Discovery and characterization of highly polymorphic ultra-short STRs for human identification via shotgun sequencing

Shotgun sequencing (SGS) enables simultaneous interrogation of a broad range of loci across the human genome, even from low-template and highly degraded DNA samples. While human identification traditionally relies on short tandem repeats (STRs) due to their high polymorphism, standard forensic STRs (100-450 bp) are poorly suited for the short read (~150 bp) constraint of SGS. The purpose of this study was to evaluate the analysis limitations of standard forensic STRs in SGS data and to identify a novel panel of STRs optimised for short-read genomic data. First, we benchmarked four STR genotyping software tools (STRait Razor, GangSTR, STRinNGS, and HipSTR) by analysing 53 standard forensic STRs in SGS data. HipSTR showed the best performance but achieved only a call rate of 64.5% and an accuracy of 83.8%, and its performance was strongly affected by STR allele length and read depth. To overcome these constraints, we screened the population-wide 1000 Genomes Project dataset and identified a panel of 265 autosomal ultra-short (< 50 bp) STRs with an effective number of alleles (Ae) ranging from 3.0 to 7.5. As few as seven of these loci were sufficient to achieve a Mean Match Probability (MMP) below 1 x 10-6. To validate these findings, we developed a custom PCR-based amplicon sequencing panel targeting 97 of the most polymorphic ultra-short STRs and evaluated these in 41 blood samples from Danish individuals. The polymorphic nature of the selected loci was confirmed (Ae ranged from 2.4 to 7.2). Our results furthermore demonstrated high concordance between the amplicon panel and SGS-derived genotypes, which substantiates that these ultra-short STRs provide a robust and highly polymorphic alternative for human identification in SGS data.