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

Area of Science:

• Genomics
• Molecular Biology
• Bioinformatics

Background:

• Microsatellites, or short tandem repeats (STRs), are crucial genetic markers with high allelic diversity.
• Current STR analysis methods, often relying on limited PCR amplicons, face throughput and accuracy limitations.
• Next-generation sequencing (NGS) offers potential for enhanced microsatellite analysis but requires optimized technologies.

Purpose of the Study:

• To introduce STR-Seq, a novel NGS technology for high-throughput and accurate microsatellite genotyping.
• To demonstrate STR-Seq's capability to analyze over 2,000 STRs in parallel.
• To showcase STR-Seq's enhanced performance in terms of throughput, accuracy, and haplotype information.

Main Methods:

• STR-Seq utilizes in vitro CRISPR-Cas9 targeted fragmentation for precise DNA molecule preparation covering complete microsatellite sequences.
• Features amplification-free library preparation for single-molecule sequencing without unique molecular barcodes.
• Employs STR-selective primers for massively parallel, targeted sequencing of extensive STR sets.

Main Results:

• STR-Seq achieves parallel analysis of over 2,000 microsatellites with high genotyping accuracy.
• The technology offers significantly higher throughput and improved accuracy compared to existing STR assays.
• STR-Seq provides a greater number of informative haplotypes, enhancing its utility in genetic studies.

Conclusions:

• STR-Seq represents a significant advancement in microsatellite analysis, enabling comprehensive and accurate genotyping.
• Its high throughput and accuracy make it suitable for diverse applications, including forensic science and population genetics.
• STR-Seq's ability to detect a 0.1% minor genome fraction in DNA mixtures opens new possibilities for mixture analysis.