A Strategy for Characterization of Single Nucleotide Polymorphisms in a Reference Material

Abstract

The advent and adoption of next generation sequencing (NGS) is enabling analysis of single nucleotide polymorphisms (SNPs) at an unprecedented scale, limited primarily by multiplexing during the PCR amplification based enrichment step used for forensic applications. Since only a single nucleotide is assayed, PCR primers may be designed to generate small amplicons, making SNP markers well-suited to forensic DNA typing. Carefully selected panels of SNP markers have been previously established for forensic applications such as one-to-one matching, estimating biogeographical ancestry, and predicting externally visible phenotype [1–6]. To support the implementation of SNPs in forensic DNA analysis, NIST has examined the HID-Ion AmpliSeq Identity Panel and the HID-Ion AmpliSeq Ancestry Panel for the Ion Torrent Personal Genome Machine (PGM). In total, over 289 SNP markers have been typed. NIST intends to use a strategy combining NGS on orthogonal platforms and Sanger sequencing for characterizing the SNP markers for varying levels of confidence. The outcome will be to report only the SNP allele calls, analogous to the mitochondrial sequence variants in NIST Standard Reference Material (SRM) 2392, and not the subsequent application of the information (e.g. prediction of ancestry or phenotype).