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      SSPACE-LongRead: scaffolding bacterial draft genomes using long read sequence information

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          Abstract

          Background

          The recent introduction of the Pacific Biosciences RS single molecule sequencing technology has opened new doors to scaffolding genome assemblies in a cost-effective manner. The long read sequence information is promised to enhance the quality of incomplete and inaccurate draft assemblies constructed from Next Generation Sequencing (NGS) data.

          Results

          Here we propose a novel hybrid assembly methodology that aims to scaffold pre-assembled contigs in an iterative manner using PacBio RS long read information as a backbone. On a test set comprising six bacterial draft genomes, assembled using either a single Illumina MiSeq or Roche 454 library, we show that even a 50× coverage of uncorrected PacBio RS long reads is sufficient to drastically reduce the number of contigs. Comparisons to the AHA scaffolder indicate our strategy is better capable of producing (nearly) complete bacterial genomes.

          Conclusions

          The current work describes our SSPACE-LongRead software which is designed to upgrade incomplete draft genomes using single molecule sequences. We conclude that the recent advances of the PacBio sequencing technology and chemistry, in combination with the limited computational resources required to run our program, allow to scaffold genomes in a fast and reliable manner.

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          Most cited references7

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          GAGE: A critical evaluation of genome assemblies and assembly algorithms.

          New sequencing technology has dramatically altered the landscape of whole-genome sequencing, allowing scientists to initiate numerous projects to decode the genomes of previously unsequenced organisms. The lowest-cost technology can generate deep coverage of most species, including mammals, in just a few days. The sequence data generated by one of these projects consist of millions or billions of short DNA sequences (reads) that range from 50 to 150 nt in length. These sequences must then be assembled de novo before most genome analyses can begin. Unfortunately, genome assembly remains a very difficult problem, made more difficult by shorter reads and unreliable long-range linking information. In this study, we evaluated several of the leading de novo assembly algorithms on four different short-read data sets, all generated by Illumina sequencers. Our results describe the relative performance of the different assemblers as well as other significant differences in assembly difficulty that appear to be inherent in the genomes themselves. Three overarching conclusions are apparent: first, that data quality, rather than the assembler itself, has a dramatic effect on the quality of an assembled genome; second, that the degree of contiguity of an assembly varies enormously among different assemblers and different genomes; and third, that the correctness of an assembly also varies widely and is not well correlated with statistics on contiguity. To enable others to replicate our results, all of our data and methods are freely available, as are all assemblers used in this study.
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            Reducing assembly complexity of microbial genomes with single-molecule sequencing

            Background The short reads output by first- and second-generation DNA sequencing instruments cannot completely reconstruct microbial chromosomes. Therefore, most genomes have been left unfinished due to the significant resources required to manually close gaps in draft assemblies. Third-generation, single-molecule sequencing addresses this problem by greatly increasing sequencing read length, which simplifies the assembly problem. Results To measure the benefit of single-molecule sequencing on microbial genome assembly, we sequenced and assembled the genomes of six bacteria and analyzed the repeat complexity of 2,267 complete bacteria and archaea. Our results indicate that the majority of known bacterial and archaeal genomes can be assembled without gaps, at finished-grade quality, using a single PacBio RS sequencing library. These single-library assemblies are also more accurate than typical short-read assemblies and hybrid assemblies of short and long reads. Conclusions Automated assembly of long, single-molecule sequencing data reduces the cost of microbial finishing to $1,000 for most genomes, and future advances in this technology are expected to drive the cost lower. This is expected to increase the number of completed genomes, improve the quality of microbial genome databases, and enable high-fidelity, population-scale studies of pan-genomes and chromosomal organization.
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              Improving PacBio Long Read Accuracy by Short Read Alignment

              The recent development of third generation sequencing (TGS) generates much longer reads than second generation sequencing (SGS) and thus provides a chance to solve problems that are difficult to study through SGS alone. However, higher raw read error rates are an intrinsic drawback in most TGS technologies. Here we present a computational method, LSC, to perform error correction of TGS long reads (LR) by SGS short reads (SR). Aiming to reduce the error rate in homopolymer runs in the main TGS platform, the PacBio® RS, LSC applies a homopolymer compression (HC) transformation strategy to increase the sensitivity of SR-LR alignment without scarifying alignment accuracy. We applied LSC to 100,000 PacBio long reads from human brain cerebellum RNA-seq data and 64 million single-end 75 bp reads from human brain RNA-seq data. The results show LSC can correct PacBio long reads to reduce the error rate by more than 3 folds. The improved accuracy greatly benefits many downstream analyses, such as directional gene isoform detection in RNA-seq study. Compared with another hybrid correction tool, LSC can achieve over double the sensitivity and similar specificity.
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                Author and article information

                Contributors
                Journal
                BMC Bioinformatics
                BMC Bioinformatics
                BMC Bioinformatics
                BioMed Central
                1471-2105
                2014
                20 June 2014
                : 15
                : 211
                Affiliations
                [1 ]BaseClear B.V., Genome analysis and technology department, Einsteinweg 5, Leiden 2333 CC, The Netherlands
                Article
                1471-2105-15-211
                10.1186/1471-2105-15-211
                4076250
                24950923
                e5d47af5-e126-46cc-ac32-e8a8b7ca0948
                Copyright © 2014 Boetzer and Pirovano; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 28 April 2014
                : 4 June 2014
                Categories
                Methodology Article

                Bioinformatics & Computational biology
                de novo assembly,scaffolding,single molecule sequencing,pacific biosciences,genome finishing

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