CRISPR-Cas9-Targeted Fragmentation and Selective Sequencing Enable Massively Parallel Microsatellite Analysis

• NCJ Number: 250611
• Author(s): GiWon Shin, Susan M. Grimes, HoJoon Lee, Billy T. Lau, Li C. Xia, Hanlee P. Ji
• Date Published: February 2017
• Length: 13 pages
• Agencies: NIJ-Sponsored
• Award: Highly Parallel Analysis of Complex Genetic Mixtures

Annotation

This article present STR-Seq, a parallel sequencing approach that generates short tandem repeats (STRs)/microsatellite-spanning sequences, that incorporates CRISPR–Cas9 to increase the number of sequenced molecules in a targeted DNA fragment.

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. In their research, the authors 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. (Publisher abstract modified)