Repeat Detector: versatile sizing of expanded tandem repeats and identification of interrupted alleles from targeted DNA sequencing

Abstract

Targeted DNA sequencing approaches will improve how the size of short tandem repeats is measured for diagnostic tests and preclinical studies. The expansion of these sequences causes dozens of disorders, with longer tracts generally leading to a more severe disease. Interrupted alleles are sometimes present within repeats and can alter disease manifestation. Determining repeat size mosaicism and identifying interruptions in targeted sequencing datasets remains a major challenge. This is in part because standard alignment tools are ill-suited for repetitive and unstable sequences. To address this, we have developed Repeat Detector (RD), a deterministic profile weighting algorithm for counting repeats in targeted sequencing data. We tested RD using blood-derived DNA samples from Huntington’s disease and Fuchs endothelial corneal dystrophy patients sequenced using either Illumina MiSeq or Pacific Biosciences single-molecule, real-time sequencing platforms. RD was highly accurate in determining repeat sizes of 609 blood-derived samples from Huntington’s disease individuals and did not require prior knowledge of the flanking sequences. Furthermore, RD can be used to identify alleles with interruptions and provide a measure of repeat instability within an individual. RD is therefore highly versatile and may find applications in the diagnosis of expanded repeat disorders and in the development of novel therapies.

Cas9 nickase-mediated contraction of CAG/CTG repeats at multiple disease loci

“…2e), which is likely due to different cell types having different rates of expansion. Together with the results in HD iPSC-derived cortical neuron cultures, this observation suggests that SMRT sequencing is robustly detecting both contractions and expansions, in line with previous work 48,54 .…”

“…2a-d). We first sorted unaligned circular consensus sequences (CCSs) with 6 to 20 repeats as determined by Repeat Detector 48 . Then we looked for rearrangements using NGMLR alignment, Picard sort and samtools indexing, followed by Sniffles2 49 (see methods).…”

“…Expanded repeats were sequenced as before using SMRT HiFi sequencing 48 . In brief, primers oVIN-1321 and oVIN-3490 were used to amplify the human HTT locus, including the CAG repeat from iPSC-derived neurons and R6/1 mouse genomes.…”