TY - JOUR
T1 - CRISPR-Cas9-targeted fragmentation and selective sequencing enable massively parallel microsatellite analysis
AU - Shin, Giwon
AU - Grimes, Susan M.
AU - Lee, Hojoon
AU - Lau, Billy T.
AU - Xia, Li C.
AU - Ji, Hanlee P.
N1 - Funding Information:
This work was supported by the National Institute of Justice grant 2013-DN-BX-K010. We acknowledge Erik Hopmans for technical advice regarding the amplification-free preparation step, and Matthew Kubit for technical assistance.
Publisher Copyright:
© 2017 The Author(s).
PY - 2017/2/7
Y1 - 2017/2/7
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85011708054&partnerID=8YFLogxK
U2 - 10.1038/ncomms14291
DO - 10.1038/ncomms14291
M3 - Article
C2 - 28169275
AN - SCOPUS:85011708054
SN - 2041-1723
VL - 8
JO - Nature communications
JF - Nature communications
M1 - 14291
ER -