An evaluation of the PacBio RS platform for sequencing and de novo assembly of a chloroplast genome

BMC Genomics

Research Article

Judson A Ward¹  Vladimir Stevanović²  Marco Ferrarini³  Duccio Cavalieri³  Lara Giongo³  Alessandro Cestaro³  Nada Šurbanovski³  Riccardo Velasco³  Daniel J Sargent³  Marco Moretto³  Roberto Viola³ 

[1] Driscoll’s, 151 Silliman Road, 95077-5045, Watsonville, CA, USA;Institute of Botany and Botanical Garden, Faculty of Biology, University of Belgrade, 11000, Takovska 43, Belgrade, Serbia;Research and Innovation Centre, Fondazione Edmund Mach, Via E. Mach 1, 38010, San Michele all’Adige, Italy;

关键词: Third-generation sequencing;    NGen;    Genomics;    Assembly;    Annotation;    Oxford nanopore;    Pacific BioSciences;    Roche 454;   

DOI  :  10.1186/1471-2164-14-670

 received in 2013-05-29, accepted in 2013-09-26,  发布年份 2013

来源: Springer

【 摘 要 】

BackgroundSecond generation sequencing has permitted detailed sequence characterisation at the whole genome level of a growing number of non-model organisms, but the data produced have short read-lengths and biased genome coverage leading to fragmented genome assemblies. The PacBio RS long-read sequencing platform offers the promise of increased read length and unbiased genome coverage and thus the potential to produce genome sequence data of a finished quality containing fewer gaps and longer contigs. However, these advantages come at a much greater cost per nucleotide and with a perceived increase in error-rate. In this investigation, we evaluated the performance of the PacBio RS sequencing platform through the sequencing and de novo assembly of the Potentilla micrantha chloroplast genome.ResultsFollowing error-correction, a total of 28,638 PacBio RS reads were recovered with a mean read length of 1,902 bp totalling 54,492,250 nucleotides and representing an average depth of coverage of 320× the chloroplast genome. The dataset covered the entire 154,959 bp of the chloroplast genome in a single contig (100% coverage) compared to seven contigs (90.59% coverage) recovered from an Illumina data, and revealed no bias in coverage of GC rich regions. Post-assembly the data were largely concordant with the Illumina data generated and allowed 187 ambiguities in the Illumina data to be resolved. The additional read length also permitted small differences in the two inverted repeat regions to be assigned unambiguously.ConclusionsThis is the first report to our knowledge of a chloroplast genome assembled de novo using PacBio sequence data. The PacBio RS data generated here were assembled into a single large contig spanning the P. micrantha chloroplast genome, with a higher degree of accuracy than an Illumina dataset generated at a much greater depth of coverage, due to longer read lengths and lower GC bias in the data. The results we present suggest PacBio data will be of immense utility for the development of genome sequence assemblies containing fewer unresolved gaps and ambiguities and a significantly smaller number of contigs than could be produced using short-read sequence data alone.

【 授权许可】

Unknown   
© Ferrarini et al.; licensee BioMed Central Ltd. 2013. This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.