STR-Seq: a massively parallel microatellite sequencing and genotyping technology

Short tandem repeats (STR) are a type of genetic variation that has the fastest mutation rate. As a result, it is one of the most informative polymorphisms found in the genome, of which one the most common application is forensics DNA fingerprinting. We developed a new high-throughput sequencing technology to analyze microsatellite motifs with high sequencing coverage and accuracy for thousands of STRs in parallel for a given sample.The analysis of STRs/microsatellites with current next generation sequencing methods methods is limited by the following: i) Only the reads which encompass an entire STR locus are informative; ii) PCR amplification during library preparation can introduce artifactual “stutter” mutations that confound accurate genotyping; iii) limits on the ability to align and determine genotypes accurately from NGS data. Current target enrichment methods such as bait-hybridization are of limited utility given that these regions are often highly repetitive and structure and yield “off-target” sequence that obfuscates genotyping. Solving these issues, we developed short tandem repeat sequencing (STR-Seq), a novel sequencing technology that generates STR-spanning reads for thousands of microsatellites.This technology uses targeting “primer-probes”; selection of target genomic sequences is mediated by polymerase extension after the primer anneals to a specific target. We demonstrate simultaneous analysis of 2,375 STR loci in a single lane with individual reads covering the entire STR. We also developed a targeted fragmentation process to improve target selection and coverage. To eliminate polymerase stutter noise, sequencing libraries were prepared by an amplification-free method, thus each read corresponds to a single DNA molecule. Finally, we developed a novel bioinformatics pipeline for quantitation of STR motifs and associated variants in phase with the STR that eliminates alignment. We demonstrate that this technology has extraordinary resolution in differentiating mixed genotypes and has enormous potential in forensic genetics as well as other areas.