생명화학공학과에서 아래와 같이 Young Investigators Seminar를 개최하오니 많은 참석 바랍니다.
- 아 래 -
◥ Speaker: Dr. GiWon Shin (Stanford School of Medicine)
◥ Date: 13 Apr, 2017 (Thu) 11:00
◥ Place: Seminar room 1 (#1101) @ W1-3 Bldg.
◥ Title: CRISPR-Cas9-targeted fragmentation and selective sequencing enable massively parallel microsatellite analysis
Microsatellites, also referred to as short tandem repeats (STRs) are multiallelic in terms of germline variation and have higher mutation rate than single nucleotide polymorphisms (SNPs). Because of the highly polymorphic nature, microsatellites are the most popular and versatile genetic marker with many applications including forensics DNA fingerprinting and population genetics. In addition, mutations in microsatellites are common in cancers that lack DNA mismatch repair mechanism, and the microsatellite instability is one of the key diagnostic marker to predict prognosis and treatment response. Despite their importance, however, the analysis of microsatellites is challenging regardless of the methods that is used. In particular, the analysis with current next generation sequencing methods is limited by the following: i) only the reads which encompass an entire microsatellite locus are informative; ii) PCR amplification during library preparation can introduce artificial “stutter” mutations that confound accurate genotyping; iii) microsatellites’ repetitive motifs complicate traditional alignment methods and lead to mapping errors. To address all of these issues, we developed STR sequencing (STR-Seq), a novel sequencing technology that generates STR-spanning reads for thousands of microsatellites.
In this study, we demonstrate simultaneous analysis of more than 2,000 microsatellites and their proximal SNPs. Particularly, STR-Seq uses paired-end sequencing reads to physically link microsatellite and SNP genotypes. Unlike other targeted sequencings, STR-Seq employs targeted in vitro CRISPR-Cas9 fragmentation, which provides extraordinary efficiency in capturing the informative DNA molecules that span the entire repetition as well as the flanking sequences. Target-selective primers enable massively parallel, targeted sequencing of large microsatellite sets. The technology eliminates PCR stutter noise because no post-capture amplification is required. Moreover, a novel bioinformatics pipeline eliminates artifacts from alignments and accurately quantifies microsatellite motifs and associated SNPs. Overall, STR-Seq has higher throughput, improved accuracy and provides a greater number of informative haplotypes compared to 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. This technology has extraordinary resolution in differentiating mixed genotypes and has enormous potential in forensics, population genetics, and cancer diagnosis.