CRISPR–Cas9-targeted fragmentation and selective sequencing enable massively parallel microsatellite analysis

GiWon Shin, Susan M. Grimes, HoJoon Lee, Billy T. Lau, Li C. Xia and Hanlee P. Ji ()
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GiWon Shin: Stanford University School of Medicine
Susan M. Grimes: Stanford Genome Technology Center, Stanford University
HoJoon Lee: Stanford University School of Medicine
Billy T. Lau: Stanford Genome Technology Center, Stanford University
Li C. Xia: Stanford University School of Medicine
Hanlee P. Ji: Stanford University School of Medicine

Nature Communications, 2017, vol. 8, issue 1, 1-13

Abstract: 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.

Date: 2017
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DOI: 10.1038/ncomms14291

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