Detection of intra-allelic sequence variants within Short Tandem Repeats using the Illumina ForenSeq DNA Signature Prep Kit and MiSeq Desktop Sequencer

Objective: The goal of this study was to identify and characterize intra-allelic sequence variants that exist inside the short tandem repeat (STR) sequences captured by the ForenSeq DNA Signature Prep Kit panel for a Hispanic sample population (n=150). Materials and methods: DNA from 150 Hispanic reference samples were used in this study. All samples were anonymized and collected according to IRB-approved protocols. The ForenSeq DNA Signature Prep Kit was used to barcode and generate sequence libraries so all 150 samples could be multiplexed. Sequencing was conducted on the MiSeq Desktop Sequencer, and data analysis was carried out using the ForenSeq Universal Analysis Software (UAS) and in-house Excel-based workbooks. Results: All 27 autosomal STRs, 24 Y-STRs and 7 X-STRs included in the ForenSeq DNA Signature Prep kit were evaluated. A total of 31 loci were found to have sequence variation in at least one nominal allele. Within that group, 21 loci exhibited variation at greater than or equal to 3 or more nominal alleles. Although most alleles had only two varying sequences per nominal allele, some loci demonstrated a larger amount of variation within their nominal alleles, increasing the diversity and discrimination power at those sites. Conclusions: These results demonstrated that intra-STR allele sequence variation can be detected and characterized for the STRs captured with the ForenSeq Signature DNA Prep kit. Furthermore, the characterization of these sequence variants in STRs offers great potential as a means of further individualizing DNA samples from one another, opportunities for improved mixture de-convolution, and kinship analyses, such as identifying victims from mass disasters.