Rapid Multiplexed Genotyping of Simple Tandem Repeats using Capture and High-Throughput Sequencing

Abstract

Although simple tandem repeats (STRs) comprise ~2% of the human genome and represent an important source of polymorphism, this class of variation remains understudied. We have developed a cost-effective strategy for performing targeted enrichment of STR regions that utilizes capture probes targeting the flanking sequences of STR loci, enabling specific capture of DNA fragments containing STRs for subsequent high-throughput sequencing. Utilizing a capture design targeting 6,243 STR loci <94bp and multiplexing eight individuals in a single Illumina HiSeq2000 sequencing lane we were able to call genotypes in at least one individual for 67.5% of the targeted STRs. We observed a strong relationship between (G+C) content and genotyping rate. STRs with moderate (G+C) content were recovered with >90% success rate, while only 12% of STRs with ≥80% (G+C) were genotyped in our assay. Analysis of a parent-offspring trio, complete hydatidiform mole samples, repeat analyses of the same individual, and Sanger sequencing-based validation indicated genotyping error rates between 7.6–12.4%. The majority of such errors were a single repeat unit at mono- or dinucleotide repeats. Altogether, our STR capture assay represents a cost-effective method that enables multiplexed genotyping of thousands of STR loci suitable for large scale population studies.

MIPSTR: a method for multiplex genotyping of germline and somatic STR variation across many individuals

“…We observed ratios as close as 49.9/50.1 (761 tag-defined read groups/764) and as far as 36/74 (40/71) for the three ascertained duplicate loci across three Col-0 siblings. These results suggest that with sufficient coverage, MIPSTR can readily identify heterozygous and somatic STR variants, which have been largely inaccessible by previous analytical or empirical methods (Gymrek et al 2012;Guilmatre et al 2013;Highnam et al 2013;Duitama et al 2014;Willems et al 2014). …”

“…To realize this potential, several recent efforts, relying on either analytical or experimental innovation, have made progress toward the ascertainment of accurate STR genotypes on a population scale (Gymrek et al 2012;Guilmatre et al 2013;Highnam et al 2013;Cao et al 2014;Duitama et al 2014). However, the STR-specific challenges for accurate genotyping-mappability and high amplification stutter-were only partially addressed.…”

“…Previous studies relied on amplification of haploid or homozygous genomes to estimate technical error for STR-containing sequencing reads (Gymrek et al 2012;Guilmatre et al 2013;Highnam et al 2013); this approach is confounded by somatic variation and high STR mutation rates. MIPSTR offers an experimental avenue for empirically ascertaining technical error for many types of STRs.…”

“…Such strategies were recently applied to the human genome, using STRtargeted microarray capture or RNA probe capture prior to sequencing (Guilmatre et al 2013;Duitama et al 2014). However, these STR capture methods produced only limited enrichment for STR-containing reads with flanking sequence, i.e., 2.2% of mappable reads (Guilmatre et al 2013) and 25% of mappable reads (Duitama et al 2014), and only marginally improved STR coverage for unit number calls (Table 1).…”

“…More than 2000 STR loci are accessible by MIPSTR in A. thaliana, and many more accessible STR loci exist in humans (Molla et al 2009;Guilmatre et al 2013;Duitama et al 2014;Willems et al 2014). Our preliminary results, considering only a fraction of the accessible A. thaliana STR loci, highlight the promise of STRs to contribute to the variation and heritability of quantitative traits (Press et al 2014).…”