NCM 2023 Houston: STRspy 2.0: unlocking the potential of long reads for forensic DNA profiles

Short tandem repeats (STR) are used in forensic studies to identify individuals. To harness the power of the MinION device, we developed STRspy 2.0 which can produce accurate forensic STR profiles from third-generation sequencing data. STRspy 2.0 is an improved version of STRspy, working as the same streamlined method capable of producing length and sequence-based allele designations from long-read data. Additionally, a new method has been implemented in STRspy 2.0, which automatically creates the autosome (auSTRs) and chrY-specific (Y-STRs) database based on the STRSeq NCBI BioProject. This expanded database (v2.0) represents the most comprehensive collection of validated sequence-based STR alleles available, enabling simultaneous profiling of auSTR (24 loci) and Y-STRs (26 loci) targeted in different next generation sequencing amplification kits. STRspy 2.0 was evaluated using four DNA controls (NIST A, B, C, and Promega 2800M) amplified and sequenced on the MinION device. The three male samples were profiled across both auSTRs and Y-STRs, whereas only auSTRs were analyzed for the female (NIST A). In auSTRs, normalized read counts were used to predict locus homozygosity or heterozygosity (reporting the top two alleles) using a threshold of 0.4. For Y-STR haplotype predictions, only the top allele was considered. The study reported a 100% concordance of each STR (auSTR and Y-STR) present in the control samples. STRspy 2.0, is the first and only third-generation sequencing platform-specific method that successfully profiles the entire autosomal STRs and Y-Chr amplified by a commercially available multiplex.