Massively parallel sequencing of 231 autosomal SNPs with a custom panel: a SNP typing assay developed for human identification with Ion Torrent PGM

Suhua Zhang,Yingnan Bian,Anqi Chen,Hancheng Zheng,Yuzhen Gao,Yiping Hou,Chengtao Li

doi:10.1080/20961790.2017.1281011

Abstract

ABSTRACTThe custom-designed single nucleotide polymorphism (SNP) panel amplified 231 autosomal SNPs in one PCR reaction and subsequently sequenced with massively parallel sequencing (MPS) technology and Ion Torrent personal genome machine (PGM). SNPs were chosen from SNPforID, IISNP, HapMap, dbSNP, and related published literatures. Full concordance was obtained between available MPS calling and Sanger sequencing with 9947A and 9948 controls. Ten SNPs (rs4606077, rs334355, rs430046, rs2920816, rs4530059, rs1478829, rs1498553, rs7141285, rs12714757 and rs2189011) with low coverage or heterozygote imbalance should be optimized or excluded from the panel. Sequence data had sufficiently high coverage and gave reliable SNP calling for the remaining 221 loci with the custom MPS–SNP panel. A default DNA input amount of 10 ng per reaction was recommended by Ampliseq technology but sensitivity testing revealed positive results from as little as 1 ng input DNA. Mixture testing with this panel is possible through analysis of the FMAR (frequency of major allele reads) values at most loci with enough high coverage depth and low level of sequencing noise. These results indicate the potential advantage of the custom MPS–SNP assays and Ion Torrent PGM platform for forensic study.

Highlights

Within forensic community, standardized methods of PCR amplification and capillary electrophoresis (CE) of short tandem repeats (STRs) are usually applied in forensic laboratories [1,2]
936 single nucleotide polymorphism (SNP) were retained after selection based on genotype frequency data with Hardy–Weinberg equilibrium (HWE) (P>0.05) and Fst < 0.06 which performed with population difference testing among African American, European American, and East Asian populations
The performance of the custom SNP panel and Ion Torrent personal genome machine (PGM) was tested by studying SNP sequencing concordance with traditional Sanger sequencing, individual sample sequencing, sensitivity and mixtures testing

Summary

Introduction

Within forensic community, standardized methods of PCR amplification and capillary electrophoresis (CE) of short tandem repeats (STRs) are usually applied in forensic laboratories [1,2]. Two commercial kits were available for sequencing of SNP loci selected for human identification with MPS technology: the HIDIon AmpliseqTM Identity Panel (currently sold under the name of “Precision ID Identity Panel” by Thermo Fisher Scientific) [9] and the ForenSeq DNA Signature Prep Kit (Illumina) [10]. Both kits type most of the SNPs in the SNPforID [11] and IISNP [12]. Evaluation reports of the two panels [9,10] reveal that there is still a need for supplementary DNA marker typing in order to increase the power to solve cases for both individual identification and complex kinship issues (e.g. half-sibling testing without parental information, relationship between uncle and nephew without reference samples, etc.)

Methods

Results

Conclusion