Sequencing Long Amplicon Microsatellite Loci Using the Oxford Nanopore Technologies MinION Device
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Purpose: Forensic DNA typing exploits the high variability of short tandem repeat (STR) markers to differentiate individuals. Typical STR workflow consists of PCR followed by separation and detection via capillary electrophoresis (CE). Despite the power and reliability of current techniques, variations in nucleotide sequences are masked in size-based DNA profiles. Nanopore sequencing has the ability to provide long-read DNA sequence data that allows for accurate alignment and identification of single nucleotide polymorphisms (SNPs) both within and around STRs of interest. Detection of hidden sequence variation significantly expands the resolving power of STRs and aids in interpretation of more challenging samples. This project aimed to evaluate the applicability of nanopore sequencing to forensically-relevant autosomal and Y STR markers. Methods: Twenty unrelated individuals, two control DNAs, and three NIST-traceable standards were evaluated for 45 STR loci. Primer sets targeting 800 base pair amplicons containing the repeat and flanking regions were designed and multiplexed. Amplicons from each sample were barcoded and sequenced on the ONT MinION device using 1D read chemistry and SpotON flow cells (vR9.4.1). Raw reads were basecalled with MinKNOW’s real-time, local base caller. Sequence data were separated by barcode, merged by sample, and mapped to the human reference sequence (GRCh37/hg19) using NextGenMap-LR (NGMLR). Variations in motif composition and flanking SNPs were detected using Sniffles and visualized with Integrative Genomics Viewer (IGV). Consensus sequences and variant reports were compiled for each sample. Size-based allelic designations were predicted and compared to those generated via CE to evaluate concordance between the STR typing approaches. Results: High quality sequencing results were obtained for all STR loci interrogated. Concordance between size-based allelic designations revealed the reliability of nanopore sequencing data analyzed using this customized pipeline. Identification of flanking SNPs within the longer amplicons added variations that could differentiate alleles with the same motif structure, enriching discrimination potential. Conclusions: Complete nucleotide sequence data for repeat and flanking regions enhances the resolving power over that of current STR typing techniques. This project sets the foundation for future development of STRs for biomedically-relevant regions and potential forensic applications.