Massively parallel sequencing of 68 insertion/deletion markers identifies novel sequence variation for utility in human identity testing

Short tandem repeat (STR) loci are traditionally used by the forensic science community for kinship, missing persons, and human identity testing. These markers hold considerable value due to their size, ability to be multiplexed, and highly polymorphic nature. However, they are unable to provide phenotypic and biogeographic ancestry estimates and are too large for use in analysis of DNA from highly compromised substrates such as explosives or human remains. Small bi-allelic polymorphisms, such as insertions/deletions (INDELs), have been of considerable interest within the forensic science community for their utility in filling such gaps. These markers range in size from 2-6 base pairs, making them ideal for highly compromised sample types. Additionally, the ease of multiplexing large INDEL panels allows for comparable discrimination power when compared to STRs. Capillary electrophoresis is a current mainstay in the forensic DNA workflow, generating fluorescent signals to detect alleles separated by size. This method is limited by number of dyes simultaneously utilized, number of loci capable of multiplexing, sample throughput, and required amplicon size. Massively parallel sequencing (MPS) provides a solution to these limitations by targeting many loci across the genomes of multiple samples simultaneously with relatively high sequence coverage. Herein, we describe the utility of MPS, using the Nextera™ Rapid Capture Custom Enrichment Kit (Illumina, Inc., San Diego, CA), to sequence 68 INDELs in four major US population groups on the Illumina MiSeq™. We also define a novel application of the STR Allele Identification Tool: Razor (STRait Razor) to analyze INDEL sequences and capture adjacent sequence variation in the form of single nucleotide polymorphisms (SNPs). This application has enabled the discovery of unique allelic variants, which increase the discrimination power and decrease the single-locus and combined random match probabilities of four well-characterized INDELs. These findings suggest that more valuable INDELs for human identification may exist elsewhere in the genome. As such, it is recommended that these four markers be included in future INDEL multiplex panels for human identification due to their enhanced individualization potential.