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Genetic analysis of Applied Biosystems™ Prec
ision ID Ancestry Panel for Seven Asian Popula
tions
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Index
1. Introduction
2. Workflow
3. Results
4. Discussion
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Asian Ancestry
Royal CD et al. Inferring Genetic Ancestry: Opportunities, Challenges, and Implications. Am J Hum Genet. 2010;86(5):661-73.
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Objects
Objects
Evaluating the performance of the Applied Biosystems™ Precision ID
Ancestry Panel for seven population in Asia
Assessing the accuracy and reliability of the HID SNP Genotyper plugin of
the Torrent Server.
Providing the SNP data of 750 people in Asia
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Workf low
• Applied Biosystems™ Precision ID Ancestry Panel
Specifications Targets 165 single-nucleotide polymorphisms (SNPs)
Amplicon length
Average 130 bp for the 55 SNPs from Dr. Kenneth Kidd Average 120 bp for the 123 SNPs from Dr. Michael Seldin
Primer pool size 165 primer pairs in 1 tube Input DNA 1 ng
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Workf low
• Solutions for forensic DNA analysis
• Simplest targeted NGS workflow
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Workf low • Asian Subgrouping
Geographical region Population sample description N Northeast Asia Han Chinese South [CHS] 99
Han Chinese in Beijing, China [CHB] 100
Japanese in Tokyo, Japan [JPT] 101
Korean from Seoul, Korea 100
Southeast Asia Kinh in Ho Chi Minh City, Vietnam [KHV] 100
Southwest Asia Nepalese 100
Indian 51
Pakistani in Pathan, Pakistan 49
Punjabi in Lahore, Pakistan 50
750 7
R e s u l t s
• SNP coverage
- Coverage varied from 193× (rs1407434) to 5369× (rs2073821)
- With an average of 1918×
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R e s u l t s
• NOC - the coverage for the locus is less than twice the standard deviation of the mean coverage for the sample
- Of the 123,750 genotypes (750people x 165 SNP markers)
- 123,431 (99.7%) were called, 6 (0.005%) with no coverage
- 318 (0.26%) called as ‘NOC’
- Average depth of 318 SNPs; 94.4x
Chrom Position Target ID Genotype Coverage A Reads C Reads G Reads T Reads
chr3 1.21E+08 rs12498138 - 0 0 0 0 0
chr12 1.09E+08 rs2070586 - 0 0 0 0 0
chr8 1.22E+08 rs2196051 - 0 0 0 0 0
chr12 17407792 rs1513056 NN 920 97 0 823 0
chr13 34847737 rs7997709 NN 1487 0 1342 0 145
chr14 20818131 rs1760921 NN 1509 2 155 1 1351
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R e s u l t s
• NOC • rs12498138 failed to type 58 of 750 with an average of 9.6×
• rs2196051 failed to type 42 of 750 with an average of 7.1×
Pop Chrom Position Target ID Genotype Coverage A Reads C Reads G Reads T Reads
Korean chr3 1.21E+08 rs12498138 - 0 0 0 0 0
Han chr3 1.21E+08 rs12498138 NN 1 0 0 1 0
India chr3 1.21E+08 rs12498138 NN 1 0 0 1 0
Pathan chr3 121459589 rs12498138 NN 21 0 0 21 0
Vietnam chr3 121459589 rs12498138 NN 22 0 0 22 0
Punjab chr3 121459589 rs12498138 NN 28 1 0 27 0
Pop Chrom Position Target ID Genotype Coverage A Reads C Reads G Reads T Reads
Korean chr8 122124302 rs2196051 - 0 0 0 0 0
India chr8 122124302 rs2196051 - 0 0 0 0 0
Vietnam chr8 122124302 rs2196051 NN 1 0 0 1 0
Pathan chr8 122124302 rs2196051 NN 4 1 0 3 0
India chr8 122124302 rs2196051 NN 5 0 0 5 0
Punjab chr8 122124302 rs2196051 NN 193 9 0 184 0
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R e s u l t s
• NOC
• 43 NOC genotypes with over 193 coverage
Pop Chrom Position Target ID Genotype Coverage A Reads C Reads G Reads T Reads
Nepal chr20 62159504 rs310644 NN 198 0 25 0 173
Korean chr15 92105708 rs8035124 NN 202 179 22 1 0
Punjab chr18 75056284 rs874299 NN 231 0 28 0 203
Beijing chr12 17407792 rs1513056 NN 920 97 0 823 0
Korean chr13 34847737 rs7997709 NN 1487 0 1342 0 145
Vietnam chr14 20818131 rs1760921 NN 1509 2 155 1 1351
- Average depth of 43 SNPs; 555.3x
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R e s u l t s
• NN - A total of 127 of the 750 individuals had at least one NN remark with a maximum of 19 locus dropouts
- Some no-call genotypes were occa
sionally observed in good quality sa
mples
Pop rs1407434 rs13400937rs12498138rs9291090 rs3811801 rs16891982rs6556352 rs192655 rs3823159
India NN NN NN NN NN NN NN NN NN
rs6990312 rs2196051 rs2306040 rs772262 rs1572018 rs7326934 rs12439433rs10512572rs6104567 rs310644
NN NN NN NN NN NN NN NN NN NN
Mapped Reads On Target Mean Depth Uniformity
23586 95.64% 141.8 78.21%
Electropherogram of PowerPlex®16 system 12
R e s u l t s
• PPC - percent of positive coverage for poor strand imbalance
- A total of 3,973 (3.21%) genotypes had the remark ‘PPC’
- 106 of 3,973 had the remark ‘PPC & NOC’
- With an average of 2277X
Pop Chrom Position Target ID Genotype Coverage A Reads C Reads G Reads T Reads Pos Cov Neg CovPerc Pos
Cov GQMaj Allele
Freq QC
Japan chr3 121459589 rs12498138 GG 1010 0 0 1010 0 827 183 81.881188 99 100 _PPC;
Vietnam chr19 52901905 rs3745099 AA 1011 1011 0 0 0 288 723 28.486647 99 100 _PPC;
Beijing chr2 37941396 rs4670767 GG 1028 0 0 1028 0 738 290 71.789883 99 100 _PPC;
Beijing chr3 121459589 rs12498138 AG 826 411 0 413 2 586 240 70.94431 99 50 _PPC;
Korean chr18 49781544 rs7238445 AG 1016 508 0 508 0 912 104 89.76378 99 50 _PPC;
Nepal chr18 49781544 rs7238445 AG 2520 1260 0 1260 0 1957 563 77.65873 99 50 _PPC;
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R e s u l t s
• Heterozygote balance (HB)
- The mean of HB ranged from 0.33(rs310644) to 3.37(rs7997709)
- rs1871534 (GG) was fixed homozygotes
- rs3827760 (GG) was homozygote except for only one people from Vietnam 14
R e s u l t s
• 35 Excluded SNPs Name ObsHET PredHET HWpval MAF
rs1426654 0.089 0.404 3.58E-100 0.281
rs3827760 0.174 0.48 9.57E-72 0.399
rs260690 0.172 0.448 5.68E-64 0.338
rs1229984 0.31 0.5 1.00E-25 0.491
rs3943253 0.298 0.479 7.11E-25 0.396
rs917115 0.192 0.315 1.13E-22 0.196
rs3811801 0.327 0.474 3.35E-17 0.387
rs7997709 0.33 0.481 4.39E-17 0.404
rs4471745 0.148 0.23 1.56E-16 0.132
rs4781011 0.279 0.4 1.03E-15 0.277
rs7745461 0.327 0.459 6.82E-15 0.357
rs4918664 0.288 0.403 4.12E-14 0.28
rs1876482 0.353 0.487 6.41E-14 0.421
rs1950993 0.298 0.413 1.18E-13 0.292
rs10496971 0.356 0.478 4.10E-12 0.395
rs174570 0.331 0.441 2.04E-11 0.328
rs1800414 0.367 0.478 2.62E-10 0.395
rs310644 0.144 0.195 4.30E-09 0.109
Name ObsHET PredHET HWpval MAF
rs4463276 0.291 0.373 7.67E-09 0.248
rs9319336 0.393 0.498 1.01E-08 0.47
rs7226659 0.388 0.491 1.11E-08 0.434
rs10512572 0.391 0.493 2.53E-08 0.44
rs1513181 0.36 0.447 1.73E-07 0.337
rs2024566 0.228 0.284 7.29E-07 0.171
rs6990312 0.142 0.178 2.44E-06 0.099
rs9522149 0.131 0.165 2.63E-06 0.091
rs10007810 0.265 0.321 8.93E-06 0.201
rs2196051 0.124 0.153 4.86E-05 0.083
rs16891982 0.066 0.083 5.35E-05 0.044
rs6754311 0.084 0.105 5.74E-05 0.055
rs4670767 0.424 0.499 6.12E-05 0.474
rs12439433 0.013 0.016 0.0871 0.008
rs4880436 0.008 0.008 1 0.004
rs1871534 0 0 1 0
rs2814778 0 0 1 0
- Of the 165 SNPs examined, 35 SNPs were excluded - high missing call rates (>5%), low minor allele frequencies (<0.01), or significant deviation
s from the Hardy-Weinberg equilibrium (HWE; p < 0.0001) were excluded for quality control 15
R e s u l t s • Northeast Asia (China, Japan and Korea); 400 individuals
- All NEA were predicted correctly as East Asian with ethnicity likelihood values
- Expect for 14 out of 400
- Those thirteen (twelve in Chinese) of 14 exceptions were observed to have the
highest values for the Lao Loum in Southeast Asia 16
R e s u l t s • Northeast Asia (China, Japan and Korea); 400 individuals
- One of the fourteen exceptions were turned as Khmer of Cambodians in South
east Asia 17
R e s u l t s • Southeast Asia (Vietnam); 100 individuals
- All Vietnamese samples were also characterized as East Asian along with multi
ple different Asian population groups
- Forty-two samples were observed as East Asian population such as the Taiwan
ese(14/42), Hakka(13/42) and other east Asian populations 18
R e s u l t s • Southeast Asia (Vietnam); 100 individuals
- One of the Vietnamese was assigned to Micronesians in Oceania
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R e s u l t s • Southwest Asia (Nepal, India and Pakistan); 250 individuals
- SWA had more complicated results than NEA and SEA
- SWA were predominantly predicted as South Asian, seven out of 250 were pr
edicted as European, 10 as East Asia, and 5 as Southwest Asia 20
R e s u l t s • Southwest Asia (Nepal, India and Pakistan); 250 individuals
- 28 cases showed it was not possible to deduce a certain ethnic group
- it was evenly distributed, and less than 50% in any one nation 21
R e s u l t s • Southwest Asia (Nepal, India and Pakistan)
- The Likelihood value with the ethnic group did not match the highest percen
tage in population prediction 22
R e s u l t s • Confidence
Sample Name Mapped Reads On Target Mean Depth Uniformity
Han-080 28624 99.23% 158.7 74.47%
Sample Name Mapped Reads On Target Mean Depth Uniformity
Han-080 283646 98.97% 1536 73.21%
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R e s u l t s • Confidence
Sample Name Mapped Reads On Target Mean Depth Uniformity
Han-082 68008 95.52% 436 93.39%
Sample Name Mapped Reads On Target Mean Depth Uniformity
Han-082 325446 95.85% 2025 90.65%
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R e s u l t s • Structure
165 SNPs 130 SNPs
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D i s c u s s i o n
• The kit is optimized to work with 1 ng of genomic DNA
• In general the Precision ID Ancestry Panel is user-friendly and performed well
- 99.7% genotypes were called
- average depth ; 1918× (200saples / 1 540 chip)
• The statistical tool used to report results and to infer ethnicity
• No linkage disequilibrium in seven populations in Asia
• Overall, the Panel is reasonable to predict Northeast Asian people
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D i s c u s s i o n
• NOC - rs12498138 and rs2196051 failed to type 58 and 42 of the 750
• 3,973 (3.21%) genotypes had the remark ‘PPC’ with an average coverage of m
ore than 2276x
• Hardy-Weinberg analyses showed that not all SNPs in the kit conformed to exp
ectations in Asians
• The HID SNP genotyper plugin does not currently allow for the inclusion of cust
omized reference populations
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A c k n o w l e d g e m e n t
• The authors thank all the volunteers who donated DNA samples used in this stu
dy
• My grateful thanks are due to Mr. Chang Hyeon Lee and his faculty for providin
g practical exposure to mechanical systems with the Thermo Fisher Scientific K
orea lab
• This research was supported by the Bio & Medical Technology Development Pr
ogram of the National Research Foundation (NRF) funded by the Ministry of Sc
ience, ICT & Future Planning(NRF-2014M3A9E1069989)
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Thank you !
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DISCLAIMER Speaker was provided travel and hotel support by Thermo Fisher Scientific for this presentation, but no remuneration When used for purposes other than Human Identification or Paternity Testing the instruments and software modules cited are for Research Use Only. Not for use in diagnostic procedures. Thermo Fisher Scientific and its affiliates are not endorsing, recommending, or promoting any use or application of Thermo Fisher Scientific products presented by third parties during this seminar. Information and materials presented or provided by third parties are provided as-is and without warranty of any kind, including regarding intellectual property rights and reported results. Parties presenting images, text and material represent they have the rights to do so.
