Abstract
Microsatellites are multi-allelic and composed of short tandem repeats (STRs) with individual motifs composed of mononucleotides, dinucleotides or higher including hexamers. Next-generation sequencing approaches and other STR assays rely on a limited number of PCR amplicons, typically in the tens. Here, we demonstrate STR-Seq, a next-generation sequencing technology that analyses over 2,000 STRs in parallel, and provides the accurate genotyping of microsatellites. STR-Seq employs in vitro CRISPR–Cas9-targeted fragmentation to produce specific DNA molecules covering the complete microsatellite sequence. Amplification-free library preparation provides single molecule sequences without unique molecular barcodes. STR-selective primers enable massively parallel, targeted sequencing of large STR sets. Overall, STR-Seq has higher throughput, improved accuracy and provides a greater number of informative haplotypes compared with other microsatellite analysis approaches. With these new features, STR-Seq can identify a 0.1% minor genome fraction in a DNA mixture composed of different, unrelated samples.
Highlights
Microsatellites are multi-allelic and composed of short tandem repeats (STRs) with individual motifs composed of mononucleotides, dinucleotides or higher including hexamers
The 13 STRs used for the Combined DNA Index System (CODIS), an important set of microsatellites used in forensic genetics, are all tetranucelotide repeats
We demonstrate that STR-Seq is highly accurate using a ground truth set of previously genotyped samples, has high efficiency in assay design and genotyping when compared to other methods such as capillary electrophoresis (CE), provides phased STR–single nucleotide polymorphisms (SNPs) haplotypes and can resolve individual-specific haplotypes at minor allelic fractions of 0.1% in genetic mixtures
Summary
Microsatellites are multi-allelic and composed of short tandem repeats (STRs) with individual motifs composed of mononucleotides, dinucleotides or higher including hexamers. STR-Seq has higher throughput, improved accuracy and provides a greater number of informative haplotypes compared with other microsatellite analysis approaches. With these new features, STR-Seq can identify a 0.1% minor genome fraction in a DNA mixture composed of different, unrelated samples. Microsatellites, otherwise called short tandem repeats (STRs), have multiple alleles that are defined by variation in the number of motif unit repeats. Given their multi-allelic characteristics, they have greater heterozygosity than single nucleotide polymorphisms (SNPs)[1]. In forensic genetic analysis, it is nearly impossible to distinguish a specific individual DNA sample amongst multiple contributors, when a specific component exists at a low ratio
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