Forensic Applications of CE and MPS

Transcription

1 Forensic Biology Polymorphic Enzyme Single Locus PCR RFLP

2 Massively Parallel Sequencing (MPS) Ion Chef System Ion S5 System

3 Ion Chef System Prepare barcoded DNA libraries with Applied Biosystems Precision ID Panels for human identitication Precision ID Ancestry Panel Precision ID Identity Panel Precision ID GlobalFiler Mixture ID Panel Applied Biosystems 7500 Real Time PCR System Quantify DNA libraries Ion Library Taqman Quantitation Kit HID Real Time PCR Analysis Software v.1.2

4 Ion Chef System Template ISPs Ion 520 Chip: 3-5 million reads Ion 530 Chip: million reads Ion S5 System Sequencing by synthesis reactions HID SNP Genotyper for Ancestry and ID Panel SNP data analysis HID Genotyper for Mixture ID Panel data analysis using Converge software Analytical 5% STR Peak Height 0.3 N- 4 bp 20%

5 Initial Testing: Database Samples Sex and ethnic origin were self reported by donor DNA extracted via SDS/PaseK DNA purified with PCI/CI DNA isolated using Millipore Microcon 100 filters Each sample quantified in duplicate using the Quantifiler Trio DNA Quantification Kit Samples were diluted to target 0.67 ng/ul and 15 ul used in library preparation using the Precision ID Ancestry Panel IonCode 96 well plates were used to barcode the samples The pooled library was quantified using the Ion Library Taqman Quantitation Kit then diluted to 20 pm for templating ISPs were templated to an Ion 530 chip The Ion S5 run plan included 500 flows

6 Precision ID Ancestry run summary data ISP Density 70% ISP Summary 29% usable reads 7,484,399 total reads Read Length Data Mean 104 bp

7 Precision ID Ancestry Run Samples 151 AISNPs Sample Sex Reported Ethnic LS1 XY European American 104 XY Southern Indian 489 XX Philippine 493 XY African American

8 LS1 - EA AA

9 104 - SI PA

10 Forensic Sample Testing Laboratory concordance DNA samples were isolated and quantified as described for the Precision ID Ancestry Panel sample set Samples were diluted to target ng/ul and 15 ul used in library preparation using the early access Precision ID GobalFiler Mixture ID Panel* IonCode 96 well plates were used to barcode the samples The pooled library was quantified using the Ion Library Taqman Quantitation Kit then diluted to 50 pm for templating ISPs were templated to an Ion 520 chip The Ion S5 run plan included 1000 flows

11 Precision ID GlobalFiler Mixture ID Panel 113 human genome targets 29 A-STRs 1 Y-STR 42 ASNPs 2 YSNPs 36 Microhaps (2-4 SNPs) Y indel Amelogenin

12 Precision ID GlobalFiler Mixture ID Panel concordance standards run summary data ISP Density 68% ISP Summary 60% usable reads 4,629,647 total reads Read Length Data Mean 139 bp Test Fragments: 23,252 reads 50AQ17 99% 100AQ17 98%

13 GlobalFiler results LS1

14 Globalfiler: CE and MPS LS1 D3S1358 vwa D16S539 CSF1PO TPOX CE 18,18 15,16 9,12 11,12 8,11 S5 18,18 15,16 9,12 11,12 8,11 Y indel Amel D8S1179 D21S11 D18S51 DYS391 CE 2 X,Y 13,14 29,31 16,18 11 S5 X,Y 13,14 29,31 16,18 11 D2S441 D19S433 TH01 FGA CE 11,15 12,16 9,9 23,23 S5 11,15 12,16 9,9 23,23 D22S1045 D5S818 D13S317 D7S820 SE33 CE 15,15 13,15 10,11 8,9 27.2,32.2 S5 15,15 13,15 10,11 8,9 D10S1248 D1S1656 D12S391 D2S1338 CE 15,17 12, ,22 16,25 S5 15,17 12, ,22 16,25

15 Concordance Standards Tested Homozygote resolution LS1 - D3S D3S D12S391 Minimum SNP coverage Sample Minimum SNP Coverage LS mean 1024

16 Forensic Sample Testing Laboratory Casework Set 1 DNA samples were isolated and quantified as described for the Ancestry panel sample set Samples were diluted to target ng/ul and 15 ul used in library preparation using the Precision ID GlobalFiler Mixture ID Panel IonCode 96 well plates were used to barcode the samples The pooled library was quantified using the Ion Library Taqman Quantitation Kit then diluted to 50 pm for templating ISPs were templated to an Ion 520 chip The Ion S5 run plan included 1000 flows

17 Precision ID GlobalFiler Mixture ID Panel Forensic case set 1 run summary data ISP Density 70% ISP Summary 61% usable reads 4,805,639 total reads Read Length Data Mean 126 bp Test Fragments: 20,443 reads 50AQ17 99% 100AQ17 98%

18 X only males I-1 SRY detected DYS393 DYS456 I-2 SRY detected No Y-STRs I-8 SRY detected DYS393 DYS456 DYS458 DYS481 DYS19

19 X only males Translocations to X without inversion with inversion locus locus position (Mb) Y1 Y2 Y-8 P arm PAR 1 SRY 2 DYS DYS Amelogenin 6.7 DYS DYS576 7 DYS DYS DYS centromere 10.2

20 SNP data - X only males I-1, I-2, I-8 Y1 Y indel Y2 Y indel Y8 Y indel

21 Non-probative bloodstain case sample 9352CQ4 bloodstain from shirt Quantifiler Trio DI = 2 complete STR profile expected

22 Non-probative bloodstain case sample 9352CQ4 Low level minor noted Mixture proportion at approximately 40:1 based on CE results

23 Non-probative bloodstain case sample 9352CQ4 Low level minor noted Mixture proportion at approximately 40:1 D3S1358 vwa D16S539 CSF1PO TPOX CE 15,16 18,18 9,12(13) 11,13 9,12 S5 15,16 18,18 9,12 11,13 9,12 Y indel Amel D8S1179 D21S11 D18S51 DYS391 CE 1 XY 10,13 (11) 31, ,14 10 S5 XY 10,13 31, ,14 10 D2S441 D19S433 TH01 FGA CE 14,14 14,14 6,9 (9.3) 22,23 S5 14,14 14,14 6,9 22,23 D22S1045 D5S818 D13S317 D7S820 SE33 CE 15,15 9,11 (12) 9,12 (13,14) 11,12 15,19 S5 15,15 9,11 (10) 9,12 11,12 D10S1248 D1S1656 D12S391 D2S1338 CE 14,17 (15) 11,17 18,18 19,24 S5 14,17 11,17 18,18 19,24

24 Forensic Sample Testing Laboratory Casework Set 2 DNA samples were isolated and quantified as described for the Precision ID Ancestry Panel sample set. Bloodstain, bone and tissue samples were extracted with a direct lysis approach Semen stains were extracted with a differential lysis approach The stamp samples were extracted using the PrepFiler BTA Forensic DNA Extraction Kit. 16 Samples were diluted to target ng/ul where available and 15 ul used in library preparation using the Precision ID GlobalFiler Mixture ID Panel IonCode 96 well plates were used to barcode the samples

25 Forensic Sample Testing The pooled library was quantified using the Ion Library Taqman Quantitation Kit then diluted to 50 pm for templating ISPs were templated to one Ion 530 chip The Ion S5 run plan included 1000 flows

26 Precision ID GlobalFiler Mixture ID Panel Forensic case set 2 run summary data ISP Density 52% ISP Summary 53% usable reads 10,241,296 total reads Read Length Data Mean 116 bp Test Fragments: 66,388 reads 50AQ17 99% 100AQ17 97%

27 Non-probative semen stain case sample 5Q5F1 stain from bed cover Quantifiler Trio DI = 5 some degradation complete STR profile expected

28 Non-probative semen stain case sample 5Q5F1

29 Non-probative semen stain case sample 5Q5F1 D3S1358 vwa D16S539 CSF1PO TPOX CE 16,18 (17) 16,19 (14,17,18) 11(12) 10,11 (12) 8 S5 16,18,17 16,19,14,17 11,12 10,11,12 8,11 Y indel Amel D8S1179 D21S11 D18S51 DYS391 CE 2 X,Y 13 (11,14) 29,30 (30.2) 12,15 (16) ND S5 X,Y 13,11,14 29,30 12,15,16 10 D2S441 D19S433 TH01 FGA CE 10,14 (11,12) 12,14 (16) 6,9.3) 21,24 (22,23) S5 10,14,11,12 12,14,16 6,9.3 21,24,22,23 D22S1045 D5S818 D13S317 D7S820 SE33 CE 15,15 (14,17) 11,12 11,11 (12) 8,9 (10,11) 16,17,27.2 S5 15,14,17 11, ,9,10 D10S1248 D1S1656 D12S391 D2S1338 CE 15,16 (13,14) 12,17.3,16 17,18,22 18 S5 15,16,13,14 12, ,18,22 18

30 Non-probative human remains sample 71BS 18 months bone-surface Quantifiler Trio DI = 8 some degradation expected

31 Non-probative human remains sample 71BS 18 months bone-surface

32 Non-probative human remains sample 71BS 18 months bone-surface

33 Non-probative human remains sample 71BS 18 months bone-surface 100% Coverage > 300

34 Non-probative human remains sample 71BS 18 months bone-surface D3S1358 vwa D16S539 CSF1PO TPOX CE 17,18 17,18 9,12 10,12 8,9 S5 17,18 17,18 9,12 10,12 8,9 Y indel Amel D8S1179 D21S11 D18S51 DYS391 CE 2 XY 12,13 29, ,18 11 S5 XY 12,13 29, ,18 11 D2S441 D19S433 TH01 FGA CE 10,11 13,13 6,7 21,24 S5 10,11 13,13 6,7 21,24 D22S1045 D5S818 D13S317 D7S820 SE33 CE 11,15 11,13 8,12 12,12 19,21 S5 11,15 11,13 8,12 12,12 D10S1248 D1S1656 D12S391 D2S1338 CE 13,15 11,14 19,24 20,25 S5 13,15 11,14 19,24 20,25

35 Non-probative human remains sample 69BB 2.5 months bone-buried Quantifiler Trio DI = 2

36 Non-probative human remains sample 69BB 2.5 months bone-buried

37 Non-probative human remains sample 69BB 2.5 months bone-buried

38 Non-probative human remains sample 69BB 2.5 months bone-buried 100% Coverage > 300

39 Non-probative human remains sample 69BB 2.5 months bone-buried D3S1358 vwa D16S539 CSF1PO TPOX CE 16,17 17,17 9,11 11,12 8,8 S5 16,17 17,17 9,11 11,12 8,8 Y indel Amel D8S1179 D21S11 D18S51 DYS391 CE ND XX 10,12 29,30 15,15 ND S5 XX 10,12 29,30 15,15 ND D2S441 D19S433 TH01 FGA CE 11,11 13,15 9.3, ,24 S5 11,11 13,15 9.3, ,24 D22S1045 D5S818 D13S317 D7S820 SE33 CE 11,16 11,12 8,11 10,11 17,20 S5 11,16 11,12 8,11 10,11 D10S1248 D1S1656 D12S391 D2S1338 CE 14,15 13,14 18,19 17,21 S5 14,15 13,14 18,19 17,21

40 Non-probative human remains sample 62TB 2 weeks soft tissue-buried Quantifiler Trio DI = 17 HL ng HS ng Degradation expected.

41 Non-probative human remains sample 62TB 2 weeks soft tissue-buried STR mh

42 Non-probative human remains sample 62TB 2 weeks soft tissue buried Quantifiler Trio DI = 2 Coverage > % Coverage > % Coverage > %

43 Non-probative human remains sample 62BS 2 weeks bone surface Quantifiler Trio DI = 2

44 Non-probative human remains sample 62BS 2 weeks bone surface

45 Non-probative human remains sample 62BS 2 weeks bone surface D3S1358 vwa D16S539 CSF1PO TPOX CE 14,17 17,18 9,11 11,12 11,11 S5 14,17 17,18 9,11 11,12 11,11 Y indel Amel D8S1179 D21S11 D18S51 DYS391 CE ND XX 12,15 27,30 13,14 ND S5 XX 12,15 27,30 13,14 ND D2S441 D19S433 TH01 FGA CE 10,11 13,13 7,9.3 24,25 S5 10,11 13,13 7,9.3 24,25 D22S1045 D5S818 D13S317 D7S820 SE33 CE 11,15 9,12 8,8 10,11 18,27.2 S5 11,15 9,12 8,8 10,11 D10S1248 D1S1656 D12S391 D2S1338 CE 14,15 12,12 19,20 18,21 S5 14,15 12,12 19,20 18,21 (20)

46 Case library 2 : Includes human remains samples

47 Non-probative stamp sample Quantifiler Trio DI = 20 HL ng HS ng Degradation expected

48 Non-probative stamp sample Quantifiler Trio DI = 20

49 Non-probative stamp sample Quantifiler Trio DI = 20

50 Non-probative stamp sample Quantifiler Trio DI = 20 No Coverage > 100

51 Conclusions Samples typically encountered in a forensic science laboratory were tested with MPS and Capillary Electrophoresis using the early access Precision ID GlobalFiler Mixture ID Panel with 113 human genome targets Results identified by MPS show overall concordance for each sample type tested when compared to results defined by CE Degraded samples showed an increase in markers detected by MPS in comparison to CE.

52 Acknowledgements Laboratory Director Sandra Brown Evidence Technician Cindy Mosley Thermo Fisher Scientific *The Precision ID GlobalFiler Mixture ID Panel is in early access development and not available for purchase. The content provided herein may relate to products that have not been officially released and is subject to change without notice. For Forensic or Paternity Use Only. When used for purposes other than Human Identification the instruments and software modules cited are for Research Use Only. Not for use in diagnostic procedures. Speaker was provided travel and hotel support by Thermo Fisher Scientific for this presentation, but no remuneration