Implementing Massively Parallel

Sequencing for Forensic DNA Analysis

Using In-house PCR Panels

Kyoung-Jin Shin, Ph.D.

Dept. of Forensic Medicine
Yonsei University College of Medicine
Seoul, Republic of Korea
 Outline of presentation

• MPS in forensic genetics

• Developed 3 in-house MPS panels
– Autosomal STR panel
– Y-STR panel
– Microhaplotype panel
• MPS data analysis of STRs
• Things for supporting forensic MPS
Application of massively parallel sequencing
to forensic genetics

Capillary electrophoresis Massively parallel sequencing

……TCTATCTGTCTGTCTGTCTATCTATCTA……
……TCTATCTGTCTGTCTGTCTATCTATCTA……
A B ……TCTATCTGTCTGTCTGTCTATCTATCTA……
……TCTATCTGTCTGTCTATCTATCTATCTA……
……TCTATCTGTCTGTCTGTCTATCTATCTA……
……TCTATCTGTCTGTCTATCTATCTATCTA……
……TCTATCTGTCTGTCTGTCTATCTATCTA……
……TCTATCTGTCTGTCTATCTATCTATCTA……
……TCTATCTGTCTGTCTGTCTATCTATCTA……
……TCTATCTGTCTGTCTATCTATCTATCTA……
……TCTATCTGTCTGTCTATCTATCTATCTA……
……TCTATCTGTCTGTCTATCTATCTATCTA……

……
A ……TCTATCTGTCTGTCTGTCTATCTATCTA…… 16 allele

B ……TCTATCTGTCTGTCTATCTATCTATCTA…… 16 allele (G>A)

Length-based genotype Sequence-based genotype

 Expectations when applying
MPS to forensic genetics

• Increased diversity of STR

– SNPs within repeat region and flaking area
– Small sized amplicon not limited to CE lane
• Simultaneous analysis of different kinds
of markers (STR, SNP and InDel, …)
• Large multiplexing of forensic markers
• Sequence based mixture deconvolution
Obstacles in application of MPS to
forensic genetics

• Limited information for currently available

commercial MPS panels
• Costly and/or time-consuming procedure in
preparing a MPS library
• The MPS data analysis is somewhat of hassle
 Limited information for currently
commercial MPS panels

• Primer sequences not reported

– Ambiguity in flanking region span of markers
– Potential mutation in primer binding area

• Limited flexibility for damaged DNA

– Sometimes qPCR is not enough to QA for library
– Need for library visualization
 Costly and/or time-consuming
procedure in preparing a MPS library

• MPS Lib. preparation > $100 / samples

• Time-consuming in adapter ligation procedure

• Cumbersome purification step needed for

individual samples
Difficulty in MPS data analysis

• MPS data analysis for commercial

NGS systems
– Platform specific
– Sometimes additional analysis needed
• Open source software
– TSSV, fdstools
– STRinNGS
– STRait Razor
※ End-user friendly ?
Development of In-house PCR panels

• Open information for PCR primers and

PCR mixture components
• Small sized amplicon as possible
• Even read count across markers
• Economic and simple library preparation
• No need of special instruments
• Platform independent data analysis
Design of multiplex PCR systems

 Criteria
- Target marker
- Basically 20 extended CODIS STR and Amelogenin + additional STRs
- 23 PowerPlex Y-STRs and Y-M175
- Microhaplptypes suggested by Kidd et al.
- Small sized amplicons is adapted as possible
- while primer is not overlapping with core region
- finally ranged in 70bp ~ 270bp
- Avoid SNP with  1% variation reported in primer binding area
 Resource
- STRBase (http://www.cstl.nist.gov/div831/strbase/)
- UCSC genome browser (http://genome.ucsc.edu/)
- Primer 3 v.0.4.0 (http://frodo.wi.mit.edu/primer3/)
Current workflow for MPS on a MiSeq system

Step 1. Step 2. Step 3. Step 4. Step 5.

PCR amplification Validate Amplicon Library preparation Validate Library Sequencing

• Template DNA • Fluorometer • TruSeq Nano DNA LT • Library Quantification • Cluster generation and
; 1 ng of Korean DNAs ; Quant-iT™ PicoGreen Sample preparation Kit ; KAPA library quantification kit sequencing on MiSeq
dsDNA assays (invitrogen) ; 2 x 250 bp (Paired-end)
• Amplicon purification
: Enzymatic purification • Agilent BioAnalyzer • Agilent BioAnalyzer

using EXO-SAP IT

* Adjustment of beads ratio

for size selection
Simplifed MPS library preparation

Fragment genomic DNA

① (PCR product)

② End repairs
①
③ Beads purifications

④ Adenylate 3’ ends

⑤ ②

⑥ Beads purifications

Beads purification Beads purification

Two-step PCR for MPS library preparation
 The first PCR using Target specific primers
 Thermal Cycle
PCR component Volume
95℃ 11 min
dH20 4.0
94℃ 20 sec
Gold ST*R 10× Buffer 2.0
5× Primer Mix-I 4.0 59℃ 60 sec × 26 cycles

5× Primer Mix-II 4.0 72℃ 45 sec (~ 27)

5× Primer Mix-III 4.0 72℃ 5 min

AmpliTaq Gold (5U/uL) 1.0 4℃ forever
Template DNA (1ng/uL) 1.0
Total 20.0

 The second PCR using Nextera XT Index primers

 Thermal Cycle
PCR component Volume
95℃ 15 min
dH20 12.5
94℃ 20 sec
Gold ST*R 10× Buffer 2.0
61℃ 30 sec × 15 cycles
Index 1 (i5) 2.0
72℃ 45 sec (~ 16)
Index 2 (i7) 2.0
72℃ 5 min
AmpliTaq Gold (5U/uL) 0.5
4℃ forever
1/10 diluted PCR product 1.0
Total 20.0
MPS library validation using Bioanalyzer
MPS library verification using CE

Autosomal STR

Y-STR

Microhaplotype
Simple Workflow of MPS on MiSeq system

Step 1. Step 2. Step 3. Step 4.

PCR amplification Library preparation Library pooling, Sequencing
purification and QC

• Template DNA • Nextera XT Index kit • Library pooling with • Cluster generation and
; 1 ng DNA samples (Illumina) equal amount (10ng/ul) sequencing on a MiSeq
; 2 x 300 bp (Paired-end)
• Amplicon 1/10 dilution • Agilent BioAnalyzer • Beads purification
; X1.1 ~ 1.2X beads ratio to
remove non specific
amplicons

• Library Quantification
; KAPA library quantification
kit for Illumina platforms
Allelic size range of 23 autosomal STRs and 2 sex markers

KplexSeq-Auto25
Average depth of coverage (DoC) for the 25 markers

Kim et al. Forensic Sci Int Genet. 2017

Average allele coverage ratio (ACR) for the 25 markers

Kim et al. Forensic Sci Int Genet. 2017

Stutter ratios of the 23 autosomal STRs observed in MPS analysis

PowerPlex
Locus MPS analysis GlobalFiler Comments
Fusion
D1S1656 14.4±3.3% 12.2% 14.2% Tetranucleotide, compound
D22S1045 13.2±5.9% 16.3% 16.4% Trinucleotide, simple
D10S1248 12.9±2.6% 11.5% 12.4% Tetranucleotide, simple
D3S1358 12.5±2.7% 11.0% 11.9% Tetranucleotide, compound
D18S51 12.0±3.4% 12.4% 14.6% Tetranucleotide, compound
D2S1338 11.6±2.7% 11.7% 13.9% Tetranucleotide, compound
vWA 11.6±2.7% 10.7% 11.2% Tetranucleotide, compound
D19S433 11.6±2.3% 10.0% 11.0% Tetranucleotide, compound
D12S391 11.5±2.9% 13.7% 15.8% Tetranucleotide, compound
FGA 10.8±2.7% 11.6% 12.1% Tetranucleotide, compound
D6S1043 10.6±2.2% - - Tetranucleotide, compound
D8S1179 9.9±2.2% 9.6% 10.9% Tetranucleotide, compound
D16S539 9.6±3.9% 9.5% 10.2% Tetranucleotide, simple
CSF1PO 9.5±2.9% 8.8% 9.5% Tetranucleotide, simple
D21S11 9.5±2.0% 10.5% 11.6% Tetranucleotide, complex
D5S818 8.5±3.0% 9.2% 9.5% Tetranucleotide, simple
D7S820 8.4±3.3% 8.3% 11.0% Tetranucleotide, simple
D2S441 6.7±2.6% 8.1% 9.2% Tetranucleotide, compound
D13S317 6.5±3.3% 9.2% 9.8% Tetranucleotide, simple
Penta E 6.1±2.4% - 7.6% Pentanucleotide, simple
TPOX 4.5±1.8% 5.6% 5.5% Tetranucleotide, simple
TH01 4.4±1.8% 4.5% 4.6% Tetranucleotide, simple
Penta D 2.1±1.0% - 6.8% Pentanucleotide, simple

Kim et al. Forensic Sci Int Genet. 2017

Discrepancy of STR genotypes between CE and MPS analysis

1) CE profile 2) MPS reads

D2S441 9 alleles
Unreported sequence variation in 5' flanking region

Repeat region

Insertion: A

Kplex-23 system * Actually, 9 allele with ‘A’ insertion in 5' flanking region

::: 20 samples in 250 Koreans were observed

Kim et al. Forensic Sci Int Genet. 2017
Number of observed alleles in 250 Koreans

Kim et al. Forensic Sci Int Genet. 2017

Forensic statistical parameters for the CE and MPS results
Loci Method Hobs Hexp MP PD PIC PE
CE 0.828 0.843 0.049 0.951 0.823 0.652
D1S1656 MPS (Repeat) 0.848 0.856 0.041 0.959 0.839 0.691
MPS (+Flanks) 0.848 0.856 0.041 0.959 0.839 0.691
CE 0.792 0.755 0.100 0.900 0.719 0.584
D2S441 MPS (Repeat) 0.816 0.788 0.070 0.930 0.763 0.629
MPS (+Flanks) 0.816 0.789 0.069 0.931 0.764 0.629
CE 0.880 0.867 0.037 0.963 0.851 0.755
D2S1338
MPS (Repeat) 0.916 0.987 0.037 0.963 0.851 0.755
CE 0.772 0.772 0.137 0.863 0.675 0.548
CSF1PO
MPS (Repeat) 0.772 0.724 0.136 0.864 0.677 0.548
CE 0.716 0.705 0.137 0.863 0.652 0.453
D3S1358
MPS (Repeat) 0.772 0.770 0.080 0.920 0.742 0.548
CE 0.840 0.791 0.085 0.915 0.757 0.675
D5S818
MPS (Flanks) 0.892 0.851 0.044 0.956 0.832 0.779
CE 0.871 0.874 0.033 0.967 0.858 0.738
D6S1043
MPS (Repeat) 0.871 0.874 0.033 0.967 0.858 0.738
CE 0.788 0.765 0.096 0.904 0.727 0.577
D7S820 MPS (Repeat) 0.784 0.765 0.096 0.904 0.727 0.570
MPS (+Flanks) 0.844 0.825 0.053 0.947 0.804 0.683
CE 0.856 0.843 0.048 0.952 0.821 0.707
D8S1179
MPS (Repeat) 0.912 0.906 0.020 0.980 0.897 0.820
CE 0.820 0.802 0.074 0.926 0.772 0.637
vWA
MPS (Repeat) 0.820 0.814 0.066 0.934 0.788 0.637
CE 0.804 0.817 0.061 0.939 0.792 0.606
D12S391
MPS (Repeat) 0.868 0.874 0.030 0.970 0.862 0.731
CE 0.808 0.804 0.072 0.928 0.774 0.614
D13S317 MPS (Repeat) 0.812 0.807 0.070 0.930 0.777 0.621
MPS (+Flanks) 0.852 0.848 0.044 0.956 0.830 0.699
CE 0.928 0.917 0.018 0.982 0.909 0.853
Penta E
MPS (Repeat) 0.928 0.918 0.018 0.982 0.910 0.853
CE 0.799 0.784 0.083 0.917 0.748 0.598
D16S539
MPS (Flanks) 0.847 0.822 0.061 0.939 0.796 0.690
CE 0.800 0.790 0.073 0.927 0.763 0.599
Kim et al.
D21S11 MPS
MPS
(Repeat)
(+Flanks)
0.924
0.924
0.911
0.956
0.017
0.017
0.983
0.983
0.903
0.903
0.845
0.845
Forensic Sci Int
Penta D
CE 0.760 0.785 0.080 0.920 0.755 0.527 Genet. 2017
MPS (Flanks) 0.764 0.786 0.079 0.921 0.756 0.534
MPS analysis of Autosomal STRs using In-house
and Commercial STR panels

• MPS run
– For 1ng of 13 Koreans male samples
– In-house panel by Kim et al. (FSIG, 2017)
• Added SE33, D4S2048 and Y-M175
• Reduced 1st round PCR cycles (29 ⇒ 26)
– Precision ID GlobalFiler NGS STR Panel
• Ion PGM Hi-Q Chef Kit
• Ion Chef and PGM
MPS analysis of Autosomal STRs using In-house
and Commercial STR panels

• Results
– All concordant genotypes with CE result
when using modified in-house panel
– Extra read observed on Precision ID panel

– Resolve isometric homozyogotes

Example of Isometric Homozygotes
Sample : NGS011 by STRait Razor v3
MPS analysis of SE33

Sample : 2800M and Koreans samples by STRait Razor v3

Analyzing sequence variants of SE33 for 315 American and Korean samples
Analysis of MPS data

Easy and speedy analysis of MPS data

Backward compatibility of STR genotypes to CE

Config file for autosomal STRs are available

on http://forensic.yonsei.ac.kr
Allelic size range of 23 Y-STRs and Y-M175

KplexSeq-Y24
Average depth of coverage (DoC) for the 23 Y-STRs

Kwon et al. Forensic Sci Int Genet. 2016

Stutter and noise ratios of the 23 Y-STRs observed in MPS analysis

Stutter
Noise
N −2 a N −1 b N+1c
Locus
Average SD Average SD Average SD Average SD
DYS19 0.0073 0.0039 0.1064 0.0198 0.0139 0.0268 0.0006 0.0008
DYS385 0.0116 0.0083 0.1211 0.0424 0.0142 0.0110 0.0008 0.0023
DYS389I 0.0067 0.0051 0.0902 0.0241 0.0108 0.0133 0.0007 0.0012
DYS389II 0.0232 0.0061 0.1982 0.0256 0.0160 0.0068 0.0011 0.0015
DYS390 0.0087 0.0085 0.1086 0.0174 0.0064 0.0028 0.0004 0.0009
DYS391 0.0063 0.0066 0.0915 0.0193 0.0111 0.0155 0.0004 0.0024
DYS392 0.0178 0.0068 0.1484 0.0245 0.0682 0.0167 0.0048 0.0049
DYS393 0.0080 0.0052 0.1074 0.0183 0.0224 0.0081 0.0009 0.0032
DYS437 0.0025 0.0016 0.0598 0.0195 0.0107 0.0085 0.0007 0.0032
DYS438 0.0020 0.0046 0.0291 0.0106 0.0051 0.0057 0.0016 0.0043
DYS439 0.0047 0.0057 0.0778 0.0115 0.0170 0.0080 0.0006 0.0023
DYS448 0.0023 0.0044 0.0222 0.0121 0.0090 0.0102 0.0021 0.0035
DYS456 0.0090 0.0028 0.1196 0.0147 0.0269 0.0059 0.0005 0.0003
DYS458 0.0133 0.0046 0.1363 0.0173 0.0165 0.0048 0.0009 0.0025
DYS481 0.0537 0.0122 0.2761 0.0309 0.0636 0.0114 0.0012 0.0019
DYS533 0.0059 0.0120 0.0912 0.0151 0.0188 0.0113 0.0004 0.0008
DYS549 0.0059 0.0065 0.0928 0.0199 0.0176 0.0060 0.0004 0.0008
DYS570 0.0113 0.0035 0.1270 0.0187 0.0244 0.0097 0.0008 0.0024
DYS576 0.0117 0.0042 0.1283 0.0181 0.0241 0.0125 0.0011 0.0038
DYS635 0.0061 0.0028 0.0946 0.0182 0.0121 0.0052 0.0005 0.0014
DYS643 0.0019 0.0030 0.0299 0.0082 0.0047 0.0044 0.0009 0.0024
YGATAH4 0.0021 0.0011 0.0950 0.0235 0.0143 0.0138 0.0008 0.0038

Kwon et al. Forensic Sci Int Genet. 2016

Number of observed alleles in 250 Koreans

Kwon et al. Forensic Sci Int Genet. 2016

Resolution of Y-STR MPS Analysis in Mixed DNA

Locus 1:1 1:3 1:6 1:9 1:14 1:19 1:29 1:49

DYS19 9/9 18/18 18/18 18/18 18/18 17/17 11/18 11/18
DYS389I 8/8 16/16 16/16 16/16 14/16 13/15 10/16 7/16
DYS389II 10/10 20/20 20/20 20/20 20/20 16/18 14/20 11/20
DYS390 6/6 12/12 12/12 12/12 11/12 11/12 8/12 7/12
DYS391 5/5 10/10 10/10 10/10 10/10 7/9 7/10 6/10
DYS392 10/10 20/20 20/20 18/20 18/20 14/18 10/20 9/20
DYS393 10/10 20/20 20/20 20/20 17/20 17/18 9/20 8/20
DYS437 6/6 12/12 12/12 11/12 11/12 9/10 7/12 3/12
DYS438 6/6 12/12 12/12 12/12 10/12 10/12 7/12 8/12
DYS439 8/8 16/16 16/16 16/16 14/16 10/15 9/16 6/16
DYS448 8/8 16/16 16/16 16/16 16/16 14/15 8/16 8/16
DYS456 6/6 12/12 12/12 12/12 12/12 11/12 5/12 5/12
DYS458 10/10 20/20 19/20 18/20 15/20 11/18 9/20 8/20
DYS481 10/10 20/20 20/20 17/20 16/20 15/18 13/20 10/20
DYS533 7/7 14/14 14/14 14/14 13/14 11/15 10/14 7/14
DYS549 6/6 12/12 12/12 10/12 10/12 8/11 9/12 6/12
DYS570 10/10 20/20 20/20 20/20 19/20 15/18 14/20 10/20
DYS576 9/9 18/18 18/18 17/18 16/18 14/17 11/18 10/18
DYS635 9/9 18/18 17/18 16/18 15/18 13/18 12/18 10/18
DYS643 10/10 20/20 20/20 20/20 20/20 16/18 13/20 12/20
GATAH4 8/8 16/16 16/16 16/16 15/16 10/16 5/16 7/16
100% 100% 99.50% 96.75% 90.90% 82.00% 52.06% 49.47%
100% ≥90% ≥80% ≥70% ≥60% ≥50% <50%
Microhaplotype

• New marker needed

– To infer biogeographic ancestry where the STR
data is limited
– To aid in mixture deconvolution and avoid the
issue of stutter
• At least three haplotypes (alleles) within a
region smaller than 200 bp
• Provide useful information when typed using
MPS
Kidd et al. FSIG, 2014 and 2017
Microhaplotype
 Simple SNP vs. Microhaplotype

Simple SNP Microhaplotype

 Bi-allelic marker  Multi-allelic marker

 Low heterozygosity  High heterozygosity

 ID or ancestry  ID and/or ancestry

Microhaplotype
 Microhaplotype study

http://medicine.yale.edu/lab/kidd/publications/publications.aspx
Microhaplotype MPS panel

 Single tube multiplex PCR system for 51 microhaplotypes

 Amplicon sizes of 51 microhaplotypes (KplexSeq-MH51)

 DNA samples
 1ng of 2800M Standard DNA and 122 Korean samples
 Results
 Observed reads per sample

 Average depth of coverage (DoC) per marker

Marker ACN9 ADH7 ARHGAP27 ATXN1 C14ORF43 CCR2 CDH4 CEP104 COG2 COL4A1 COL4A3 COMT CPNE4

Read depth 13753 10502 16976 11374 15277 9113 11331 9065 9547 13051 10447 13875 5318

상대적 비율(%)
2.41 1.84 2.97 1.99 2.67 1.59 1.98 1.59 1.67 2.28 1.83 2.43 0.93

Marker D13S169 D18S1122 D21S1263 D22S1159 D5S1970 DLEU1 DRD3 EDAR FAM99A FAT1 FRMD4A GATA4 GFI1B

Read depth 13642 12341 15300 12394 10665 5702 9363 10958 10063 5305 12382 10136 13795

상대적 비율(%)
2.39 2.16 2.68 2.17 1.87 1.00 1.64 1.92 1.76 0.93 2.17 1.77 2.41
 Results

 Phase-known haplotyping of microhaplotype marker

Example) D13S169

rs9536429
rs1927847 T/C rs7492234 rs9536430
T/C T/C C/T

Haplotype

2800M T T T T 1 - TTTT
C C C T 2 - CCCT
T T T T 1 - TTTT
Sample
T T C T 2 - TTCT
Reference seq.
Results
 Genotypes of 143 SNPs in 2800M standard DNA
Marker SNPs_Included Haplotype Marker SNPs_Included Haplotype Marker SNPs_Included Haplotype
D13S169 rs1927847/rs9536429/rs7492234/rs9536430 1 TTTT FRMD4A rs10796164/rs10796165/rs17154765/rs10796166 1 GCTG TAS2R1 rs41461/rs41462 1 CC
2 CCCT 2 ACTG 2 TC
D5S1970 rs74865590/rs438055/rs370672/rs6555108 1 CAAG GFI1B rs606141/rs8193001/rs56256724/rs633153 1 GTCC DLEU1 rs806301/rs2066700 1 CC
2 CAGA 2 GCCT 2 TC
D21S1263 rs8126597/rs6517970/rs8131148/rs6517971 1 GACT CPNE4 rs1225051/rs1225050/rs1225049/rs1225048 1 AATA TTC12 rs2288159/rs10891537 1 GT
2 GCTC 2 AACA 2 TT
LINC00111 rs2838081/rs2838082/rs78902658/rs2838083 1 AACG SUDS3 rs1503767/rs11068953 1 TA CCR2 rs4513489/rs6441961 1 TC
2 AACG 2 TA 2 TC
FAM99A rs12802112/rs28631755/rs7112918/rs4752777 1 ACTG ADH7 rs4699748/rs2584461/rs1442492 1 ACA GATA4 rs1390950/rs2898295 1 TA
2 GCCG 2 GCA 2 TA
LRRC63 rs9562648/rs9562649/rs2765614 1 GCG C14ORF43 rs12717560/rs12878166 1 GT NPEPPS rs3760370/rs3760371 1 CT
2 GCG 2 GT 2 TC
LRRN2 rs17413714/rs2772234/rs1610401/rs1610400 1 CACC COG2 rs2296796/rs2296797/rs2296798 1 GGG LRRC2 rs6808142/rs17030627 1 CT
2 CACC 2 GGG 2 TG
COL4A1 rs1192204/rs1192205/rs3825483/rs3825481 1 CGTT ARHGAP27 rs1059504/rs8327 1 AA ACN9 rs17168174/rs10246622 1 CG
2 CGCT 2 AA 2 CG
IGSF21 rs11810587/rs1336130/rs1533623/rs1533622 1 TTAA RXRA rs3118582/rs10776839 1 TG TYRP1 rs1408800/rs1408801 1 AA
2 TCAG 2 TG 2 AA
LINC01233 rs4932999/rs4932769/rs2361019/rs2860462 1 TGAA OR52S1P1 rs10500616/rs2499936 1 CG CDH4 rs10854214/rs10854215 1 AA
2 TGAA 2 CG 2 GG
LOC642852 rs6518223/rs2838868/rs7279250/rs8133697 1 TCAG GNGT2 rs2233362/rs634370 1 AA PAPD7 rs870348/rs870347 1 TA
2 TCTG 2 AA 2 AA
COL4A3 rs6714835/rs6756898/rs12617010 1 GTC CEP104 rs4648344/rs6663840 1 CA DRD3 rs3732783/rs6280 1 TT
2 TCC 2 CA 2 TT
SGCG rs8181845/rs679482/rs9510616 1 TCA FAT1 rs1280100/rs1280099 1 AG ATXN1 rs4565296/rs4431439/rs179939 1 GCA
2 TCA 2 GA 2 GTG
PLCG2 rs16956011/rs3934955/rs3934956/rs4073828 1 ACCG PLIN3 rs1055919/rs2271057 1 TA EDAR rs260694/rs11123719/rs11691107 1 TTC
2 GACA 2 CA 2 TTC
ITGB6 rs3101043/rs3111398/rs72623112 1 TTG COMT rs4818/rs4680 1 CA USH2A rs4528199/ rs6604596 1 GA
2 CTG 2 GG 2 AA
KIF16B rs6044080/rs17674942/rs6044081/rs16997830 1 CTGA LYPD6B rs2170607/rs10497052 1 AG
2 CTGA 2 GG
D18S1122 rs621320/rs621340/rs678179/rs621766 1 AGAA PAH rs2133298/rs3817446 1 TT
2 AGAA 2 TC
D22S1159 rs763040/rs5764924/rs763041 1 GAA NCAM1 rs2303377/rs2303378 1 TG
2 GAA 2 CG
Results
 Genotypes of 143 SNPs in 2800M standard DNA
Marker SNPs_Included Haplotype Marker SNPs_Included Haplotype Marker SNPs_Included Haplotype
D13S169 rs1927847/rs9536429/rs7492234/rs9536430 1 TTTT FRMD4A rs10796164/rs10796165/rs17154765/rs10796166 1 GCTG TAS2R1 rs41461/rs41462 1 CC
2 CCCT 2 ACTG 2 TC
D5S1970 rs74865590/rs438055/rs370672/rs6555108 1 CAAG GFI1B rs606141/rs8193001/rs56256724/rs633153 1 GTCC DLEU1 rs806301/rs2066700 1 CC
2 CAGA 2 GCCT 2 TC
D21S1263 rs8126597/rs6517970/rs8131148/rs6517971 1 GACT CPNE4 rs1225051/rs1225050/rs1225049/rs1225048 1 AATA TTC12 rs2288159/rs10891537 1 GT
2 GCTC 2 AACA 2 TT
LINC00111 rs2838081/rs2838082/rs78902658/rs2838083 1 AACG SUDS3 rs1503767/rs11068953 1 TA CCR2 rs4513489/rs6441961 1 TC
2 AACG 2 TA 2 TC
FAM99A rs12802112/rs28631755/rs7112918/rs4752777 1 ACTG ADH7 rs4699748/rs2584461/rs1442492 1 ACA GATA4 rs1390950/rs2898295 1 TA
2 GCCG 2 GCA 2 TA
LRRC63 rs9562648/rs9562649/rs2765614 1 GCG C14ORF43 rs12717560/rs12878166 1 GT NPEPPS rs3760370/rs3760371 1 CT
2 GCG 2 GT 2 TC
LRRN2 rs17413714/rs2772234/rs1610401/rs1610400 1 CACC COG2 rs2296796/rs2296797/rs2296798 1 GGG LRRC2 rs6808142/rs17030627 1 CT
2 CACC 2 GGG 2 TG
COL4A1 rs1192204/rs1192205/rs3825483/rs3825481 1 CGTT ARHGAP27 rs1059504/rs8327 1 AA ACN9 rs17168174/rs10246622 1 CG
2 CGCT 2 AA 2 CG
IGSF21 rs11810587/rs1336130/rs1533623/rs1533622 1 TTAA RXRA rs3118582/rs10776839 1 TG TYRP1 rs1408800/rs1408801 1 AA
2 TCAG 2 TG 2 AA
LINC01233 rs4932999/rs4932769/rs2361019/rs2860462 1 TGAA OR52S1P1 rs10500616/rs2499936 1 CG CDH4 rs10854214/rs10854215 1 AA
2 TGAA 2 CG 2 GG
LOC642852 rs6518223/rs2838868/rs7279250/rs8133697 1 TCAG GNGT2 rs2233362/rs634370 1 AA PAPD7 rs870348/rs870347 1 TA
2 TCTG 2 AA 2 AA
COL4A3 rs6714835/rs6756898/rs12617010 1 GTC CEP104 rs4648344/rs6663840 1 CA DRD3 rs3732783/rs6280 1 TT
2 TCC 2 CA 2 TT
SGCG rs8181845/rs679482/rs9510616 1 TCA FAT1 rs1280100/rs1280099 1 AG ATXN1 rs4565296/rs4431439/rs179939 1 GCA
2 TCA 2 GA 2 GTG
PLCG2 rs16956011/rs3934955/rs3934956/rs4073828 1 ACCG PLIN3 rs1055919/rs2271057 1 TA EDAR rs260694/rs11123719/rs11691107 1 TTC
2 GACA 2 CA 2 TTC
ITGB6 rs3101043/rs3111398/rs72623112 1 TTG COMT rs4818/rs4680 1 CA USH2A rs4528199/ rs6604596 1 GA
2 CTG 2 GG 2 AA
KIF16B rs6044080/rs17674942/rs6044081/rs16997830 1 CTGA LYPD6B rs2170607/rs10497052 1 AG
2 CTGA 2 GG
D18S1122 rs621320/rs621340/rs678179/rs621766 1 AGAA PAH rs2133298/rs3817446 1 TT
2 AGAA 2 TC
D22S1159 rs763040/rs5764924/rs763041 1 GAA NCAM1 rs2303377/rs2303378 1 TG
2 GAA 2 CG
 FRMD4A 0.639 0.611 0.169 0.831 0.590 0.341 1.39

Results
D22S1159
GFI1B
0.631
0.656
0.654
0.655
0.176
0.180
0.824
0.820
0.591 0.330
Preparing
0.595 manuscript…
0.363 1.45
1.36

FAT1 0.623 0.657 0.188 0.812 0.580 0.319 1.33

COMT 0.664 0.667 0.189 0.811 0.590 0.375 1.49
 Forensic
LYPD6B statistical
0.631 parameters
0.656 0.190 for 51
0.810MHs in
0.579Koreans
0.330 1.36
RXRA 0.648 0.656 0.193 0.807 0.579 0.352 1.42
Forensic Statistics Paternity Statistics
ITGB6 Hobs
0.639 Hexp
0.630 0.193 0.807 0.581 0.341 1.39
MP PD PIC PE TPI
GNGT2 0.623 0.654 0.194 0.806 0.578 0.319 1.33
D13S169 0.869 0.875 0.035 0.965 0.857 0.732 3.81
ARHGAP27 0.648 0.648 0.201 0.799 0.573 0.352 1.42
D5S1970 0.885 0.870 0.037 0.963 0.852 0.765 4.36
OR52S1P1 0.648 0.655 0.202 0.798 0.577 0.352 1.42
D21S1263 0.811 0.818 0.060 0.940 0.793 0.621 2.65
PAPD7 0.656 0.643 0.206 0.794 0.567 0.363 1.45
LINC00111 0.811 0.819 0.067 0.933 0.790 0.621 2.65
NPEPPS 0.615 0.632 0.208 0.792 0.552 0.309 1.30
COL4A1 0.779 0.801 0.074 0.926 0.767 0.560 2.26
CCR2 0.664 0.633 0.217 0.783 0.557 0.375 1.49
PLCG2 0.779 0.783 0.080 0.920 0.747 0.560 2.26
TAS2R1 0.557 0.570 0.249 0.751 0.505 0.243 1.13
LRRN2 0.828 0.780 0.083 0.917 0.748 0.652 2.90
ACN9 0.623 0.581 0.268 0.732 0.508 0.319 1.33
LRRC63 0.787 0.769 0.093 0.907 0.730 0.575 2.35
C14ORF43 0.492 0.542 0.287 0.713 0.461 0.180 0.98
COL4A3 0.811 0.772 0.098 0.902 0.732 0.621 2.65
GATA4 0.492 0.499 0.304 0.696 0.445 0.180 0.98
KIF16B 0.697 0.759 0.099 0.901 0.715 0.423 1.65
USH2A
IGSF21
DLEU1
0.443
0.664
0.484
0.501
0.766
0.487
Match Probability
0.306
0.102
0.316
0.694
0.898
0.684
0.446
0.725
0.435
0.142
0.375
0.174
0.90
1.49
0.97
FAM99A 0.738 0.733 0.110 0.890 0.694 0.489 1.91
TTC12
• 0.541 0.533 0.334 0.666 0.421 0.226 1.09

= 3.82 × 1037
SUDS3
•
0.623 0.741 0.112 0.888 0.690 0.319 1.33
PLIN3
• 0.459 0.514 0.336 0.664 0.393 0.154 0.92
LINC01233 0.762 0.741 0.114 0.886 0.695 0.531 2.10
NCAM1 0.508 0.495 0.336 0.664 0.422 0.195 1.02
SGCG 0.803 0.753 0.116 0.884 0.709 0.605 2.54
CDH4 0.525 0.479 0.341 0.659 0.417 0.210 1.05
LOC642852 0.689 0.717 0.125 0.875 0.669 0.411 1.61
CEP104 0.549 0.525 0.345 0.655 0.416 0.234 1.11
D18S1122 0.664 0.710 0.126 0.874 0.666 0.375 1.49
LRRC2 0.426 0.475 0.370 0.630 0.365 0.131 0.87
CPNE4 0.705 0.704 0.146 0.854 0.644 0.436 1.69
PAH 0.402 0.369 0.427 0.573 0.334 0.115 0.84
COG2 0.680 0.681 0.162 0.838 0.619 0.398 1.56
DRD3 0.385 0.380 0.440 0.560 0.326 0.105 0.81
FRMD4A 0.639 0.611 0.169 0.831 0.590 0.341 1.39
ADH7 0.344 0.343 0.456 0.544 0.316 0.083 0.76
D22S1159 0.631 0.654 0.176 0.824 0.591 0.330 1.36
TYRP1 0.344 0.332 0.492 0.508 0.283 0.083 0.76
GFI1B 0.656 0.655 0.180 0.820 0.595 0.363 1.45
EDAR 0.246 0.245 0.592 0.408 0.233 0.044 0.66
FAT1 0.623 0.657 0.188 0.812 0.580 0.319 1.33
ATXN1 0.074 0.103 0.832 0.168 0.100 0.005 0.54
COMT 0.664 0.667 0.189 0.811 0.590 0.375 1.49
Future work

• Validation study
– Sensitivity and specificity

• Resolution in mixture deconvolution

• Application to complex kinship case

• Evaluation of biogeographic inference
Things for supporting forensic MPS

• For STR analysis

– Automatically extracting repeat structure
– Variant calling in the franking region
– Database for sequence based allele

• For Microhaplotype analysis

– Automatic haplotype generation
– Cryptic variation consideration
– Database for sequence based haplotype
Concluding remarks

• Three in-house MPS panels were developed

• Sequence-based genotyping was performed

• MPS data could be analyzed by open

source software

• Bioinformatics tools should be upgraded

Acknowledgements

 Yonsei DNA profiling group

Eun Young Lee, MS
Eun Hye Kim, MS
So Yeun Kwon, MS
Sang Eun Jung, MS
Hwan Young Lee, Ph.D.

 Yonsei Genome Center

In Seok Yang, Ph.D.
Sora Kim, Ph.D. candidate
Sang Woo Kim, Ph.D.