Piercing the dark matter: bioinformatics of long-range sequencing and mapping

Abstract

Several new genomics technologies have become available that offer long-read sequencing or long-range mapping with higher throughput and higher resolution analysis than ever before. These long-range technologies are rapidly advancing the field with improved reference genomes, more comprehensive variant identification and more complete views of transcriptomes and epigenomes. However, they also require new bioinformatics approaches to take full advantage of their unique characteristics while overcoming their complex errors and modalities. Here, we discuss several of the most important applications of the new technologies, focusing on both the currently available bioinformatics tools and opportunities for future research.

How complete are “complete” genome assemblies?—An avian perspective

“…At last, it is important to keep in mind that birds are on the low end of repeat content among vertebrates (Sotero‐Caio, Platt, Suh, & Ray, ). Given that difficulty of genome assembly increases with repeat content (Sedlazeck, Lee, Darby, & Schatz, ), our case study on avian genomes might be a good starting point to illustrate that even sequencing genomes with relatively low repeat content is far from trivial and should not be labelled as “complete” yet. While avian genomes show a repeat density of only 4%–10% with a maximum of 22% in the downy woodpecker (Zhang et al., ), other vertebrates, invertebrates and plants often reach a repeat density of more than 50% (e.g., human genome 50%–69%, Cordaux & Batzer, ; de Koning, Gu, Castoe, Batzer, & Pollock, ; Locusta migratoria ~59%, Wu, Twort, Crowhurst, Newcomb, & Buckley, ; Fritillaria spp.…”

Can a New Microscopy Platform Help to Improve Clinical Outcomes?

“…With nanoscale resolution, the alignment of single-molecule maps becomes far more computationally straightforward than if labels are localized with multi-kb ambiguity, yielding reduced processing time and cost for assembling a consensus map [11]. Given its single-molecule sensitivity, 15bp accuracy, and no amplification requirement, our method is amenable to small sample sizes unlike single-molecule sequencing, which necessitates a clinically burdensome 10g of DNA [2]. …”

“…At significantly lower cost and testing time, optical mapping techniques (e.g.,Opgen andBioNano) can detect the locations of fluorescent probes along extended strands up to 250kb, and then compare the resulting map against a reference to determine the presence and character of indels, repeats or other variants [7]. Optical mapping has proved an important complement to sequencing that can essentially serve as a more detailed version of fluorescent in situ hybridization (FISH), but it suffers from limitations of its own: the template is only sparsely sampled, the nicking required to introduce fluorescent biomarkers can make heavily labeled portions prone to breakage, and most significantly, experimental and optical limitations typically render variants shorter than 10kb inaccessible to mapping [2]. …”

“…Single-molecule, third-generation sequencers (e.g.,PacBio and Oxford Nanopore) can produce reads of 10kb or more, enabling the sequencing of previously inaccessible portions of the genome despite stochastic error rates of about 15. Between these third-generation approaches, NGS improvements, and hybrid second- and third-generation assemblies, extremely accurate whole-genome or targeted sequencing efforts are now possible [2]. The effective translation of these techniques into a clinical setting is hindered by their time and cost, however: a whole-genome NGS run is, at best, currently priced at multiple thousands of USdollars, without considering computing or instrument costs, third-generation techniques are significantly more expensive, and both require several days and substantial computing power.…”

“…In the clinical sphere, NGS spurred the dream of precision medicine (PM) or personalized medicine, in which the genome of each patient (or pre-patient) is sequenced, and an optimal treatment is designed based on one's genetic code, lifestyle and environment [1]. Despite these gains and hopes, NGS is limited by its short-read approach and per-patient cost [2], and PM has yet to exhibit transformative reach, with only a small proportion of US patients currently receiving genome-informed therapy [3]. In order for PM to become a standard of care, there is an urgent need for fast, cheap and reliable genetic diagnostics to complement existing techniques.…”