Resolving the Full Spectrum of Human Genome Variation using Linked-Reads

Abstract

Large-scale population based 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, standard short-read approaches, used primarily due to accuracy, throughput and costs, fail to give a complete picture of a genome. They struggle to identify large, balanced structural events, cannot access repetitive regions of the genome and fail to resolve the human genome into its two haplotypes. Here we describe an approach that retains long range information while harnessing the advantages of short reads. Starting from only~ ng of DNA, we produce barcoded short read libraries. The use of novel informatic approaches allows for the barcoded short reads to be associated with the long molecules of origin producing a novel datatype known as 'Linked-Reads'. This approach allows for simultaneous detection of small and large variants from a single Linked-Read library. We have previously demonstrated the utility of whole genome Linked-Reads (lrWGS) for performing diploid, de novo assembly of individual genomes (Weisenfeld et al. ). In this manuscript, weshow the advantages of Linked-Reads over standard short read approaches for reference based analysis. We demonstrate the ability of Linked-Reads to reconstruct megabase scale haplotypes and to recover parts of the genome that are typically inaccessible to short reads, including phenotypically important genes such as STRC, SMN and SMN . We demonstrate the ability of both lrWGS and Linked-Read Whole Exome Sequencing (lrWES) to identify complex structural variations, including balanced events, single exon deletions, and single exon duplications. The data presented here show that Linked-Reads provide a scalable approach for comprehensive genome analysis that is not possible using short reads alone.

Haplotype-resolved and integrated genome analysis of the cancer cell line HepG2

“…The 10X Genomics linked-reads dataset was used to phase haplotypes as well as to identify, assemble, and phase primarily large (>30kb) SVs that include translocations (Spies et al 2017;Zheng et al 2016;Marks et al 2018). The 3kb-mate pair was used to validate the large SVs resolved from using the linked-read sequencing data and also to identify additional SVs, mostly in the medium size-range (1kb-100kb).…”

“…SVs can also be assigned to specific haplotypes if the breakpoint-supporting reads contain phased SNVs or indels Marks et al 2018). Using this approach (implemented by the Long Ranger software from 10X Genomics), we identified 97 large SVs >30 kb (99% phased) (Dataset 3) and 3,473 deletions between 50 bp and 30 kb (78% phased) (Dataset 4).…”

“…We phased the heterozygous SNVs and indels in the HepG2 genome by performing 10X Genomics Chromium linked-read library preparation and sequencing Marks et al 2018). Post sequencing quality control analysis show that 1.49 ng or approximately 447 genomic equivalents of high molecular weight (HMW) genomic DNA fragments (mean=68 kb, 96.1% > 20 kb, 22.0% >100 kb) were partitioned .…”

“…Paired-end linked-reads (median insert size 396 bp, duplication rate 7.68%, Q30 Read1 78.7%, Q30 Read2 63.1%) were aligned to hg19 (alignment rate 90.4%, mean coverage 67.0x, zero coverage 0.117%) and analyzed using the Long Ranger Software (version 2.1.5) from 10x Genomics Marks et al 2018) (Pleasanton, CA, USA). Segmental duplications, reference gaps, unplaced contigs, regions with assembly issues, and highly polymorphic sites (http://cf.10xgenomics.com/supp/genome/hg19/sv_blacklist.bed, http://cf.10xgenomics.com/supp/genome/hg19/segdups.bedpe) were excluded from the analysis.…”

“…HepG2 genomic DNA sample (~35 kb-80 kb) was size selected on the BluePippin instrument (Sage Science, Beverly, MA, USA) using the manufacturer's U1 Maker 30 kb High Pass protocol and then diluted to 1 ng/µl and used as input for the Chromium reagent delivery system Marks et al 2018) from 10x Genomics (Pleasanton, CA, USA) where HMW DNA fragments are partitioned into >1 million droplets, uniquely barcoded (16 bp) within each droplet, and subjected to random priming and isothermal amplification following standard manufacturer's protocol. Afterwards, the emulsion was broken, and the barcoded DNA molecules were released and converted to a Chromium linked-read library in which each library molecule retains its "HMW fragment barcode".…”