Dataset Information

REXTAL: Regional Extension of Assemblies Using Linked-Reads.

ABSTRACT: It is currently impossible to get complete de-novo assembly of segmentally duplicated genome regions using genome-wide short-read datasets. Here, we devise a new computational method called Regional Extension of Assemblies Using Linked-Reads (REXTAL) for improved region-specific assembly of segmental duplication-containing DNA, leveraging genomic short-read datasets generated from large DNA molecules partitioned and barcoded using the "Gel Bead in Emulsion" (GEM) microfluidic method (Zheng et al., 2016). We show that using REXTAL, it is possible to extend assembly of single-copy diploid DNA into adjacent, otherwise inaccessible subtelomere segmental duplication regions and other subtelomeric gap regions. Moreover, REXTAL is computationally more efficient for the directed assembly of such regions from multiple genomes (e.g., for the comparison of structural variation) than genome-wide assembly approaches.

SUBMITTER: Islam T

PROVIDER: S-EPMC6996091 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Publications

REXTAL: Regional Extension of Assemblies Using Linked-Reads.

Islam Tunazzina T Ranjan Desh D Young Eleanor E Xiao Ming M Zubair Mohammad M Riethman Harold H

Bioinformatics research and applications : 14th International Symposium, ISBRA 2018, Beijing, China, June 8-11, 2018, Proceedings. ISBRA (Conference) (14th : 2018 : Beijing, China) 20180601

It is currently impossible to get complete de-novo assembly of segmentally duplicated genome regions using genome-wide short-read datasets. Here, we devise a new computational method called Regional Extension of Assemblies Using Linked-Reads (REXTAL) for improved region-specific assembly of segmental duplication-containing DNA, leveraging genomic short-read datasets generated from large DNA molecules partitioned and barcoded using the "Gel Bead in Emulsion" (GEM) microfluidic method (Zheng et al ...[more]

PMID: 32016171

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Project description:BACKGROUND:Genome sequencing yields the sequence of many short snippets of DNA (reads) from a genome. Genome assembly attempts to reconstruct the original genome from which these reads were derived. This task is difficult due to gaps and errors in the sequencing data, repetitive sequence in the underlying genome, and heterozygosity. As a result, assembly errors are common. In the absence of a reference genome, these misassemblies may be identified by comparing the sequencing data to the assembly and looking for discrepancies between the two. Once identified, these misassemblies may be corrected, improving the quality of the assembled sequence. Although tools exist to identify and correct misassemblies using Illumina paired-end and mate-pair sequencing, no such tool yet exists that makes use of the long distance information of the large molecules provided by linked reads, such as those offered by the 10x Genomics Chromium platform. We have developed the tool Tigmint to address this gap. RESULTS:To demonstrate the effectiveness of Tigmint, we applied it to assemblies of a human genome using short reads assembled with ABySS 2.0 and other assemblers. Tigmint reduced the number of misassemblies identified by QUAST in the ABySS assembly by 216 (27%). While scaffolding with ARCS alone more than doubled the scaffold NGA50 of the assembly from 3 to 8 Mbp, the combination of Tigmint and ARCS improved the scaffold NGA50 of the assembly over five-fold to 16.4 Mbp. This notable improvement in contiguity highlights the utility of assembly correction in refining assemblies. We demonstrate the utility of Tigmint in correcting the assemblies of multiple tools, as well as in using Chromium reads to correct and scaffold assemblies of long single-molecule sequencing. CONCLUSIONS:Scaffolding an assembly that has been corrected with Tigmint yields a final assembly that is both more correct and substantially more contiguous than an assembly that has not been corrected. Using single-molecule sequencing in combination with linked reads enables a genome sequence assembly that achieves both a high sequence contiguity as well as high scaffold contiguity, a feat not currently achievable with either technology alone.

Project description:Because of the short read length of high throughput sequencing data, assembly errors are introduced in genome assembly, which may have adverse impact to the downstream data analysis. Several tools have been developed to eliminate these errors by either 1) comparing the assembled sequences with some similar reference genome, or 2) analyzing paired-end reads aligned to the assembled sequences and determining inconsistent features alone mis-assembled sequences. However, the former approach cannot distinguish real structural variations between the target genome and the reference genome while the latter approach could have many false positive detections (correctly assembled sequence being considered as mis-assembled sequence).We present misFinder, a tool that aims to identify the assembly errors with high accuracy in an unbiased way and correct these errors at their mis-assembled positions to improve the assembly accuracy for downstream analysis. It combines the information of reference (or close related reference) genome and aligned paired-end reads to the assembled sequence. Assembly errors and correct assemblies corresponding to structural variations can be detected by comparing the genome reference and assembled sequence. Different types of assembly errors can then be distinguished from the mis-assembled sequence by analyzing the aligned paired-end reads using multiple features derived from coverage and consistence of insert distance to obtain high confident error calls.We tested the performance of misFinder on both simulated and real paired-end reads data, and misFinder gave accurate error calls with only very few miscalls. And, we further compared misFinder with QUAST and REAPR. misFinder outperformed QUAST and REAPR by 1) identified more true positive mis-assemblies with very few false positives and false negatives, and 2) distinguished the correct assemblies corresponding to structural variations from mis-assembled sequence. misFinder can be freely downloaded from https://github.com/hitbio/misFinder.

Project description:BackgroundWhole genome sequencing of cultured pathogens is the state of the art public health response for the bioinformatic source tracking of illness outbreaks. Quasimetagenomics can substantially reduce the amount of culturing needed before a high quality genome can be recovered. Highly accurate short read data is analyzed for single nucleotide polymorphisms and multi-locus sequence types to differentiate strains but cannot span many genomic repeats, resulting in highly fragmented assemblies. Long reads can span repeats, resulting in much more contiguous assemblies, but have lower accuracy than short reads.ResultsWe evaluated the accuracy of Listeria monocytogenes assemblies from enrichments (quasimetagenomes) of naturally-contaminated ice cream using long read (Oxford Nanopore) and short read (Illumina) sequencing data. Accuracy of ten assembly approaches, over a range of sequencing depths, was evaluated by comparing sequence similarity of genes in assemblies to a complete reference genome. Long read assemblies reconstructed a circularized genome as well as a 71 kbp plasmid after 24 h of enrichment; however, high error rates prevented high fidelity gene assembly, even at 150X depth of coverage. Short read assemblies accurately reconstructed the core genes after 28 h of enrichment but produced highly fragmented genomes. Hybrid approaches demonstrated promising results but had biases based upon the initial assembly strategy. Short read assemblies scaffolded with long reads accurately assembled the core genes after just 24 h of enrichment, but were highly fragmented. Long read assemblies polished with short reads reconstructed a circularized genome and plasmid and assembled all the genes after 24 h enrichment but with less fidelity for the core genes than the short read assemblies.ConclusionThe integration of long and short read sequencing of quasimetagenomes expedited the reconstruction of a high quality pathogen genome compared to either platform alone. A new and more complete level of information about genome structure, gene order and mobile elements can be added to the public health response by incorporating long read analyses with the standard short read WGS outbreak response.

Dataset Information

REXTAL: Regional Extension of Assemblies Using Linked-Reads.

Publications

REXTAL: Regional Extension of Assemblies Using Linked-Reads.

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