2017-09-05 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 1
2017-09-05 Application of massively parallel sequencing to forensic genetics Capillary electrophoresis Massively parallel sequencing ……TCTATCTGTCTGTCT G TCTATCTATCTA…… ……TCTATCTGTCTGTCT G TCTATCTATCTA…… ……TCTATCTGTCTGTCT G TCTATCTATCTA…… ……TCTATCTGTCTGTCT G TCTATCTATCTA…… ……TCTATCTGTCTGTCT G TCTATCTATCTA…… ……TCTATCTGTCTGTCT G TCTATCTATCTA…… ……TCTATCTGTCTGTCT G TCTATCTATCTA…… A B ……TCTATCTGTCTGTCT G TCTATCTATCTA…… ……TCTATCTGTCTGTCT G TCTATCTATCTA…… ……TCTATCTGTCTGTCT G TCTATCTATCTA…… ……TCTATCTGTCTGTCT G TCTATCTATCTA…… ……TCTATCTGTCTGTCT G TCTATCTATCTA…… ……TCTATCTGTCTGTCT G TCTATCTATCTA…… ……TCTATCTGTCTGTCT A TCTATCTATCTA…… ……TCTATCTGTCTGTCT G TCTATCTATCTA…… ……TCTATCTGTCTGTCT G TCTATCTATCTA…… ……TCTATCTGTCTGTCT G TCTATCTATCTA…… ……TCTATCTGTCTGTCT G TCTATCTATCTA…… ……TCTATCTGTCTGTCT G TCTATCTATCTA…… ……TCTATCTGTCTGTCT G TCTATCTATCTA…… ……TCTATCTGTCTGTCT A TCTATCTATCTA…… ……TCTATCTGTCTGTCT A TCTATCTATCTA…… ……TCTATCTGTCTGTCT A TCTATCTATCTA…… ……TCTATCTGTCTGTCT A TCTATCTATCTA…… ……TCTATCTGTCTGTCT A TCTATCTATCTA…… ……TCTATCTGTCTGTCT A TCTATCTATCTA…… ……TCTATCTGTCTGTCT G TCTATCTATCTA…… ……TCTATCTGTCTGTCT G TCTATCTATCTA…… ……TCTATCTGTCTGTCT G TCTATCTATCTA…… ……TCTATCTGTCTGTCT G TCTATCTATCTA…… ……TCTATCTGTCTGTCT G TCTATCTATCTA…… ……TCTATCTGTCTGTCT G TCTATCTATCTA…… ……TCTATCTGTCTGTCT A TCTATCTATCTA…… ……TCTATCTGTCTGTCT A TCTATCTATCTA…… ……TCTATCTGTCTGTCT A TCTATCTATCTA…… ……TCTATCTGTCTGTCT A TCTATCTATCTA…… ……TCTATCTGTCTGTCT A TCTATCTATCTA…… ……TCTATCTGTCTGTCT A TCTATCTATCTA…… ……TCTATCTGTCTGTCT G TCTATCTATCTA…… ……TCTATCTGTCTGTCT G TCTATCTATCTA…… ……TCTATCTGTCTGTCT A TCTATCTATCTA…… ……TCTATCTGTCTGTCT A TCTATCTATCTA…… ……TCTATCTGTCTGTCT A TCTATCTATCTA…… ……TCTATCTGTCTGTCT A TCTATCTATCTA…… ……TCTATCTGTCTGTCT A TCTATCTATCTA…… ……TCTATCTGTCTGTCT A TCTATCTATCTA…… ……TCTATCTGTCTGTCT A TCTATCTATCTA…… ……TCTATCTGTCTGTCT A TCTATCTATCTA…… ……TCTATCTGTCTGTCT A TCTATCTATCTA…… ……TCTATCTGTCTGTCT A TCTATCTATCTA…… ……TCTATCTGTCTGTCT A TCTATCTATCTA…… ……TCTATCTGTCTGTCT A TCTATCTATCTA…… ……TCTATCTGTCTGTCT A TCTATCTATCTA…… ……TCTATCTGTCTGTCT A TCTATCTATCTA…… …… A ……TCTATCTGTCTGTCT G TCTATCTATCTA…… 16 allele 16 allele (G>A) ……TCTATCTGTCTGTCT A TCTATCTATCTA…… B 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 2
2017-09-05 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 3
2017-09-05 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 ? 4
2017-09-05 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 u 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 ³ 1% variation reported in primer binding area u 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/) 5
2017-09-05 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 • Agilent BioAnalyzer Agilent BioAnalyzer • • : Enzymatic purification using EXO-SAP IT * Adjustment of beads ratio for size selection Simplifed MPS library preparation Fragment genomic DNA ① (PCR product) ② ① End repairs ③ Beads purifications ④ Adenylate 3’ ends ② ⑤ ⑥ Beads purifications ⑦ Beads purification Beads purification 6
2017-09-05 Two-step PCR for MPS library preparation u The first PCR using Target specific primers u Thermal Cycle PCR component Volume 95℃ 11 min dH 2 0 4.0 94℃ 20 sec Gold ST*R 10× Buffer 2.0 59℃ 60 sec × 26 cycles 5× Primer Mix-I 4.0 72℃ 45 sec (~ 27) 5× Primer Mix-II 4.0 72℃ 5 min 5× Primer Mix-III 4.0 4℃ forever AmpliTaq Gold (5U/uL) 1.0 Template DNA (1ng/uL) 1.0 Total 20.0 u The second PCR using Nextera XT Index primers u Thermal Cycle PCR component Volume 95℃ 15 min dH 2 0 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 7
2017-09-05 MPS library verification using CE Autosomal STR Y-STR Microhaplotype Simple Workflow of MPS on MiSeq system Step 3. Step 1. Step 2. Step 4. Library pooling, PCR amplification Library preparation 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 8
2017-09-05 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 9
2017-09-05 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 10
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