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

Abstract

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.

Organelle DNA rearrangement mapping reveals U-turn-like inversions as a major source of genomic instability in Arabidopsis and humans

“…Results indicate that the coverage in WT plants has a similar pattern to those previously reported in the literature for plastid genomes (Wu et al 2012;Ferrarini et al 2013) and to that of all single and double mutant lines used in this study (Supplemental Fig. S4).…”

Graphene nanodevices for DNA sequencing

“…Although sizeable error rates (∼13%) have been reported [8], these errors are random, in contrast to context-specific errors (e.g. palindromic sequences or GC rich contents) that are generally observed in other techniques, such that multiple lower-quality base calls can be aligned to derive high-quality (de-novo) sequence data [9,10].…”

Genomic characterisation of Australian wild rice species

“…The first report of total plant DNA analysis for chloroplast sequencing (Nock et al 2011) used short Illumina reads (36 bp) and relied on a reference genome for successful assembly. More recent studies based upon longer reads (Ferrarini et al 2013;McPherson et al 2013) have reported more success with de novo assembly.…”

“…Various NGS technologies are now available for the rapid sequencing of whole genomes (Ferrarini et al 2013;Rothberg et al 2011;Shendure & Ji 2008). The choice of selecting one or more NGS technologies depends on the yield of data required, read length required, cost per data point and accuracy of the sequence data (Quail et al 2012).…”