Analysis of Y-STR Profiles in Mixed DNA using Next Generation Sequencing

Transcription

1 Analysis of Y-STR Profiles in Mixed DNA using Next Generation Sequencing So Yeun Kwon, Hwan Young Lee, and Kyoung-Jin Shin Department of Forensic Medicine, Yonsei University College of Medicine, Seoul, Korea DNA analysis in Forensic genetics - Capillary electrophoresis (CE) is a gold standard method for STR typing based on fragment analysis. - CE method is size-based analysis. - Relative florescent unit (RFU) is used for peak height and represents relative quantity of DNA detected by CE. - Stutter products result from PCR process due to slippage of DNA polymerase in STR typing. 1

2 NGS in Forensic genetics Current mixed DNA analysis SWGDAM (Scientific Working Group on DNA Analysis Method), ISFG (International Society for Forensic Genetics) 2

3 Advantages of NGS analysis - Artifacts such as minus A peak, pull-up peak, off ladder alleles, and dye blobs would not be produced and can be excluded for interpretation. - Complete sequences can be identified including primer region, repeat sequence region and flanking sequence region, therefore, lead to accurate information of true alleles. - Higher detection sensitivity of NGS Objective Evaluation of effectiveness and utility of NGS based mixed DNA analysis targeting Y chromosome STRs Stutter ratio and noise ratio in NGS system Analytical threshold/interpretational threshold Determination of genotypes in mixed DNAs Estimation of mixture ratios 3

4 Materials and Methods DNA samples - 18 unrelated Korean males (single-source) two-person mixed DNAs (from 13 males combination) (A:B - 1:1, 1:3, 1:6, 1:9, 1:14, 1:19, 1:29, 1:49 and 3:1, 6:1, 9:1, 14:1, 19:1, 29:1, 49:1) No. of repeat difference a I 389II c 392 b c c b Total Y GATA H4 Target Y-STRs - PowerPlex Y23 loci except for 385ab The developed Y-STR NGS analysis system Step 1. PCR amplification PowerPlex Y23 loci and 1 Y-SNP Investigation into 23 Y-STR loci with massively parallel sequencing - Submitted to Forensic Science International; Genetics (2016, in progress) 4

5 The developed Y-STR NGS analysis system Step 2. Library preparation - Template DNA : 1 ng of DNAs - 1 st PCR 27 cycles Amplicon 1/10 fold dilution - 2 nd PCR 15 cycles - Nextera XT index kit (Illumina) Lee et al. Forensic. Sci. Int. Gent. 22 (2016) The developed Y-STR NGS analysis system Step 3. Validation of library - Library quantification ; KAPA library quantification kit - Agilent Bioanalyzer - Library pooling 5

6 The developed Y-STR NGS analysis system Step 4. Sequencing - Miseq System (Ilumina) - Miseq reagent kit v3 (2 x 300 bp) - Lab genomics (requested) The developed Y-STR NGS analysis system Sample Step 5. Data analysis STRiNGS developed by Kim SR, Yonsei Genome Center #Fastq format STRiNGS program Data arrangement with Microsoft Excel Size-based analysis Allelic size ranges Final report Y-STR genotyping (Allele, read counts) 6

7 Mixed DNA analysis NGS result and coverage NGS data generation (Miseq reagent kit v3) - Total : 12.7 GB - Q>30, 7.6 GB - Average read counts : 234,184 Average read counts of the 23 Y-STRs and Y-SNP (M175) Stutter ratio Stutter ratio of single source DNA of 18 Korean males Locus N-3 N-2 N-1 N+1 AVE SD AVE SD AVE SD AVE SD I II GATAH

8 Noise ratio Noise ratio of single source DNA of 18 Korean males % % 0.12% 0.13% % 0.07% 0.07% 0.08% 0.08% 0.09% 0.09% 0.04% 0.04% 0.05% 0.05% 0.05% 0.06% 0.06% 0.06% 0.06% 0.03% 0 Mixed DNA analysis Genotyping results of NGS data Noise average ratio + 3SD True allele + stutter Analytical threshold ratio Average = 0.44%, 0.08% ATR 0.84% Stutter average ratio + 3SD True allele Interpretational threshold ratio Average ratios N-3 : 1.57%, N-2 : 2.24%, N-1 : 17.50%, N+1 : 3.07% 8

9 Resolution power of Y-STRs in Mixed DNA Locus 1:1 1:3 1:6 1:9 1:14 1:19 1:29 1: /9 18/18 18/18 18/18 18/18 17/17 11/18 11/18 389I 8/8 16/16 16/16 16/16 14/16 13/15 10/16 7/16 389II 10/10 20/20 20/20 20/20 20/20 16/18 14/20 11/ /6 12/12 12/12 12/12 11/12 11/12 8/12 7/ /5 10/10 10/10 10/10 10/10 7/9 7/10 6/ /10 20/20 20/20 18/20 18/20 14/18 10/20 9/ /10 20/20 20/20 20/20 17/20 17/18 9/20 8/ /6 12/12 12/12 11/12 11/12 9/10 7/12 3/ /6 12/12 12/12 12/12 10/12 10/12 7/12 8/ /8 16/16 16/16 16/16 14/16 10/15 9/16 6/ /8 16/16 16/16 16/16 16/16 14/15 8/16 8/ /6 12/12 12/12 12/12 12/12 11/12 5/12 5/ /10 20/20 19/20 18/20 15/20 11/18 9/20 8/ /10 20/20 20/20 17/20 16/20 15/18 13/20 10/ /7 14/14 14/14 14/14 13/14 11/15 10/14 7/ /6 12/12 12/12 10/12 10/12 8/11 9/12 6/ /10 20/20 20/20 20/20 19/20 15/18 14/20 10/ /9 18/18 18/18 17/18 16/18 14/17 11/18 10/ /9 18/18 17/18 16/18 15/18 13/18 12/18 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% Mixture ratio estimation Mixture ratio estimation by the number of repeat difference (1:1, 1:3, 1:6, 1:9, 1:14) 9

10 Conclusion Single source DNA analysis Noise ratios of 21 Y-STRs in this NGS system Analytical threshold ratio Average = 0.44%, 0.08% ATR 0.84% Stutter ratios of 21 Y-STRs in this NGS system Interpretation threshold ratio Average ratios N-3 : 1.57%, N-2 : 2.42%, N-1 : 17.50%, N+1 : 3.07% Conclusion Mixed DNA analysis The different resolution power for minor component detection in mixed DNAs ; 1:19 19, 1:6-458 and 635. Most Y-STRs ( 90%) were determined by the ratio of 1:14. Read counts are not completely correlated with mixture ratios. Considerations for STR analysis using PCR-based NGS system ; Inevitable stutter generation of STR and Stochastic effect of low template DNA 10

11 Further study Sequence-based NGS data analysis - Re-investigation of stutter characteristics and noise ratios Re-evaluate the utility of NGS based mixed DNA analysis NGS Data Analysis Pipeline for STR typing - Required to accumulate various types of NGS data - Integration of stutter and noise ratio analysis, and sequence-based STR typing Acknowledgements This study was supported by a faculty research grant from Yonsei University College of Medicine in Seoul, Korea for 2015 ( ) 11