High-throughput multiplexed tandem repeat genotyping using targeted long-read sequencing

Abstract

3Tandem repeats (TRs) are highly prone to variation in copy numbers due to their repetitive and 2 4 unstable nature, which makes them a major source of genomic variation between individuals. 5However, population variation of TRs have not been widely explored due to the limitations of 2 6 existing tools, which are either low-throughput or restricted to a small subset of TRs. Here, we 2 7used SureSelect targeted sequencing approach combined with Nanopore sequencing to 2 8 overcome these limitations. We achieved an average of 3062-fold target enrichment on a panel 2 9 of 142 TR loci, generating an average of 97X sequence coverage on 7 samples utilizing 2 3 0 MinION flow-cells with 200ng of input DNA per sample. We identified a subset of 110 TR loci 3 1with length less than 2kb, and GC content greater than 25% for which we achieved an average 3 2 genotyping rate of 75% and increasing to 91% for the highest-coverage sample. Alleles 3 3 estimated from targeted long-read sequencing were concordant with gold standard PCR sizing 3 4 analysis and moreover highly correlated with alleles estimated from whole genome long-read 3 5sequencing. We demonstrate a targeted long-read sequencing approach that enables 3 6

STRsearch: a new pipeline for targeted profiling of short tandem repeats in massively parallel sequencing data

“…For the second challenge, a previous study [10] performed a comprehensive survey and then demonstrated that Stampy [12] was the most accurate with regards to mapping reads in STR regions, while Novoalign (http:// www.novocraft.com), Bowtie2 [13] and BWA [14] consumed much shorter running times. For the third challenge, research demonstrated that long-read sequencing technologies (such as Nanopore or PacBio) could potentially sequence through larger repeat loci with accuracy and effective cost [15]. Furthermore, short paired-end reads with sequence overlaps can be assembled to create longer sequences, and assembled reads will span the full length of the original DNA fragment.…”