Resolving the full spectrum of human genome variation using Linked-Reads

Abstract

Large-scale population analyses coupled with advances in technology have demonstrated that the human genome is more diverse than originally thought. To date, this diversity has largely been uncovered using short-read whole-genome sequencing. However, these short-read approaches fail to give a complete picture of a genome. They struggle to identify structural events, cannot access repetitive regions, and fail to resolve the human genome into haplotypes. Here, we describe an approach that retains long range information while maintaining the advantages of short reads. Starting from ∼1 ng of high molecular weight DNA, we produce barcoded short-read libraries. Novel informatic approaches allow for the barcoded short reads to be associated with their original long molecules producing a novel data type known as "Linked-Reads". This approach allows for simultaneous detection of small and large variants from a single library. In this manuscript, we show the advantages of Linked-Reads over standard short-read approaches for reference-based analysis. Linked-Reads allow mapping to 38 Mb of sequence not accessible to short reads, adding sequence in 423 difficult-to-sequence genes including disease-relevant genes STRC, SMN1, and SMN2. Both Linked-Read whole-genome and whole-exome sequencing identify complex structural variations, including balanced events and single exon deletions and duplications. Further, Linked-Reads extend the region of high-confidence calls by 68.9 Mb. The data presented here show that Linked-Reads provide a scalable approach for comprehensive genome analysis that is not possible using short reads alone.

A Comparison between Hi-C and 10X Genomics Linked Read Sequencing for Whole Genome Phasing in Hanwoo Cattle

“…First, the reads were aligned to the bovine reference genome (UMD 3.1) using the Lariat aligner; subsequently, SNPs and insertion-deletion polymorphisms (indels) were called using the GATK mode (version 3.8) [26] within Long Ranger pipeline. The identified SNPs (Single nucleotide polymorphisms) were phased using two methods: first, using the phasing method implemented in Long Ranger, which builds on the Markov chain Monte Carlo (MCMC) algorithm-based phasing method proposed by Bansal et al [27] by extending the probabilistic model to be robust to mixed fragments containing alleles from both haplotypes [28]. In the second method, the variants (SNPs) were phased using Hapcut2 [18] using default options.…”

Long-Read Sequencing Emerging in Medical Genetics

“…Similar technologies have recently been further optimized and commercialized by 10× Genomics Inc. (Weisenfeld et al, 2017). These technologies have already provided haplotype aware SR-WGS (Zheng et al, 2016) and improved SV calling from SR-WGS data (Nazaryan-Petersen et al, 2018; Marks et al, 2019). While synthetic long-reads leverage advantages of LRS, some short-read issues may persist, e.g., PCR-bias and intra-read complexity may not always be fully resolved.…”

An integrated Asian human SNV and indel benchmark established using multiple sequencing methods

“…to process stLFR reads for human germline variant calling and phasing 37 . Using a high accuracy pipeline to call variants of CCS long reads, which was also used in the previous study of human HG002/NA24385 with high precision and recall values of variant-calling 38 .…”