Linked-Read sequencing resolves complex structural variants

Abstract

SummaryLarge genomic structural variants (>50bp) are important contributors to disease, yet they remain one of the most difficult types of variation to accurately ascertain, in part because they tend to cluster in duplicated and repetitive regions, but also because the various signals for these events can be challenging to detect with short reads. Clinically, aCGH and karyotype remain the most commonly used assays for genome-wide structural variant (SV) detection, though there is clear potential benefit to an NGS-based assay that accurately detects both SVs and single nucleotide variants. Linked-Read sequencing is a relatively simple, fast, and cost-effective method that is applicable to both genome and targeted assays. Linked-Reads are generated by performing haplotype-level dilution of long input DNA molecules into >1 million barcoded partitions, generating barcoded short reads within those partitions, and then performing short read sequencing in bulk. We performed 30x Linked-Read genome sequencing on a set of 23 samples with known balanced or unbalanced SVs. Twenty-seven of the 29 known events were detected and another event was called as a candidate. Sequence downsampling was performed on a subset to determine the lowest sequence depth required to detect variations. Copy-number variants can be called with as little as 1-2x sequencing depth (5-10Gb) while balanced events require on the order of 10x coverage for variant calls to be made, although specific signal is clearly present at 1-2x sequencing depth. In addition to detecting a full spectrum of variant types with a single test, Linked-Read sequencing provides base-level resolution of breakpoints, enabling complete resolution of even the most complex chromosomal rearrangements.

Genome sequencing in cytogenetics: Comparison of short‐read and linked‐read approaches for germline structural variant detection and characterization

“…The involvement of repetitive elements in SV is not uncommon (Chiang et al, 2012;Higgins et al, 2008;Schluth-Bolard et al, 2013) (Table S4) and is known to be an important limitation in SV detection using short-read sequencing (Elyanow et al, 2018). Thus, linked-read technology has already been described as a good alternative in SVs' detection in repetitive elements (Elyanow et al, 2018;Garcia et al, 2017).…”

Genome graphs detect human polymorphisms in active epigenomic state during influenza infection

“…This provides long-range positional information in regions of the human genome that cannot be reached by short reads alone. Linked reads have been used to genotype, 13 , 14 , 15 identify SVs, 16 , 17 , 18 detect MEI polymorphisms, 19 and assemble genomes. 20 , 21 …”

Methods to Improve Molecular Diagnosis in Genomic Cold Cases in Pediatric Neurology

“…Artificial long-read sequencing methods, also called synthetic long-read sequencing methods, work on the premise that they barcode long genomic DNA molecules prior to short-read sequencing, therefore retaining long-read information [ 181 , 182 ]. One such technology is 10× Genomics linked-read sequencing, which leverages microfluidics to partition and barcode high molecular weight DNA prior to short-read sequencing [ 181 , 182 ]. The second relatively newer option is the single tube long fragment reads (stLFR) method [ 183 ], that allows the barcoding of subfragments of long genomic DNA molecules via microbeads.…”