Sequence-based short tandem repeat (STR) analysis for forensic human identity testing enables detection of intra-allelic sequence variation beyond length-based resolution, increasing the discriminatory power of alleles for genotyping and statistical determinations. However, increased resolution in the form of unique alleles introduces complexity in locus characterizations due to potential repeat motif variation and sequence polymorphisms within both repeat and flanking regions, including single nucleotide polymorphisms (SNPs) and insertion/deletion events, necessitating robust interpretation and nomenclature frameworks. Herein, we have re-evaluated existing public STR data to re-characterize a subset of loci within a 73-plex panel using the published string sequence information to evaluate overall forensic utility, and to assess whether increased sequence-level variation at non-CODIS loci may improve discriminatory performance when compared to sequence-based CODIS STR markers alone.

Using sequence-based allele data derived from 440 individuals comprising three population groups (African American, Caucasian, and Hispanic) in the United States, a re-characterization and alignment was conducted to enumerate the totality of complexity within each locus, as defined by the allele spread and variability, and qualitative measures were employed to assess repeat region integrity, possible microvariant structure (if any), and the totality of SNP and/or indel variability in the amplicon. Loci were further evaluated for atypical sequence patterns suggestive of alignment inconsistencies or sequence assembly artifacts.

Consistent with previous studies, our preliminary observations demonstrate substantial heterogeneity in sequence-based allele diversity across loci, with allele counts ranging from fewer than 20 to potentially tens to hundreds of distinct sequence-based alleles per locus. Highly polymorphic loci, including D3S2406 and D2S1360, exhibit extensive sequence heterogeneity driven by complex repeat architectures and SNP-defined isoalleles (i.e., alleles of identical length that differ in sequence composition), while other loci exhibit lower sequence diversity Some loci have presented unique patterns of SNP diversity, demonstrating how linked SNPs may inherently impact STR patterns of diversity. 

Forensically, these findings highlight both the increased discriminatory potential of sequence-based STRs and the challenges associated with interpretation, standardization, and locus selection within new multiplex designs.

Authors

• Shivani Gami (Institute of Forensic Science, Department of Anthropology, University of Toronto Mississauga, Mississauga, Ontario, Canada ; School of Natural Sciences, Laurentian University, Canada)
• Krista Currie (School of Natural Sciences, Laurentian University, Canada)
• Nicole Novroski (IFS-DA-UTM, Ontario, Canada ;SNS-LU, Sudbury, Canada; DMI&G-SBTS-UNTHSC, Texas, USA; Center for Human Identification, University of North Texas Health Science Center at Fort Worth, United States of America)