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Forensic Applications of CE and MPS

• Forensic Biology

Polymorphic Enzyme Single Locus PCR RFLP

Illinois State PoliceResearch and Development Laboratory


• Massively Parallel Sequencing (MPS)

– Ion Chef™ System

– Ion S5™ System



• 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



• Ion Chef System

• Template ISPs– Ion 520™ Chip: 3-5 million reads

– Ion 530™ Chip: 15-20 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 threshold @ 5%

– STR Peak Height Ratio @ 0.3

– N- 4 bp stutter @ 20%



• 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



• 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



• 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


• LS1 - EA

• 493 - AA



• 104 - SI

• 489 - PA



• 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 0.067 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





• 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



• Precision ID GlobalFiler Mixture ID Panel concordance standards run summary data

– ISP Density • 68%




– Test Fragments: 23,252 reads

• 50AQ17 99%

• 100AQ17 98%Illinois State Police

Research and Development Laboratory


• GlobalFiler results

– LS1



• 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 12,17.3 17,22 16,25

S5 15,17 12,17.3 17,22 16,25


• Concordance Standards Tested– Homozygote resolution

• LS1 - D3S1358

• 486 - D3S1358

• 496 - D12S391

– Minimum SNP coverage


Sample Minimum SNP Coverage

LS1 1489

486 1063

496 849

498 1212

495 907

490 769

493 1039

494 866

mean 1024


• 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 0.067 ng/ul and 15 ul used in library preparation using the Precision ID GlobalFiler Mixture ID Panel


• The pooled library was quantified using the Ion Library Taqman Quantitation Kit then diluted to 50 pM for templating





• Precision ID GlobalFiler Mixture ID Panel Forensic case set 1 run summary data

– ISP Density • 70%





• 50AQ17 99%

• 100AQ17 98%



• X only males

• I-1– SRY detected

– DYS393

– DYS456


– No Y-STRs


– DYS393

– DYS456

– DYS458

– DYS481

– DYS19 Illinois State PoliceResearch and Development Laboratory


• X only males


Translocations to X

without inversion with inversion

locus locus position (Mb) Y1 Y2 Y-8

P arm PAR

1

SRY 2

DYS393 3.1

DYS456 4.2

5

Amelogenin 6.7

DYS570 6.8

DYS576 7

DYS458 7.8

DYS481 8.4

DYS19 9.5

10

centromere 10.2


SNP data - X only males I-1, I-2, I-8


Y1

Y8

Y2

Y indel

Y indel

Y indel


• Non-probative bloodstain case sample 9352CQ4 – bloodstain from shirt

• Quantifiler Trio DI = 2 – complete STR profile expected



• Non-probative bloodstain case sample 9352CQ4– Low level minor noted

– Mixture proportion at approximately 40:1 based on CE results



• Non-probative bloodstain case sample 9352CQ4– Low level minor noted

– Mixture proportion at approximately 40:1



CE 15,16 18,18 9,12(13) 11,13 9,12

S5 15,16 18,18 9,12 11,13 9,12


CE 1 XY 10,13 (11) 31,33.2 12,14 10

S5 XY 10,13 31,33.2 12,14 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


• 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 0.067 ng/ul where available and 15 ul used in library preparation using the Precision ID GlobalFilerMixture ID Panel




• 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




• Precision ID GlobalFiler Mixture ID Panel Forensic case set 2 run summary data

– ISP Density• 52%





• 50AQ17 99%

• 100AQ17 97% Illinois State PoliceResearch and Development Laboratory


• Non-probative semen stain case sample 5Q5F1 – stain from bed cover

• Quantifiler Trio DI = 5 – some degradation

– complete STR profile expected


• Non-probative semen stain case sample 5Q5F1



• Non-probative semen stain case sample 5Q5F1



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


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 11,12 11 8,9,10

D10S1248 D1S1656 D12S391 D2S1338

CE 15,16 (13,14) 12,17.3,16 17,18,22 18

S5 15,16,13,14 12,17.3 17,18,22 18


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

• Quantifiler Trio DI = 8

– some degradation expected



• 100% Coverage > 300






CE 17,18 17,18 9,12 10,12 8,9

S5 17,18 17,18 9,12 10,12 8,9


CE 2 XY 12,13 29,30.2 12,18 11

S5 XY 12,13 29,30.2 12,18 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


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




• 100% Coverage > 300






CE 16,17 17,17 9,11 11,12 8,8

S5 16,17 17,17 9,11 11,12 8,8


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 9.3,9.3 22.2,24

S5 11,11 13,15 9.3,9.3 22.2,24

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


• Non-probative human remains sample 62TB – 2 weeks soft tissue-buried

• Quantifiler Trio DI = 17– HL 0.0002 ng

– HS 0.0030 ng

• Degradation expected.


• Non-probative human remains sample 62TB – 2 weeks soft tissue-buried

STR

mh


• Non-probative human remains sample 62TB – 2 weeks soft tissue buried


• Coverage > 300 62%





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





CE 14,17 17,18 9,11 11,12 11,11

S5 14,17 17,18 9,11 11,12 11,11


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)



• Case library 2 : Includes human remains samples



• Non-probative stamp sample

• Quantifiler Trio DI = 20 – HL 0.0007 ng

– HS 0.0029 ng

• Degradation expected




• No Coverage > 100



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.



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

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