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

Abstract

Short tandem repeats (STRs) are highly mutable genetic elements that often reside in regulatory and coding DNA. The cumulative evidence of genetic studies on individual STRs suggests that STR variation profoundly affects phenotype and contributes to trait heritability. Despite recent advances in sequencing technology, STR variation has remained largely inaccessible across many individuals compared to single nucleotide variation or copy number variation. STR genotyping with short-read sequence data is confounded by (1) the difficulty of uniquely mapping short, low-complexity reads; and (2) the high rate of STR amplification stutter. Here, we present MIPSTR, a robust, scalable, and affordable method that addresses these challenges. MIPSTR uses targeted capture of STR loci by single-molecule Molecular Inversion Probes (smMIPs) and a unique mapping strategy. Targeted capture and our mapping strategy resolve the first challenge; the use of single molecule information resolves the second challenge. Unlike previous methods, MIPSTR is capable of distinguishing technical error due to amplification stutter from somatic STR mutations. In proof-of-principle experiments, we use MIPSTR to determine germline STR genotypes for 102 STR loci with high accuracy across diverse populations of the plant A. thaliana. We show that putatively functional STRs may be identified by deviation from predicted STR variation and by association with quantitative phenotypes. Using DNA mixing experiments and a mutant deficient in DNA repair, we demonstrate that MIPSTR can detect low-frequency somatic STR variants. MIPSTR is applicable to any organism with a high-quality reference genome and is scalable to genotyping many thousands of STR loci in thousands of individuals.

Tandem repeat variation in human and great ape populations and its impact on gene expression divergence

“…Recent advances in sequencing technology, together with the development of computational tools, have finally made it affordable to accurately genotype up to millions of TRs at the genome-wide level and to circumvent much of the constraints that traditional repeat genotyping methods entail (Gymrek et al 2012;Guilmatre et al 2013;Highnam et al 2013;Duitama et al 2014;Willems et al 2014;Carlson et al 2015). Furthermore, quantitative deduction of gene expression divergence based on distance trees allowed reconstructing global evolutionary trends in more detail, as they are highly consistent with the known phylogeny.…”

“…In the future, studies of the repeat landscape will be facilitated not only by the use of longer reads but also by a thorough subsequent genotyping of a subset of repeats, using one of the recently developed methods that specifically target repeats (Guilmatre et al 2013;Duitama et al 2014;Carlson et al 2015). These methods rely on a presequencing enrichment step for repeats.…”

A generic, cost-effective, and scalable cell lineage analysis platform

“…CNVs pose a harder challenge, as they are in fact a duplication of the exact locus and therefore may have a closer MS signal and a similar flanking region. Utilization of unique molecular identifiers (UMIs) (Carlson et al 2015) may reduce the generated noise; however, due to the need for SC WGA, a background noise will remain. Future plans focus on improving the MS calling algorithm to input non-X and CNV loci, thus increasing the platform accuracy for both normal cells and specifically for cancer cells which exhibit substantial CNVs (Navin et al 2011).…”

“…Our future development is focused mainly on development and improving targeted enrichment one-pot multiplexed reactions, such as molecular inversion probes (MIP) or targeted capture (Leung et al 2016), in which per-probe cost is significantly lower due to multiplexed synthesis in advanced microarray technologies. MIPs were previously validated for low-scale MS analysis (Carlson et al 2015) and for a large number of targets (Li et al 2009), proving the feasibility of such protocols for massive MS analysis from SC WGA products. MIPs also allow for a precise targeting, rather than random shearing, which can split the MS sequence, as previously discussed.…”

0309 Systematic profiling of short tandem repeats in the cattle genome

“…In contrast to calling methods for single nucleotide polymorphisms (SNPs), insertions, deletions (indels) and copy number variations (CNVs), STRs are substantially more difficult to detect based on short reads produced by next generation sequencing (NGS); however, numerous methods have been recently developed to identify STR variants in human (Gymrek et al 2012;Tae et al 2013;Highnam et al 2013;Anvar et al 2014;Cao et al 2014;Fungtammasan et al 2015;Carlson et al 2015). These programs enable STR detection in NGS data by, among other features, carefully adjusting for high mismatch/indel levels in STRs during the mapping step (lobSTR) and guiding genotyping of STRs using informed error profiles (RepeatSeq) (Gymrek et al 2012;Highnam et al 2013).…”