CRISPR-Cas9-targeted fragmentation and selective sequencing enable massively parallel microsatellite analysis.
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.
Division of Oncology, Department of Medicine, Stanford University School of Medicine, CCSR 1115, 269 Campus Drive, Stanford, California 94305, USA.
GiWon
G
Shin
Stanford Genome Technology Center, Stanford University, 3165 Porter Drive, Palo Alto, California 94304, USA.
Susan M
SM
Grimes
Division of Oncology, Department of Medicine, Stanford University School of Medicine, CCSR 1115, 269 Campus Drive, Stanford, California 94305, USA.
HoJoon
H
Lee
Stanford Genome Technology Center, Stanford University, 3165 Porter Drive, Palo Alto, California 94304, USA.
Billy T
BT
Lau
Division of Oncology, Department of Medicine, Stanford University School of Medicine, CCSR 1115, 269 Campus Drive, Stanford, California 94305, USA.
Li C
LC
Xia
Division of Oncology, Department of Medicine, Stanford University School of Medicine, CCSR 1115, 269 Campus Drive, Stanford, California 94305, USA.|Stanford Genome Technology Center, Stanford University, 3165 Porter Drive, Palo Alto, California 94304, USA.
Hanlee P
HP
Ji
D000483:Alleles; D064113:CRISPR-Cas Systems; D015698:Genomic Library; D006239:Haplotypes / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D016045:Human Genome Project; D006801:Humans; D018895:Microsatellite Repeats / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA / Q000379:methods*