Testing novel CLC pipeline to evaluate next generation sequencing concordance of forensically relevant single nucleotide polymorphisms

Forensic laboratories have been analyzing DNA for over 30 years, however only recently has the introduction of Single Nucleotide Polymorphism (SNP) analysis using Massively Parallel Sequencing (MPS) become a possibility.... [ view full abstract ]

Forensic laboratories have been analyzing DNA for over 30 years, however only recently has the introduction of Single Nucleotide Polymorphism (SNP) analysis using Massively Parallel Sequencing (MPS) become a possibility. Illumina along with ThermoFisher Scientific manufacture kits that can simultaneously genotype hundreds of forensically relevant (i.e. ancestry, identity and/or phenotypic) SNPs on their MPS platforms (MiSeq FGx and PGM^™ or S5, respectively). Additionally, both companies provide proprietary software suites (Universal Analysis Software for the MiSeq and the HID Genotyper Plugin for either ThermoFisher instrument), that analyze and report SNP genotypes. There are a number of identity (n, 83) and ancestry (n, 55) SNPs that overlap between Illumina’s ForenSeq^™ Signature DNA Prep Kit and the Precision ID Identity and Ancestry Panels from ThermoFisher. To simplify the data analysis while removing software bias from multiple MPS platforms, a universal workflow was developed in CLC Genomics Workbench (v.9.0) to process FASTQ and BAM files produced by the sequencing instruments. The CLC pipeline subjects all sequencing files to quality trimming, alignment to an abridged version of the Hg19 genome, and exports the read coverage of SNPs found in all three panels. The pipeline can assess the concordance in genotypes generated from the independent CLC analysis to the report files from the UAS and the HID Genotyper Plugin for a given sample. The pipeline was tested using data collected from all three kits for five commercially available DNAs (9947A, 9948, HeLa, K562, Raji) and six forensic-type samples (blood, buccal swab and sexual fluid) obtained from both a male and female donor (total n, 11). This poster will outline the details of the developed pipeline, along with results from the initial testing of the pipeline using data from both the ForenSeq ^™ Signature Prep Kit (n, 11) and Precision ID Ancestry Panel (n, 29) and the Precision ID Identity Panel (n, 33).