 D2 20,23 CSF 10,13 Penta D 12,13 THO1 9 6 9 9.3 6,9 vWA 17 15 15,1 7 D21 29 30,30.2 D7 11 10 ,10 D5 10,11 TPOX 8,11 DYS391 N/A D8 7,11 13,16 13 10 10,12 D12 19 20 18,21 D19 13 14 14,15.2 FGA 21,25 D22 15 ,15 191 Table H19 Locus 69 - 1.45 69 - 3.45 69 - 4.45 69 - 1 .22 69 - 3.22 69 - 4.22 R Amel X X X X X ,X D3 16 16 16,17 D1 12 17.3 12,18.3 D2 11 11.3 11 ,11 D10 13,14 D13 12 ,12 Penta E 14 10,14 D16 12,14 D18 17 17,18 D2 20,23 CSF 10 10,13 Penta D 12,13 THO1 6 6,9 6 9 8 6,9 vWA 18 17 15,17 D21 30,30.2 D7 10 ,10 D5 11 10,11 TPOX 11 8,11 DYS391 N/A D8 10 12 10 10 10 10,12 D12 19 18,21 D19 14 15 14,15.2 FGA 21,25 D22 15 ,15 192 Table H20 . Fusi on profiles generated from spent cartridge casings loaded by individual OOO. Locus 70 - 3.45a 70 - 3.45b 70 - 3.45c 70 - 3.22a 70 - 3.22b 70 - 3.22c OOO Amel Y X X X , Y D3 14 14 14 14 ,14 D1 16.3,17.3 D2 11,11.3 D10 15 16 15,16 D13 12 12 10 10,12 Penta E 5,14 D16 12 11,12 D18 16,17 D2 20 20,22 CSF 10,11 Penta D 11,12 THO1 9 9,9.3, 11 9 9.3 9,9.3 vWA 16 20 12 16,18 D21 32.2 28,32.2 D7 11 12 11,12 D5 12 ,12 TPOX 8 ,8 DYS391 11 D8 12, 13 9 9,12 D12 21 21,23 D19 12,14 FGA 22 22 21.2,22 D22 11,16 193 Table H20 Locus 70 - 1.45 70 - 2.45 70 - 4.45 70 - 1.22 70 - 2.22 70 - 4.22 OOO Amel X , Y Y X , Y X Y X X , Y D3 14, 16 14, 17 14 14 ,14 D1 11 ,16.3 16 .3,17.3 16.3,17.3 16.3,17.3 D2 10 11,11.3 11,11.3 11,11.3 D10 16 16 15,16 D13 10 10 10,12 Penta E 5,14 5,14 D16 9 ,11 11,12, 13 11,12 11,12 11,12 D18 16 16,17 17 16 16,17 D2 20 20 22 20,22 CSF 10,11 11 10,11 Penta D 12 11,12 THO1 9.3 9,9.3 9,9.3 9 9.3 9,9.3 vWA 16,18 16,18 15 ,16,18 16 18 16,18 D21 28,32.2 32.2 28,32.2 D7 12 12 11,12 D5 12 12 12 ,12 TPOX 8 8 ,8 DYS391 11 11 11 D8 12 9,12 9,12 9 12 12 ,13, 16 9,12 D12 23 23 21, 22 ,23 21 21, 23 21,23 D19 12, 15 12 12, 13 12,14 FGA 21.2 21.2 21.2,22 22 21.2,22 D22 16 11,16 194 APPENDIX I. HANDLER AND NON - HANDLER ALLELES AMPLIFIED WITH FUSION FROM COLLECTION 2 Table I1. Summary of number of handler (H) alleles, non - handler (NH) alleles , and percent profile produced using Fusion from the individually swabbed 0.45 casings from Collection 2. Sample # H Alleles # NH Alleles % Profile 62 - 3.45b 21 1 51.2 60 - 4.45c 25 2 61.0 62 - 3.45c 14 2 34.1 53 - 4.45a 21 2 52.5 67 - 1.45b 1 2 2.4 58 - 3.45b 5 17 12.8 52 - 3.45b 28 2 63.6 53 - 4.45b 19 0 47.5 58 - 3.45a 8 1 20.5 39 - 4.45c 13 0 32.5 65 - 2.45b 11 7 26.8 70 - 3.45c 11 2 25.0 58 - 3.45c 3 5 7.7 63 - 2.45a 4 4 8.7 53 - 4.45c 9 0 22.5 63 - 2.45c 6 2 13.0 63 - 2.45b 2 1 4.3 66 - 1.45a 18 4 46.2 67 - 1.45a 2 1 4 .9 60 - 4.45b 10 0 24.4 52 - 3.45a 12 3 27.3 66 - 1.45b 15 1 38.5 51 - 2.45c 21 1 51.2 59 - 2.45a 4 7 9.1 61 - 4.45c 6 1 15.8 60 - 4.45a 18 3 43.9 39 - 4.45b 15 2 37.5 65 - 2.45c 1 0 2.4 52 - 3.45c 12 1 27.3 59 - 2.45b 9 3 20.5 70 - 3.45b 2 0 4.5 61 - 4.45b 5 11 13.2 65 - 2.45a 9 8 22.0 39 - 4.45a 11 1 27.5 195 70 - 3.45a 6 0 13.6 67 - 1.45c 6 2 14.6 57 - 1.45a 3 6 7.0 55 - 1.45c 2 1 4.8 51 - 2.45b 11 4 26.8 62 - 3.45a 6 1 14.6 59 - 2.45c 10 1 22.7 68 - 1.45b 3 5 7.1 57 - 1.45b 1 4 2.3 69 - 2.45a 2 9 4.9 56 - 4.45a 3 0 6.8 54 - 3.45a 17 1 44.7 55 - 1.45b 5 0 11.9 55 - 1.45a 1 0 2.4 51 - 2.45a 15 3 36.6 66 - 1.45c 6 1 15.4 54 - 3.45c 7 3 18.4 68 - 1.45c 3 1 7.1 61 - 4.45a 6 7 15.8 54 - 3.45b 9 2 23.7 69 - 2.45b 4 1 9.8 57 - 1.45c 1 0 2.3 68 - 1.45a 4 2 9.5 69 - 2.45c 0 2 0.0 56 - 4.45 b 2 0 4.5 56 - 4.45c 2 2 4.5 196 Table I2. Summary of number of handler (H) alleles, non - handler (NH) alleles, and percent profile produced using Fusion from the individually swabbed 0.22 casings from Collection 2. Sample # H Alleles # NH Alleles % P rofile 60 - 4.22c 14 1 34.1 60 - 4.22b 16 1 39.0 62 - 3.22b 11 1 26.8 63 - 2.22b 1 0 2.2 63 - 2.22c 2 1 4.3 62 - 3.22a 11 10 26.8 55 - 1.22b 0 0 0.0 70 - 3.22a 7 0 15.9 67 - 1.22a 1 2 2.4 70 - 3.22c 0 1 0.0 39 - 4.22a 13 1 32.5 63 - 2.22a 0 1 0.0 53 - 4.22a 4 1 10.0 5 5 - 1.22a 4 3 9.5 70 - 3.22b 1 1 2.3 59 - 2.22a 18 4 40.9 56 - 4.22c 2 1 4.5 67 - 1.22b 3 0 7.3 56 - 4.22a 1 0 2.3 59 - 2.22b 10 5 22.7 67 - 1.22c 3 2 7.3 65 - 2.22a 5 4 12.2 60 - 4.22b 14 1 34.1 56 - 4.22b 1 0 2.3 53 - 4.22b 0 1 0.0 54 - 3.22b 6 2 15.8 52 - 3.22b 11 2 2 5.0 54 - 3.22a 7 1 18.4 59 - 2.22c 16 0 36.4 39 - 4.22b 8 4 20.0 62 - 3.22c 9 3 22.0 58 - 3.22c 2 0 5.1 65 - 2.22c 9 5 22.0 54 - 3.22c 6 2 15.8 65 - 2.22b 7 1 17.1 53 - 4.22c 1 0 2.5 52 - 3.22c 5 0 11.4 197 55 - 1.22c 0 1 0.0 68 - 1.22c 3 5 7.1 66 - 1.22a 9 2 23.1 69 - 2.22b 5 1 12.2 51 - 2.22c 11 1 26.8 68 - 1.22a 5 8 11.9 51 - 2.22a 5 1 12.2 58 - 3.22a 5 1 12.8 51 - 2.22b 7 0 17.1 57 - 1.22b 3 3 7.0 69 - 2.22c 3 1 7.3 66 - 1.22b 2 1 5.1 69 - 2.22a 0 2 0.0 61 - 4.22a 3 3 7.9 57 - 1.22a 2 0 4.7 58 - 3.22b 3 2 7.7 52 - 3 .22a 2 0 4.5 66 - 1.22c 4 0 10.3 57 - 1.22c 1 2 2.3 61 - 4.22c 4 1 10.5 68 - 1.22b 0 0 0.0 61 - 4.22b 2 1 5.3 198 Table I3. Summary of number of handler (H) alleles, non - handler (NH) alleles, and percent profile produced using Fusion from the cumulativ ely swabbed 0.45 casings from Collection 2. Sample # H Alleles # NH Alleles % Profile 52 - 1.45 24 20 54.5 54 - 1.45 35 2 92.1 53 - 3.45 36 0 90.0 53 - 1.45 27 9 67.5 66 - 3.45 38 0 97.4 67 - 4.45 14 25 34.1 57 - 2.45 43 1 100.0 65 - 3.45 39 2 95.1 65 - 4.45 22 15 53.7 62 - 2.45 34 4 82.9 65 - 1.45 20 5 48.8 54 - 2.45 20 2 52.6 62 - 4.45 22 7 53.7 52 - 4.45 32 0 72.7 60 - 3.45 35 11 85.4 62 - 1.45 29 1 70.7 70 - 4.45 28 3 63.6 53 - 2.45 30 1 75.0 54 - 4.45 26 7 68.4 39 - 2.45 29 12 72.5 52 - 2.45 24 1 54.5 66 - 2.45 24 7 61.5 5 6 - 3.45 10 9 22.7 68 - 2.45 7 9 16.7 39 - 3.45 28 20 70.0 66 - 4.45 12 11 30.8 70 - 2.45 27 2 61.4 55 - 3.45 30 11 71.4 58 - 2.45 19 9 48.7 70 - 1.45 18 4 40.9 58 - 4.45 14 8 35.9 58 - 1.45 12 19 30.8 61 - 2.45 11 3 28.9 39 - 1.45 24 5 60.0 55 - 4.45 17 8 40.5 61 - 1.45 10 9 26.3 199 60 - 1.45 23 4 56.1 56 - 2.45 4 12 9.1 60 - 2.45 22 8 53.7 63 - 4.45 5 6 10.9 56 - 1.45 17 2 41.5 63 - 3.45 9 8 19.6 61 - 3.45 14 2 36.8 69 - 4.45 5 0 12.2 67 - 3.45 3 0 7.3 63 - 1.45 9 10 19.6 59 - 1.45 22 12 50.0 55 - 2.45 10 7 23.8 51 - 4.45 15 2 36.6 57 - 4.45 15 3 34.9 69 - 3.45 5 0 12.2 51 - 3.45 17 5 41.5 59 - 4.45 13 3 29.5 67 - 2.45 3 4 7.3 68 - 3.45 6 4 14.3 69 - 1.45 4 1 9.8 68 - 4.45 9 3 21.4 59 - 3.45 12 3 27.3 57 - 3.45 6 2 14.0 51 - 1.45 11 6 26.8 200 Table I4. Summary of number of handler (H) alleles, non - handler (NH) alleles, and percent profile produced using Fusion from the cumulatively swabbed 0.22 casings from Collection 2. Sample # H Alleles # NH Alleles % Profile 59 - 3.22 41 2 93.2 60 - 1.22 41 0 100.0 60 - 2.22 31 1 75.6 59 - 4.22 30 3 68.2 60 - 3.22 26 5 63.4 54 - 1.22 11 16 28.9 65 - 3.22 11 6 26.8 68 - 3.22 8 10 19.0 62 - 1.22 4 0 9.8 39 - 1.22 21 7 52.5 54 - 2.22 22 3 57.9 39 - 3.22 18 5 45.0 39 - 2.22 24 8 60.0 65 - 4.22 9 2 22.0 56 - 1.22 5 10 11.4 54 - 4.22 17 4 44.7 56 - 2.22 3 5 6.8 63 - 4.22 12 2 26.1 63 - 1.22 8 6 17.4 67 - 2.22 5 8 12.2 63 - 3.22 4 2 8.7 52 - 2.22 20 1 45.5 52 - 1.22 20 2 45.5 51 - 3.22 9 2 22.0 66 - 3.22 10 4 25.6 53 - 3.22 6 4 15.0 62 - 2.22 7 1 17.1 53 - 1.22 6 6 15.0 56 - 3.22 9 3 20.5 61 - 3.22 3 2 7.9 66 - 2.22 14 2 35.9 58 - 1.22 7 10 17.9 62 - 4.22 15 1 36.6 51 - 4.22 13 5 31.7 53 - 2.22 9 2 22.5 61 - 2.22 6 0 15.8 201 69 - 4.22 5 0 12.2 68 - 4.22 7 5 17.1 55 - 2.22 9 13 21.4 67 - 3.22 3 3 7.3 61 - 1.22 4 2 10.5 70 - 2.22 8 1 18.2 70 - 4.22 10 2 22.7 51 - 1.22 10 1 24.4 67 - 4.22 3 2 7.3 55 - 3.22 6 3 14.3 65 - 1.22 8 3 19.5 69 - 3.22 2 4 4.9 52 - 4.22 19 1 43.2 58 - 2.22 9 1 23.1 69 - 1.22 4 1 9.8 70 - 1.22 3 0 6.8 58 - 4.22 15 19 38.5 57 - 4.22 2 6 4.7 68 - 2.22 1 2 2.4 57 - 3.22 4 3 9.3 66 - 4.22 3 0 7.7 55 - 4.22 7 4 16.7 5 9 - 1.22 18 2 40.9 57 - 2.22 3 2 7.0 202 APPENDIX J. MTDNA PROFILES GENERATED FROM SPENT CARTRIDGE CASINGS Red font: polymorphism not consistent with handler Blank: no polymorphisms A: adenine T: thymine C: cytosine G: guanine Y: mixture between cytosine a nd thymine R: mixture between adenine and guanine M: mixture between adenine and cytosine S: mixture between cytosine and guanine Table J1. MtDNA profiles generated from spent cartridge casings loaded by individual NN. Sample HV1 HV2 mtDNA Classification Quantification Level NN 16293G,16311T 195C, 263G, 309.1C, 315.1C ------ ------ 51 - 1.45 16293G, 16311T (73 not sequenced) 195C, 263G, 309.1C, 315.1C Consistent Low 51 - 4.22 16069T, 16126C, 16160G 73G, 185A , 263G, 295T , 315.1C, 462T Inconsistent Medium 203 Table J2. MtDNA profiles generated from spent cartridge casings loaded by individual ZZZ. Sample HV1 HV2 mtDNA Classification Quantification Level ZZZ 16126C, 16294T, 16296T, 16304C 73G, 263G, 315.1C ------ ------ 52 - 1.45 16256T 204C , 263G, 315.1C Inconsistent High 52 - 3.22a 16126C, 16294T, 16296T, 16304C 73G, 263G, 315.1C Consistent Low 52 - 3.22b 16185T, 16223T, 16355A, 16362C 73G, 263G, 315.1C Inconsistent Medium 52 - 3.45b 16126C, 16294T, 16296T, 16304C 73G, 263G, 315.1C Consistent High 52 - 3.45c 16126C, 16294T, 16296T, 16304C 73R, 263G (315.1 not sequenced) Mixed - Consistent Medium Table J3. MtDNA profiles generated from spent cartridge casings loaded by individual B. Sample HV1 HV2 mtDNA Classification Quantification Level B 16069T, 16126C, 16 160G, 16222T 73G, 185A, 263G, 295T, 315.1C, 462T ------ ------ 53 - 1.22 16069Y, 16126Y, 16160R, 16222Y 73G, 185A, 263G, 295T, 315.1C, 462T Mixed - Consistent Medium 53 - 1.45 16069Y, 16126C, 16160R, 16222Y 73G, 185A, 263G, 295T, 315.1C, 462T Mixed - Consistent High 53 - 3.45 16069T, 16126C, 16160G, 16222T 73G, 185A, 263G, 295T, 315.1C, 462T Consistent High 53 - 4.45a 16069T, 16126C, 16160G, 16222T 73G, 185A, 263G, 295T, 315.1C, 462T Consistent High 53 - 4.45b 16069T, 16126C, 16160G, 16222T 73G, 185A, 263G, 295T, 31 5.1C (462 not sequenced) Consistent High 204 Table J4. MtDNA profiles generated from spent cartridge casings loaded by individual BBB. Sample HV1 HV2 mtDNA Classification Quantification Level BBB 66T, 152C, 263G, 315.1C ------ ------ 54 - 1.22 263G, 315.1C Inconsistent High 54 - 1.45 66T, 152C, 263G, 315.1C Consistent High 54 - 3.22a 66T, 152C, 263G, 315.1C Consistent Medium 54 - 3.22c 66T, 152C, 263G, 315.1C Consistent Medium 54 - 3.45b 66T, 152C, 263G, 315.1C Consistent Low Table J5. MtDNA profile s generated from spent cartridge casings loaded by individual C. Sample HV1 HV2 mtDNA Classification Quantification Level C 16192T, 16256T, 16270T 73G, 263G, 315.1C ------ ------ 55 - 1.22b 263G, 315.1C Inconsistent High 55 - 3.45 16192T, 16256T, 16270T (7 3 not sequenced) 263G, 315.1C Consistent Medium 55 - 4.22 263G, 315.1C Inconsistent Low Table J6. MtDNA profiles generated from spent cartridge casings loaded by individual AA. Sample HV1 HV2 mtDNA Classification Quantification Level AA 16104T, 16126C, 16294T, 16304C 73G, 152C, 263G, 315.1C ------ ------ 56 - 3.22 16104Y, 16126Y, 16294Y, 16304Y 73G, 152Y, 263G, 315.1C Mixed - Consistent Medium 56 - 4.45b 16069Y, 16126Y, 16160R, 16222Y 73G, 152C, 263G, 315.1C Mixed - Inconsistent Low 56 - 4.45c 16104T, 16126C, 16294T, 16304C 73G, 152C, 263G, 315.1C Consistent Low 205 Table J7. MtDNA profiles generated from spent cartridge casings loaded by individual A. Sample HV1 HV2 mtDNA Classification Quantification Level A 16051G, 16129C, 16183C, 16189C 73G, 152C, 217C, 263 G, 315.1C ------ ------ 57 - 1.22a 16051G, 16162G 263G, 315.1C Inconsistent Low 57 - 1.22c 16093C , 16189C 73G, 263G (315 not sequenced) Inconsistent Low 57 - 1.45c 16051R, 16126Y, 16129S, 16183M, 16189Y, 16294Y, 16296Y 73G, 152Y, 217Y, 263G, 315.1C Mixed - Cons istent Low 57 - 2.22 16051R, 16126Y, 16129S, 16183M, 16189C, 16294Y, 16296Y 73G, 152C, 217Y, 263G, 315.1C Mixed - Consistent Low 57 - 2.45 16051G, 16129C, 16183C, 16189C 73G, 152C, 217C, 263G, 315.1C Consistent High 57 - 3.22 16051R, 16126Y, 16129S, 16183M, 161 89Y, 16294Y, 16296Y 73G, 185R, 263G, 295Y , 315.1C Mixed - Consistent Low 57 - 3.45 16051G, 16126Y, 16129S, 16183C, 16189Y 73G, 152C, 263G, 315.1C Mixed - Consistent Low 57 - 4.22 16051R, 16126Y, 16129S, 16183M, 16189Y, 16294Y, 16296Y 73G, 152Y, 217Y, 263G, 315.1 C Mixed - Consistent Low 206 Table J8. MtDNA profiles generated from spent cartridge casings loaded by individual J. Sample HV1 HV2 mtDNA Classification Quantification Level J 263G, 309.2C, 315.1C ------ ------ 58 - 1.22 263G (309, 315 not sequenced ) Consistent Medium 58 - 1.45 16069Y, 16126Y, 16160R, 16222Y 73R , 185R, 263G (309, 315 not sequenced) Mixed - Consistent Medium 58 - 2.45 16126Y, 16222Y 263G, 309.2C, 315.1C Mixed - Consistent Medium 58 - 3.22b 263G, 309.2C, 315.1C Consistent Low 58 - 3.22c not sequenced Consistent Medium 58 - 3.45b 263G (309, 315 not sequenced) Consistent High 58 - 4.22 73G , 185A , 263G, 295T , 315.1C, 462T Inconsistent Low 58 - 4.45 73R, 152Y, 263G (309, 315 not sequenced) Mixed - Consistent Medium Table J9. MtDNA profiles genera ted from spent cartridge casings loaded by individual KKK. Sample HV1 HV2 mtDNA Classification Quantification Level KKK 16311C 93G, 263G, 315.1C ------ ------ 59 - 1.22 16311C 93R, 263G, 315.1C Mixed - Consistent Low 59 - 2.22c 16311C 93G, 263G, 315.1C Consis tent Medium 59 - 2.45b 16311C 73R, 93R, 263G, 315.1C Mixed - Consistent Medium 59 - 3.22 16311C 93G, 263G, 315.1C Consistent High 59 - 3.45 16311C 73R, 93R, 263G, 295Y, 315.1C Mixed - Consistent Low 59 - 4.22 16311C 93G, 263G, 315.1C Consistent High 59 - 4.45 16311 C 73R, 93R, 263G, 315.1C Mixed - Consistent Low 207 Table J10. MtDNA profiles generated from spent cartridge casings loaded by individual JJJ. Sample HV1 HV2 mtDNA Classification Quantification Level JJJ 16126C, 16294T, 16296T 73G, 263G, 315.1C ------ --- --- 60 - 1.22 16126C, 16294T, 16296T 73G, 263G, 315.1C Consistent High 60 - 2.22 16126C, 16294T, 16296T 73G, 263G, 315.1C Consistent High 60 - 3.22 16126C, 16294T, 16296T 73G, 263G, 315.1C Consistent High 60 - 4.22b 16126C, 16294T, 16296T 73G, 263G, 315.1C Con sistent High 60 - 4.22c 16126C, 16294T, 16296T 73G, 263G, 315.1C Consistent High 60 - 4.45c 16126C, 16294T, 16296T 73G, 263G, 315.1C Consistent High Table J11. MtDNA profiles generated from spent cartridge casings loaded by individual I. Sample HV1 HV2 mtD NA Classification Quantification Level I 16192T, 16256T, 16270T, 16291T 73G, 263G, 315.1C ------ ------ 61 - 2.45 16192T, 16256T, 16270T, 16291T 73G, 263G, 315.1C Consistent Medium 61 - 3.22 16192T, 16246T, 16270T, 16291T 73G, 263G, 315.1C Consistent Medium 61 - 4.22c 16192T, 16256T, 16270T, 16291T 73G, 263G, 315.1C Consistent Low 61 - 4.45a 16192T, 16256T, 16270T, 16291T 73G, 263G, 315.1C Consistent Low 61 - 4.45b 16104Y, 16126Y, 16192Y, 16256Y, 16270Y, 16291Y, 16294Y, 16304Y 73G, 263G, 315.1C Mixed - Consistent Medium 208 Table J12. MtDNA profiles generated from spent cartridge casings loaded by individual YYY. Sample HV1 HV2 mtDNA Classification Quantification Level YYY 16126C, 16294T, 16296T, 16304C 73G, 263G, 309.1C, 315.1C, 458T ------ ------ 62 - 2.22 1606 9Y , 16126Y, 16160R, 16162R, 16222Y, 16294T, 16296T, 16304C 73G, 263G, 309.1C, 315.1C, 458T Mixed - Consistent Medium 62 - 3.22a 16093C , 16104T , 16126C, 16294T 73G, 152C , 263G, 315.1C Inconsistent High 62 - 3.22b 16126C, 16294T, 16296T, 16304C 73G, 263G, 309.1C , 315.1C, 458T Consistent High 62 - 3.22c 16126C, 16294T, 16296T, 16304C 73G, 263G, 309.1C, 315.1C, 458T Consistent Medium 62 - 3.45b 16126C, 16294T, 16296T, 16304C 73G, 263G, 309.1C, 315.1C, 458T Consistent High 62 - 3.45c 16051R, 16126Y, 16126R, 16294T, 162 96T, 16304C 73G, 263G, 309.1C, 315.1C, 458T Mixed - Consistent High 62 - 4.22 16093C , 16192T , 16294T, 16296T, 16304C 73G, 263G, 315.1C Inconsistent Medium Table J13. MtDNA profiles generated from spent cartridge casings loaded by individual EE. Sample HV1 H V2 mtDNA Classification Quantification Level EE 16126C, 16189C, 16294T, 16296T, 16298C 73G, 195C, 263G, 315.1C ------ ------ 63 - 2.22b 16126Y, 16189Y, 16294Y, 16296Y, 16304Y 73R, 195Y, 263G (315 not sequenced) Mixed - Consistent High 63 - 2.22c 16126Y, 16294 Y, 16296Y 73R, 263G, 315.1C Mixed - Inconsistent High 209 Table J14. MtDNA profiles generated from spent cartridge casings loaded by individual JJ. Sample HV1 HV2 mtDNA Classification Quantification Level JJ 16147T 263G, 309.2C, 315.1C ------ ------ 65 - 2 .22c 16147T 263G (309, 315 not sequenced) Consistent Medium 65 - 2.45a 16126Y, 16222Y 73G, 242Y, 263G, 295Y , 315.1C Mixed - Inconsistent Medium 65 - 2.45c 16147T 263G, 309.2C, 315.1C Consistent Medium 65 - 3.22 16126Y, 16147Y, 16294Y 73R, 263G, 309.1Y, 315.1C M ixed - Consistent High 65 - 3.45 16147T 263G, 309.2C, 315.1C Consistent High Table J15. MtDNA profiles generated from spent cartridge casings loaded by individual DDD. Sample HV1 HV2 mtDNA Classification Quantification Level DDD 16051G, 16162G 73G, 263G, 3 15.1C ------ ------ 66 - 1.22c 16051G, 16162G 73G, 263G, 315.1C Consistent Low 66 - 2.22 16051G, 16162G 73G, 263G, 315.1C Consistent Medium 66 - 3.45 16051G, 16162G, 73G, 263G, 315.1C Consistent High 66 - 4.22 16051G, 16162G 73R, 263G, 315.1C Mixed - Consistent Low Table J16. MtDNA profiles generated from spent cartridge casings loaded by individual VVV. Sample HV1 HV2 mtDNA Classification Quantification Level VVV 16189C 73G, 150T, 263G, 315.1C ------ ------ 67 - 1.45b 16189C 73G, 150T, 263G, 315.1C Consistent High 67 - 2.45 16189Y 73G, 150Y, 242Y, 263G, 295Y, 315.1C Mixed - Consistent Low 67 - 4.45 16179T, 16242T 73G, 150T, 195Y , 263G, 315.1C Inconsistent High 210 Table J17. MtDNA profiles generated from spent cartridge casings loaded by individual XXX. Sample HV 1 HV2 mtDNA Classification Quantification Level XXX 16093C, 16189C 263G, 315.1C ------ ------ 68 - 1.45a 16093C, 16189C 263G, 315.1C Consistent Low 68 - 1.22b 16051G , 16129C , 16183C , 16189C, 73G , 152C , 217C , 263G, 315.1C Inconsistent Low 68 - 2.22 16093T/Y, 16189T/Y 263G, 315.1C Mixed - Consistent Low 68 - 3.22 263G, 315.1C Inconsistent High 68 - 3.45 16069T, 16126C, 16160G, 16222T 73G , 185A , 263G, 295T , 315.1C, 462T Inconsistent Low 68 - 4.45 16093Y, 16189Y 263G, 315.1C Mixed - Consistent Low Table J18. MtDNA pr ofiles generated from spent cartridge casings loaded by individual R. Sample HV1 HV2 mtDNA Classification Quantification Level R 16093C, 16192T, 16256T, 16270T, 16291T 73G, 263G, 315.1C ------ ------ 69 - 1.45 16104T, 16126C, 16294T, 16304C 73G, 152C , 263G , 315.1C Inconsistent Low 69 - 2.45b 16093C, 16192T, 16256T, 16270T, 16291T 73G, 263G, 315.1C Consistent Low 69 - 2.45c 16256Y 73G, 152C , 195C , 263G, 309.1C , 315.1C Inconsistent Low 211 Table J19. MtDNA profiles generated from spent cartridge casings lo aded by individual OOO. Sample HV1 HV2 mtDNA Classification Quantification Level OOO 263G, 309.1C, 315.1C ------ ------ 70 - 1.45 263G, 309.1C, 315.1C Consistent Medium 70 - 2.45 263G, 309.1C, 315.1C Consistent Medium 70 - 3.22a 263G, 309.1C, 315.1C Cons istent High 70 - 3.45b 16051G, 16129C, 16183C, 16189C 73G, 152C, 217C, 263G, 315.1C Inconsistent Medium 212 APPENDIX K. COMPARISON OF HANDLER AND NON - HANDLER ALLELES AMPLIFIED WITH FUSION AND MTDNA PROFILE CLASSIFICATIONS Table K1. Number of alleles both consistent with (H) and not consistent with (NH) the handler and the corresponding mtDNA profile result. Sample Quantitation Level mtDNA Result #H Alleles #NH Alleles 52 - 1.45 High Inconsistent 24 20 54 - 1.45 High Consistent 35 4 53 - 3.45 High Consistent 36 2 53 - 1.45 High Mixed - Consistent 27 9 66 - 3.45 High Consistent 38 0 67 - 4.45 High Inconsistent 14 27 57 - 2.45 High Consistent 43 1 65 - 3.45 High Consistent 39 2 59 - 3.22 High Consistent 41 4 60 - 1.22 High Consistent 41 0 60 - 2.22 High Consistent 31 2 5 9 - 4.22 High Consistent 30 4 60 - 3.22 High Consistent 26 5 54 - 1.22 High Inconsistent 11 17 65 - 3.22 High Mixed - Consistent 11 8 68 - 3.22 High Inconsistent 8 11 62 - 3.45b High Consistent 21 3 60 - 4.45c High Consistent 25 4 62 - 3.45c High Mixed - Consistent 13 3 53 - 4.45a High Consistent 21 3 67 - 1.45b High Consistent 1 3 58 - 3.45b High Consistent 5 17 52 - 3.45b High Consistent 28 2 53 - 4.45b High Consistent 20 4 60 - 4.22c High Consistent 14 3 60 - 4.22b High Consistent 17 1 62 - 3.22b High Consistent 11 2 63 - 2.2 2b High Mixed - Consistent 1 3 63 - 2.22c High Mixed - Inconsistent 2 3 62 - 3.22a High Inconsistent 11 10 55 - 1.22b High Inconsistent 2 4 70 - 3.22a High Consistent 7 1 70 - 2.45 Medium Consistent 27 2 213 55 - 3.45 Medium Consistent 30 14 58 - 2.45 M edium Mixed - Consistent 19 9 70 - 1.45 Medium Consistent 18 4 58 - 4.45 Medium Mixed - Consistent 14 10 58 - 1.45 Medium Mixed - Consistent 12 19 61 - 2.45 Medium Consistent 11 5 39 - 1.45 Medium Consistent 24 5 62 - 2.22 Medium Mixed - Consistent 7 1 53 - 1.22 Medium M ixed - Consistent 6 7 56 - 3.22 Medium Mixed - Consistent 9 3 61 - 3.22 Medium Consistent 3 3 66 - 2.22 Medium Consistent 14 2 58 - 1.22 Medium Consistent 7 10 62 - 4.22 Medium Inconsistent 15 2 51 - 4.22 Medium Inconsistent 13 6 39 - 4.45b Medium Consistent 15 5 65 - 2.45c Medium Consistent 0 4 52 - 3.45c Medium Mixed - Consistent 12 3 59 - 2.45b Medium Mixed - Consistent 9 3 70 - 3.45b Medium Inconsistent 2 1 61 - 4.45b Medium Mixed - Consistent 5 12 65 - 2.45a Medium Mixed - Inconsistent 9 9 39 - 4.45a Medium Consistent 11 2 52 - 3.22b Medium Inconsistent 11 3 54 - 3.22a Medium Consistent 7 2 59 - 2.22c Medium Consistent 16 0 39 - 4.22b Medium Consistent 8 4 62 - 3.22c Medium Consistent 9 4 58 - 3.22c Medium Consistent 2 3 65 - 2.22c Medium Consistent 9 5 54 - 3.22c Medium Consistent 6 5 59 - 4.45 Low Mixed - Consistent 12 3 67 - 2.45 Low Mixed - Consistent 3 5 68 - 3.45 Low Inconsistent 6 7 69 - 1.45 Low Inconsistent 4 3 68 - 4.45 Low Mixed - Consistent 9 5 59 - 3.45 Low Mixed - Consistent 12 6 57 - 3.45 Low Mixed - Consistent 6 2 51 - 1.45 Low Consistent 11 7 214 58 - 4.22 Low Inconsistent 15 19 57 - 4.22 Low Mixed - Consistent 2 8 68 - 2.22 Low Mixed - Consistent 1 2 57 - 3.22 Low Mixed - Consistent 4 4 66 - 4.22 Low Mixed - Consistent 3 4 55 - 4.22 Low Inconsistent 7 4 59 - 1.22 Low Mixed - Consistent 18 4 57 - 2.22 Low Mixed - Consistent 3 3 61 - 4.45a Low Consistent 6 11 54 - 3.45b Low Consistent 9 7 69 - 2.45b Low Mixed - Consistent 4 2 57 - 1.45c Low Consistent 1 6 68 - 1.45a Low Consistent 4 5 69 - 2.45c Low Inconsistent 0 5 56 - 4.45b Low Mixed - Inconsistent 2 2 5 6 - 4.45c Low Consistent 2 5 57 - 1.22a Low Inconsistent 2 3 58 - 3.22b Low Consistent 3 2 52 - 3.22a Low Consistent 2 3 66 - 1.22c Low Consistent 4 2 57 - 1.22c Low Inconsistent 1 8 61 - 4.22c Low Consistent 4 2 68 - 1.22b Low Inconsistent 0 2 61 - 4.22b Low Consis tent 2 5 215 REFERENCES 216 REFERENCES Anderson S, Bankier AT, Barrell BG, de Bruijn MH, Coulson AR, Drouin J et al. 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