张素华

(Suhua Zhang)

司法部司法鉴定科学技术研究所

(Institute of Forensic Science, Ministry of Justice, China)

2016.11.17

Pilot Study of Forensic Genetic Markers

with Ion Torrent PGM Platform

Trio-Case:

BM AF

D

Goldeneye 20A

Locus Sample CE genotyping

vWA BM 14

D 14/19

AF 17/18

FGA BM 25

D 24/25 CPI(trios):378.361

AF 23 CPI(duos):0.980

Investigator Argus X-12, Goldeneye 17X

Goldeneye 22NC, HDplex, AGCU 21+1

Locus Sample CE genotyping

D3S4529 BM 14/15

D 14/15

AF 13

Locus Sample CE genotyping

DXS10101 BM 31.2/33

D 33

AF 32

DXS10146 BM 26/32

D 26?

AF 34?

CPI(trios):9722553059889.670

CPI(duos):1422268.818

Demanding for more markers and new technology

Commercial Panel testing：

1. HID-Ion AmpliSeq™ Identity Panel SNP-124 (Evaluation in HAN population)

Cov depth<100：rs2342747, rs12997453

Heterozygous imbalance：rs7520386、rs4530059、rs214955、rs1523537、rs2342747、rs576261

Commercial Panel testing：

1. HID-Ion AmpliSeq™ Identity Panel SNP-124 (Evaluation in HAN population)

Sensitivity testing

Commercial Panel testing：

2. Early Access STR Kit v1 (Evaluation in African, European and Asian)

STR

A B C

ACR SD ACR SD ACR SD

CSF1PO 90.76 7.05 85.53 0.87 88.33 5.17

D10S1248 79.33 18.11 82.15 9.19 77.76 7.14

D13S317 92.56 0.36 94.62 3.52 88.62 2.11

D14S1434 86.70 6.37 82.71 8.51 85.87 3.71

D16S539 84.32 4.16 92.91 1.20 75.76 6.75

D19S433 85.72 2.13 81.86 19.47 62.41 15.45

D1S1656 99.47 0.16 88.62 2.56 76.41 7.47

D1S1677 79.58 0.21 84.42 0.23 62.31 2.54

D21S11 93.18 0.35 95.34 1.25 79.00 21.21

D2S1338 92.37 2.62 67.81 10.20 79.38 3.75

D2S1776 96.50 1.27 85.24 1.26 88.65 0.00

D2S441 84.00 10.88 63.00 10.33 70.76 15.34

D3S1358 90.85 3.80 84.63 12.88 94.22 2.55

D4S2408 84.06 9.25 84.02 0.19 74.36 13.26

D5S2500 64.52 2.58 75.24 4.25 56.25 1.21

D5S818 85.25 4.52 95.24 4.21 76.32 4.21

D6S1043 83.80 8.83 83.44 7.52 92.76 3.55

D6S474 83.29 25.22 87.97 5.64 80.24 9.25

D7S820 77.07 22.48 100.70 34.35 76.61 13.30

D8S1179 78.28 9.84 82.96 5.24 58.44 18.89

D9S2157 69.06 51.56 69.43 11.45 64.25 8.54

TH01 84.18 15.16 96.51 3.46 96.94 0.54

TPOX 96.83 1.67 87.44 12.84 91.26 3.73

vWA 79.79 7.73 85.18 14.89 76.53 7.04

Note: A-Full library reaction volume with 1 ng DNA

B: Half library reaction volume with 1 ng DNA

C: Half library reaction volume with 0.5 ng DNA

SD: standard deviation

Commercial Panel testing：

2. Early Access STR Kit v1 (Evaluation in African, European and Asian)

STR Chromosome BED file provided by Early Access STR Kit v1 ISFG recommendation [23]

Motif GRCh37 coordinates-start GRCh37 coordinates-stop Motif GRCh37 coordinates-start GRCh37 coordinates-stop

D1S1677 1 [TTCC] 163559816 163559875 [TTCC] 163559816 163559875

D1S1656 1 [TAGA][TGA][TAGG][TG] (Reverse)

230905362 230905429 [CCTA] [TCTA] 230905362 230905429

TPOX 2 [AATG] 1493425 1493456 [AATG] 1493425 1493456

D2S441 2 [TCTA] 68239079 68239126 [TCTA] 68239079 68239126

D2S1776 2 [AGAT] 169645403 169645446 [AGAT] 169645403 169645446

D2S1338 2 [TGCC][TTCC] (Reverse) 218879582 218879673 [GGAA] [GGCA] 218879582 218879673

D3S1358 3 [TCTA][TCTG] 45582231 45582294 TCTA [TCTG] [TCTA] 45582231 45582294

D4S2408 4 [ATCT] 31304420 31304455 [ATCT] 31304420 31304455

D5S2500 (AC008791) [12]

5 [GGTA] [GACA] [GATA] [GATT] 58698958 58699025 [GGTA] [GACA] [GATA] [GATT]

58698958 58699025

D5S818 5 [AGAT] (Reverse) 123111250 123111293 [ATCT] 123111250 123111293

CSF1PO 5 [AGAT] (Reverse) 149455887 149455938 [ATCT] 149455887 149455938

D6S1043 6 [AGAT] ACAT [AGAT] (Reverse) 92449943 92449990 [ATCT] ATGT 0-1 [ATCT] 92449943 92449990

D6S474 6 [AGAT][GATA] 112879153 112879220 [AGAT] [GATA] [GGTA] [GACA]

112879153 112879220

D7S820 7 [GATA] (Reverse) 83789542 83789593 [TATC] 83789542 83789593

D8S1179 8 [TCTA] [TCTG] [TCTA] 125907107 125907158 [TCTA] [TCTG]0-2 [TCTA] 125907107 125907158

D9S2157 9 [ATA] 136035669 136035698 [ATA] 136035669 136035698

D10S1248 10 [GGAA] 131092508 131092559 [GGAA] 131092508 131092559

TH01 11 [TCAT]CAT[TCAT] (Reverse) 2192319 2192346 [AATG] ATG 0-1 [AATG] 2192318 2192345

vWA 12 [TCTA][TCTG](Recerse) 6093143 6093210 [TCTA] [TCTG] [TCTA] 6093143 6093210

D13S317 13 [TATC] 82722160 82722203 [TATC] 82722160 82722203

D14S1434 14 [CTGT][CTAT] 95308391 95308442 [CTGT] [CTAT] * 95308391 95308442

D16S539 16 [GATA] 86386308 86386351 [GATA] 86386308 86386351

D19S433 19 [AAGG](AAGGTAGG) (Reverse) 30417142 30417197 [CCTT]1 CCTA [CCTT]1 CTTT [CCTT]

30417142 30417205

D21S11 21 [TCTA][TCTG]{TA}{TCA}{TCCATA} 20554291 20554417 [TCTA] [TCTG] [TCTA] TA [TCTA] TCA [TCTA] TCCATA [TCTA]

20554291 20554417

Study one : A custom MPS-SNP panel including 273 SNPs for forensic application

Marker selection and panel optimization:

Study one : A custom MPS-SNP panel including 273 SNPs for forensic application

Panel No. SNP Performance

r080 rs1029047 Heterozygote imbalance （average FMAR%>60）

r114 rs3808378 Heterozygote imbalance （average FMAR%>60）

r116 rs4606077 Heterozygote imbalance （average FMAR%>60）

r123 rs334355 Heterozygote imbalance （average FMAR%>60）

r192 rs430046 Heterozygote imbalance （average FMAR%>60）

r212 rs1108414 Heterozygote imbalance （average FMAR%>60）

r157 rs2920816 Heterozygote imbalance （average FMAR%>60）

r178 rs4530059 Heterozygote imbalance （average FMAR%>60）

r087 rs1478829 Heterozygote imbalance （average FMAR%>60）and coverage reads<20

r143 rs1498553 Heterozygote imbalance （average FMAR%>60）and coverage reads<20

r279 rs9786401 Coverage reads<100（Y-SNP）

r271 rs9786043 Coverage reads<100（Y-SNP）

r250 rs5915052 Coverage reads<100（X-SNP）but with heterozygote balance

r174 rs7141285 Coverage reads<100 but with heterozygote balance

r043 rs12714757 Coverage reads<100 but with heterozygote balance

r102 rs2189011 Coverage reads<100 but with heterozygote balance

Sequencing performance:

Study one : A custom MPS-SNP panel including 273 SNPs for forensic application

Sequencing performance:

Study one : A custom MPS-SNP panel including 273 SNPs for forensic application

Sensitivity evaluation (10 ng-0.2 ng)

Study one : A custom MPS-SNP panel including 273 SNPs for forensic application

rs1029047

[A/G/T]

Study one : A custom MPS-SNP panel including 273 SNPs for forensic application

rs3808378

Study one : A custom MPS-SNP panel including 273 SNPs for forensic application

Data quality control:

Mixture analysis (X-SNP)

表型1 表型2 混合比例 理论FMAR计算公式 理论FMAR值

aa b 1:1 2a/(2a+b) 0.6667

ab a 1:1 2a/(b+2a) 0.6667

aa b 1:5 5b/(2a+5b) 0.7143

ab a 1:5 6a/(6a+b) 0.8571

aa b 5:1 10a/(10a+b) 0.9091

ab a 5:1 6a/(6a+5b) 0.5455

aa b 1:10 10b/(2a+10b) 0.8333

ab a 1:10 11a/(11a+b) 0.9167

aa b 10:1 20a/(20a+b) 0.9524

ab a 10:1 11a/(11a+10b) 0.5238

aa b 1:100 100b/(2a+100b) 0.9804

ab a 1:100 101a/(101a+1b) 0.9902

aa b 100:1 200a/(200a+b) 0.9950

ab a 100:1 101a/(101a+100b) 0.5025

Study one : A custom MPS-SNP panel including 273 SNPs for forensic application

Marker selection:

1. STRBase：CODIS Core STRs 和European Core Loci （SE33 excluded）；

2. SF/Z JD0105002-2014

3. Kits： Goldeneye 20A, Goldeneye 18NC, Qiagen HDplex, AGCU 21+1, A 26plex Assay (Butler)

Investigator Argus X-12

AmpFLSTR® Yfiler®

Goldeneye 17X

Goldeneye 26Y

Study Two : A custom MPS panel including SNPs and STRs for forensic application

“STR-Panel ”

• “WG-Panel-P1”

• “WG-Panel-P2”

109 STRs (57 auto STRs, 28 X-STRs and 24 Y-STRs)

Study Two : A custom MPS panel including SNPs and STRs for forensic application

Concordance study——MPS VS CE

Reasons for Inconsistent results:

• Indels

• Allele drop-out

• Analysis method

Study Two : A custom MPS panel including SNPs and STRs for forensic application

“STR-Panel” performance:

• No sequence reads：DXS10146、DYS389II、DYS458、DYS447

• Too low reads： DYS438、 D1S1656、D8S1132

• Mapping problem： D1S1656、D8S1132、 DXS10135、DXS10101

• Imbalance：D2S441、D7S1517、D12S391、D14S608、D18S51、GATA165B12

• Allele drop-out：D8S1179、D13S325、D15S659、D1GATA113、D12ATA63、D14S1434

Study Two : A custom MPS panel including SNPs and STRs for forensic application

ACR analysis

D21S2055

Study Two : A custom MPS panel including SNPs and STRs for forensic application

Coverage analysis: D19S253

2288X

DYS437 46X

Study Two : A custom MPS panel including SNPs and STRs for forensic application

Noticeable STRs

Study Two : A custom MPS panel including SNPs and STRs for forensic application

Thanks!

E-mail:zsh-daisy@163.com