The Development and Validation of Massively Parallel Sequencing Marker Panels for use within a New Zealand Population in Forensic Science

Abstract

The MiSeq FGx™ Forensic Genomics Solution is a massively parallel sequencing workflow that sequences 231 forensic DNA markers, including autosomal STRs (aSTRs), Y chromosome STRs, and ancestry and phenotype informative SNPs. This research aims to determine whether the method is robust and reliable for application in the New Zealand criminal justice sector. The New Zealand population is diverse, comprising indigenous Māori and more recent immigrants. Much of the publicly available allele frequency information and biogeographical ancestry and phenotype prediction tools lack data representing Oceania and the New Zealand population. The research contributes to internal validation studies of the MiSeq FGx™ Forensic Genomics Solution. The workflow was very sensitive; 10 pg/μL input DNA generated accurate STR results. The SNP loci required at least 20 pg/μL. The workflow was compatible with a laser microdissection cell collection method. Complete aSTR profiles could be obtained from 50 epithelial cells and 100 spermatozoa. Repeatability and reproducibility studies found occasional variability in locus calling rates between library preparation batches. 550 DNA samples from the New Zealand population were sequenced. An STR and SNP allele frequency database was created. This study is the first to report the New Zealand sequence-based allele diversity in these STR. The sequence-based alleles improved the aSTR heterozygosity and random match probabilities. The ForenSeq™ Universal Analysis Software was unsuitable for predicting phenotype and ancestry in the New Zealand population. Eye and hair colour can be accurately predicted using the HIrisPlex webtool. The Snipper Suite ancestry tool, Structure and Frog-kb, can be used to predict 90% of the samples' ancestry accurately. By combining the results of multiple tools, we improve the overall picture generated about an individual and avoid the limitations of each tool. Ancestry, eye and hair colour could be accurately predicted in Oceanian samples. The ancestry and phenotype predictions can generate intelligence information for a criminal investigation. The ForenSeq™ STR profile can then be compared to a suspect’s reference sample. This research demonstrates that the MiSeq FGx™ Forensic Genomics Solution and ancestry and phenotype prediction is fit for purpose. This research will support the implementation of the method into forensic casework.