BACKGROUND
the ability to perform sequencing and de novo assembly of genomes has been greatly facilitated in recent years thanks to the advent of second-generation sequencing technologies, and as such is becoming relatively routine for genome analysis of all but the largest and most complex genomes.
the range of platforms available for sequencing is increasing, and novel ‘third-generation’ technologies promising advantages over the more established ‘second-generation’ short read sequencing platforms have recently been brought to market.
the ‘second-generation’ sequencing revolution, which began with the release of the  <dig> pyro-sequencing platform  <cit> , has been dominated in recent years by illumina, who deliver up to  <dig> gb of sequence data per run with the hiseq <dig>  illumina’s technology employs sequencing-by-synthesis  <cit>  in which fluorescently labelled reversible terminator nucleotides are imaged as they are incorporated into growing dna strands synthesised from clonally amplified dna templates that are immobilised onto the surface of a glass flow-cell.
the hiseq platform has become the industry standard for high throughput dna sequencing in terms of throughput and accuracy; however, the technology is limited by the number of nucleotides that can be sequenced from a given dna template, currently less than ~ <dig> bases, and amplification of the dna template by pcr is typically required before sequencing, leading to a base-composition bias in genome coverage due to the chemical-physical properties of the dna template  <cit> .
recently, pacific biosciences released their pacbio rs sequencing platform which offers real-time sequencing from single polymerase molecules  <cit> .
the procedure, termed single-molecule real-time  sequencing, utilises dna polymerase molecules bound to  <dig> nm-wide nanophotonic structures in an array slide which pacific biosciences have called ‘zero-mode waveguides’ .
the polymerases synthesise dna from a template using four fluorescently-labelled nucleotides within the zmws and thus sequencing requires no prior amplification of the dna template.
the width of the zmws permits light to enter and excite the fluorophore that is being incorporated into the growing dna strand, but not to propagate through the wave-guide, enabling single-fluorophore detection simultaneously in each zmw on the array in real-time as the dna strand is synthesised.
the data produced from the ‘third-generation’ pacbio rs sequencing platform has a significantly longer read length than that of ‘second-generation’ technologies such as the illumina hiseq <dig>  and maximum read lengths of  <dig>  bp have been reported in the literature, with current average read lengths reaching  <dig>  kbp  <cit> .
however, the raw data generated from the pacbio rs platform is inherently error-prone, with up to  <dig> % errors having been reported  <cit> , the majority being indel events, caused by incorporation events or the intervals between them being too short to be reliably detected  <cit> .
despite this drawback, context-specific error modes affecting short-read sequencing platforms  <cit>  are nearly absent from pacbio data.
instead, the error model of pacbio data is random, and thus with sufficient depth of coverage, up to  <dig> % consensus accuracy can be achieved from sequencing and de novo assembly using pacbio rs sequencing data  <cit> .
this lack of context-specific error combined with pacbio’s long single-molecule derived reads has allowed sequencing through both plant and animal long tandem repeats  <cit> , which are very difficult to resolve with any other platform.
additionally, the recent release of the hierarchical genome assembly process  workflow of the smrt-analysis pipeline  <cit>  permits error-correction of continuous long reads to be performed without the need for additional circular consensus pacbio sequencing data, or short-read sequencing data from other platforms.
mitochondrial and chloroplast genomes make interesting targets for evaluation of the pacbio system because despite the fact that plastid genomes are relatively small, they are rarely completely assembled from second generation sequencing technologies unless specifically targeted, and even then assemblies are often fragmented into relatively large numbers of contigs even at high levels of coverage  <cit> .
assembly of plastid genomes with pacbio data would also allow for the evaluation of the platform to resolve long inverted repeats that are characteristic of chloroplast genomes and which are difficult to resolve with other sequencing platforms.
in this investigation, the performance of the pacbio rs sequencing platform for the sequencing and de novo assembly of the chloroplast genome of a member of the rosaceae, potentilla micrantha, was evaluated.
to our knowledge this is the first report of a chloroplast genome sequenced using pacbio rs data.
since data generated using the illumina hiseq <dig> platform are considered to be of very high quality, the relative performance of the pacbio sequence data was evaluated in relation to a de novo assembly of the same genome performed with data generated from a single illumina library sequenced on a single lane of illumina hiseq <dig>  the performance of the data generated from the pacbio rs platform is discussed.
RESULTS
data output from illumina hiseq <dig> and pacbio rs platforms
following extraction of reads containing only chloroplast genome sequence data and prior to error-correction, pacbio rs reads with a mean length of  <dig> .
<dig> bp were recovered, totalling  <dig> , <dig> nucleotides.
post hgap error-correction  <cit>  ,  <dig>  pacbio rs reads were recovered with a mean read length of  <dig> .
<dig> bp totalling  <dig> , <dig> bp.
following trimming,  <dig> , <dig> paired illumina reads with a mean length of  <dig>  bp were recovered containing a total of  <dig> , <dig>  nucleotides.
assembly of the chloroplast genome sequence
pacbio rs
a total of  <dig> overlapping contigs were obtained from the celera assembly of the chloroplast reads of the hgap-corrected pacbio dataset, which were merged into a single contiguous sequence using minimus <dig> and seqman .
the pacbio contig contained a total of  <dig>  nucleotides.
the two irs in the pacbio dataset differed at three nucleotide positions which allowed the two irs to be resolved across  <dig>  nucleotides.
the remaining  <dig>  bp section of the inverted repeat  was identical in both irs and thus the total length for the p. micrantha chloroplast genome was  <dig>  bp.
illumina hiseq2000
the chloroplast reads extracted from the illumina dataset were assembled into a total of seven contigs containing  <dig>  nucleotides, including a single  <dig>  bp inverted repeat .
since the chloroplasts of angiosperms contain a large sequence repeated once in reverse polarity  <cit> , the sequence was resolved manually based on read depth within the region and comparison to the ir of the fragaria chloroplast genome to identify ir borders , in line with the methodology used to defined the chloroplast genomes of other plant species  <cit> , to give a total length of  <dig>  bp .
contigs had a minimum length of  <dig>  bp, a maximum length of  <dig>  and a mean and n <dig> length of  <dig>  and  <dig>  respectively.
the gaps in the illumina assembly had a minimum length of  <dig> bp, a maximum length of  <dig> and a mean length of  <dig> bp.
the average gc content of the gaps in the assembly was  <dig> %, compared to an average gc content of the chloroplast consensus sequence of  <dig> %.
a summary of the data generated and the assemblies produced from the pacbio rs data in comparison to the data generated from the illumina hiseq <dig> platform is given table  <dig>
summary statistics for the assembly of the p. micrantha chloroplast genome using pacbio rs and illumina hiseq <dig> sequencing data.
1trimmed illumina reads.
2error-corrected pacbio reads and raw illumina reads.
3in comparison to the chloroplast consensus sequence.
depth of coverage and gc bias
both the pacbio and illumina reads covered the majority of the p. micrantha chloroplast genome, with 100% and  <dig> % of the genome covered by pacbio and illumina respectively following alignment of all reads from each dataset to the assembled chloroplast consensus sequence using blat.
the high percentage coverage of both datasets following the blat alignment supports the use of closely related chloroplast genomes to extract chloroplast-containing reads from the raw datasets generated from both the illumina and pacbio platforms, and suggests this process did not bias the data towards longer pacbio reads.
low read coverage in certain regions of the illumina assembly  meant that the seven contigs resolved covered just  <dig> % of the chloroplast consensus sequence , whilst the pacbio data were significantly more evenly distributed  and were assembled into a single contig which formed the basis of the chloroplast consensus sequence presented here .
blat aligned a total of  <dig>  reads containing a total of  <dig> , <dig> bp from the pacbio rs dataset and  <dig> , <dig> reads containing  <dig> , <dig>  bp from the illumina dataset.
thus, the average depth of coverage of the p. micrantha chloroplast genome represented by the error-corrected pacbio rs data was 320×, whilst the average depth of coverage of the illumina reads was  <dig> ×.
figure  <dig> shows the base per base coverage of the reads aligned by blat for both the pacbio and illumina datasets across the p. micrantha chloroplast genome, showing a more uniform coverage of genome by the pacbio rs dataset.
to determine whether a gc bias existed in the two sequencing datasets, the pearson correlation coefficient was computed between mean coverage and percentage gc content in  <dig> contiguous non-overlapping windows of  <dig> nucleotides.
for the purposes of the calculation, data from the two inverted repeat regions was excluded.
the calculated pearson correlation coefficients were  <dig>   and  <dig>   respectively for the pacbio and illumina datasets.
thus, a much stronger positive dependency between the mean coverage against percentage gc content was observed in the illumina dataset than in the pacbio data .
error rates
the mean pre-assembly error rate in the pacbio rs reads in comparison to the p. micrantha chloroplast consensus sequence was  <dig> %, whilst the mean error rate in the illumina reads was  <dig> % compared to the chloroplast consensus sequence.
post-assembly, the two assemblies were generally in concordance however,  <dig> nucleotide sites could not be discriminated unambiguously in the illumina assembly .
performing error-correction prior to assembly using coral  <cit>  on the illumina reads did not help resolve the ambiguities at these  <dig> sites .
however, inspection of coverage and base calling at those sites in the pacbio rs data showed a clear single nucleotide consensus and thus all  <dig> nucleotides were resolved unambiguously in the chloroplast consensus sequence.
chloroplast genome assemblies at different depths of sequence coverage
to determine the effect of depth of sequence coverage on the assembly of the p. micrantha genome using pacbio rs data, a titration of sequence depths was performed with data sampled at 10×, 20×, 35×, 50×, 100×, 150× and 200× depth of coverage, following which the genome was assembled de novo from each dataset using the procedure described for the full datasets.
of the seven assemblies performed, five  returned a single contig spanning the chloroplast genome, whilst the assembly performed at 20× returned four contigs spanning  <dig> % of the genome and the assembly at 10× returned  <dig> contigs spanning  <dig> % of the chloroplast genome.
for comparison, illumina data were sampled at the same seven depths of coverage as the pacbio data and assemblies were performed, however, none returned more complete assemblies than that performed with 9111× depth of coverage.
structural organisation of the potentilla micrantha chloroplast genome
the assembled chloroplast genome of potentilla micrantha was  <dig>  bp in length .
the inverted repeats  were  <dig>  bp in length each, whilst the large single copy  and small single copy  regions were  <dig>  bp and  <dig>  bp in length respectively.
the p. micrantha chloroplast contains  <dig> genes,  <dig> of which are duplicated in the irs, giving a total of  <dig> genes of known function.
of these genes,  <dig> were trna coding genes, of which seven were located in the ir.
a comparison with the f. vesca chloroplast genome sequence, the closest relative to p. micrantha for which a fully-sequenced chloroplast genome is available, revealed that the gene number and order within the genomes was identical between the two species.
data relating to this project have been submitted to the ena sequence read archive of the embl database under the project accession number prjeb <dig>
discussion
here we present the first report of the sequencing and de novo assembly of a chloroplast genome using the pacbio rs sequencing platform in which we recovered a single contig containing  <dig>  bp that covered the entire p. micrantha chloroplast genome.
to enable an evaluation of the relative performance of the pacbio rs sequencing platform for sequencing and de novo assembly of the p. micrantha chloroplast genome, we compared the results obtained to an assembly performed with a single library from the illumina hiseq <dig> platform.
since the data from the two platforms were assembled by necessity using different assembly programs and assembly parameters, the results obtained clearly cannot be compared on a like-for-like basis, and the experimental design did not provide for the ‘optimal’ results that could be obtained for the assembly of a chloroplast genome using the illumina hiseq <dig> platform.
nevertheless, illumina data are recognised as being of immense utility to sequencing and de novo assembly of draft genome sequences, and thus, whilst the comparison is not intended to be a reflection of the performance of the hiseq <dig> platform per se, the resultant illumina assembly provided a useful yardstick with which to judge the relative merits and short-comings of the pacbio rs sequencing platform.
short-read sequencing platforms, including the illumina hiseq <dig>  derive sequencing reads from template dna that has undergone pre-sequencing amplification by pcr  <cit> .
this amplification step results in sequencing bias, and thus poor or no sequencing coverage in certain regions of the genome, and a strong positive correlation between %gc content and read coverage  <cit> .
this lack of coverage is evident even when average depths of sequence coverage are high.
such bias leads to regions of no sequence coverage within sequencing datasets and thus assemblies that contain multiple small gaps, leading to a large number of contigs and scaffolds even in modest sized genomes such as those of bacteria  <cit>  and chloroplast genomes  <cit> .
in this investigation, the p. micrantha chloroplast genome was sequenced on the hiseq <dig> platform to an average depth of  <dig> × from a single illumina truseq library, but despite this depth of coverage, there remained a total of  <dig>   nucleotides of the genome which were not assembled from the illumina data and thus seven contigs were recovered from the genome assembly.
the gap regions contained a much lower average gc content than the entire chloroplast genome, in line with other studies that have reported a lower gc content in low coverage and gapped regions in illumina assemblies  <cit>  and reinforcing evidence of a strong positive dependency between coverage and gc content observed in the illumina data set.
in contrast, despite a lower depth of sequence coverage  achieved following error-correction, data from the pacbio rs platform were assembled into a single contig spanning the entire p. micrantha chloroplast genome.
coverage of pacbio reads across the entire chloroplast consensus sequence was relatively even, demonstrating that data from this platform does not suffer from % gc and other context-specific biases affecting data produced by short-read ‘second-generation’ sequencing platforms  <cit> .
our data were also in accord with the recently reported findings of tang et al  <cit>  who recovered two contigs spanning the mitochondrial genome of tomato in an assembly using 122 × of pacbio data, in contrast to  <dig> scaffolds covering the same genome using 4098× of illumina data, suggesting longer read length and less genome coverage bias can result in significantly longer contigs in de novo plastid genome assemblies.
it is possible that if multiple illumina libraries, including mate-pair libraries and overlapping fragment libraries, were sequenced, then a single scaffold covering the chloroplast genome would have been recovered.
however, due to the inherent biases in the pcr amplification performed prior to sequencing, it is likely that the scaffold would still have contained gaps associated with the regions of poor and no coverage as was found in this investigation and in other studies of chloroplast assembly using second generation sequencing platforms  <cit> .
indeed, assemblies performed following a titration of sequence depths for both pacbio and illumina datasets demonstrated that the high depth of coverage of the illumina dataset did not confound the assembly process, and no assembly at a lower depth of coverage performed better than the assembly utilising the entire illumina dataset.
pacbio assemblies at depths of coverage of 35× and above, recovered a single contig spanning the chloroplast genome, suggesting that de novo non-hybrid assemblies with pacbio data could be possible at relatively low depths of sequencing coverage.
error-rates from single read data generated from the pacbio rs platform have been reported to be relatively high, in the region of  <dig>  –  <dig> %  <cit> .
however, since sequencing errors are introduced randomly into the reads generated and are thus largely non-context specific  <cit> , they are likely to have minimal effect on the final assembled sequence if sufficient depth of coverage is achieved and error-correction is performed prior to assembly.
since data generated from the illumina hiseq <dig> platform has been established as the ‘gold standard’ for second-generation sequencing technologies, we evaluated the error-rate in the assembly of the pacbio rs data by comparison to illumina data and where both assemblies resolved the same result for a nucleotide, we took this as an indication that the base had been called correctly in both assemblies.
in this investigation, error rates of  <dig> % were observed in the pacbio rs data following processing and error correction using hgap  <cit>  when compared to the chloroplast consensus sequence.
illumina sequencing data has been shown to contain non-random distribution of errors, with 3% of all error positions accounting for  <dig> % of all substitution errors in one study  <cit>  and no universal motif responsible for the occurrence of these error-prone positions.
this type of error was observed at  <dig> nucleotide sites in the contigs derived from the illumina assembly of the p. micrantha chloroplast genome in this investigation which despite high sequence coverage, returned ambiguous base calls following assembly.
in all cases however, these ambiguous nucleotides were unambiguously called in the assembly derived from pacbio rs data as one of the alternative bases in the illumina assembly.
the pacbio and illumina assemblies were concordant at all other bases within the assemblies, indicating that post-error correction and assembly pacbio data are potentially as robust as data derived from other sequencing platforms if sufficient depth of coverage is achieved to permit reliable error-correction.
indeed, recent reports suggest that with the latest chemistry and the most recent version of the hgap algorithm, accuracy rates in pacbio rs datasets post-error-correction as high as  <dig> % could be achieved  <cit> .
it is important to highlight here however that the analyses performed for creating the consensus sequence favour the pacbio assembly since it contains more nucleotides than the illumina assembly.
thus where no illumina data were available for comparison, the pacbio data may contain a low percentage of errors that could not be verified in this study.
in previous studies, pacbio rs data have been reported to contain maximum read lengths of up to  <dig>  nucleotides and median lengths of  <dig>  nucleotides  <cit> .
such read lengths have been shown to significantly improve the quality of sequence assemblies when used for hybrid assemblies  <cit> .
in this investigation, the maximum and mean un-corrected read lengths were  <dig>  and  <dig>  nucleotides respectively, with an average read length following error-correction of  <dig> .
the data generated using the pacbio rs platform covered a greater proportion of the chloroplast genome and was able to resolve the small percentage of ambiguities that were present in the illumina assembly.
thus the data from the chloroplast assembly reported here supports previous findings that pacbio rs data can produce high quality sequence assemblies covering a greater proportion of the genome than can be achieved by illumina sequencing alone  <cit> .
pacbio rs data is significantly less expensive to generate than data from traditional sanger sequencing, and reports indicate that for targeted exon sequencing, for use in genomic profiling of tumor biopsies, pacbio rs sequence data was in 100% concordance with traditional sanger sequencing  <cit> .
additionally, other researchers demonstrated the utility of pacbio rs data for snp validation in medical re-sequencing projects, where sanger sequencing has traditionally been employed  <cit> .
however, the additional read length of pacbio rs data comes at the cost of a higher cost per base than ‘second generation’ short read technologies  <cit> , and higher single molecule error-rates necessitates the need for a greater depth of sequence coverage to be achieved to permit consensus accuracies of an acceptable level for de novo sequence assembly with currently available software.
additionally, since the pacbio sequencing platform performs real-time sequencing from single molecules, a greater quantity of dna is required than second generation sequencing platforms, which could be a limiting factor for sequencing from organisms from which dna is hard to obtain or which are difficult to culture.
despite the advantages to the use of pacbio rs sequencing data, and recent significant increases in throughput, the cost per base for de novo sequencing and assembly of larger genomes, such as those of plants are still significantly more expensive than data derived from the illumina hiseq platform  <cit> .
thus de novo assemblies of the genomes of minority species at the time of writing may be best served through the combination of pacbio data with data from other platforms.
koren et al.
<cit>  demonstrated that the addition of a modest amount of illumina error-corrected pacbio data to supplement  <dig> sequencing data from multiple libraries resulted in a 32% increase in n <dig> sizes in the parrot  genome sequence assembly and other researchers have demonstrated the utility of pacbio sequence data for gap filling and genome finishing  <cit> .
the data presented here support the findings of those previous studies and illustrate the power and utility of pacbio rs sequencing data for sequencing and de novo assembly, as well as demonstrating that despite high initial single read error rates, following error-correction and assembly, the data produced by the platform are robust and reliable.
CONCLUSIONS
as part of an on-going effort to sequence the nuclear genome of p. micrantha, we are employing pacbio sequence data in combination with illumina small insert and mate pair sequencing libraries and initial data suggest that, as with the chloroplast data presented here and by other authors, pacbio rs sequencing data show great promise in scaffolding, gap filling and genome sequence finishing.
in addition, if the current trend in increased throughput and reliability continue, it is reasonable to speculate that the technology may be able to deliver affordable high quality finished genomes for a variety of eukaryotic organisms.

 the ability to perform sequencing and de novo assembly of genomes has been greatly facilitated in recent years thanks to the advent of second-generation sequencing technologies, and as such is becoming relatively routine for genome analysis of all but the largest and most complex genomes. the range of platforms available for sequencing is increasing, and novel ‘third-generation’ technologies promising advantages over the more established ‘second-generation’ short read sequencing platforms have recently been brought to market. the ‘second-generation’ sequencing revolution, which began with the release of the pyro-sequencing platform, has been dominated in recent years by illumina, who deliver up to gb of sequence data per run with the hiseq illumina’s technology employs sequencing-by-synthesis in which fluorescently labelled reversible terminator nucleotides are imaged as they are incorporated into growing dna strands synthesised from clonally amplified dna templates that are immobilised onto the surface of a glass flow-cell. the hiseq platform has become the industry standard for high throughput dna sequencing in terms of throughput and accuracy; however, the technology is limited by the number of nucleotides that can be sequenced from a given dna template, currently less than ~ bases, and amplification of the dna template by pcr is typically required before sequencing, leading to a base-composition bias in genome coverage due to the chemical-physical properties of the dna template. recently, pacific biosciences released their pacbio rs sequencing platform which offers real-time sequencing from single polymerase molecules. the procedure, termed single-molecule real-time sequencing, utilises dna polymerase molecules bound to nm-wide nanophotonic structures in an array slide which pacific biosciences have called ‘zero-mode waveguides’. the polymerases synthesise dna from a template using four fluorescently-labelled nucleotides within the zmws and thus sequencing requires no prior amplification of the dna template. the width of the zmws permits light to enter and excite the fluorophore that is being incorporated into the growing dna strand, but not to propagate through the wave-guide, enabling single-fluorophore detection simultaneously in each zmw on the array in real-time as the dna strand is synthesised. the data produced from the ‘third-generation’ pacbio rs sequencing platform has a significantly longer read length than that of ‘second-generation’ technologies such as the illumina hiseq and maximum read lengths of bp have been reported in the literature, with current average read lengths reaching kbp. however, the raw data generated from the pacbio rs platform is inherently error-prone, with up to % errors having been reported, the majority being indel events, caused by incorporation events or the intervals between them being too short to be reliably detected. despite this drawback, context-specific error modes affecting short-read sequencing platforms are nearly absent from pacbio data. instead, the error model of pacbio data is random, and thus with sufficient depth of coverage, up to % consensus accuracy can be achieved from sequencing and de novo assembly using pacbio rs sequencing data. this lack of context-specific error combined with pacbio’s long single-molecule derived reads has allowed sequencing through both plant and animal long tandem repeats, which are very difficult to resolve with any other platform. additionally, the recent release of the hierarchical genome assembly process workflow of the smrt-analysis pipeline permits error-correction of continuous long reads to be performed without the need for additional circular consensus pacbio sequencing data, or short-read sequencing data from other platforms. mitochondrial and chloroplast genomes make interesting targets for evaluation of the pacbio system because despite the fact that plastid genomes are relatively small, they are rarely completely assembled from second generation sequencing technologies unless specifically targeted, and even then assemblies are often fragmented into relatively large numbers of contigs even at high levels of coverage. assembly of plastid genomes with pacbio data would also allow for the evaluation of the platform to resolve long inverted repeats that are characteristic of chloroplast genomes and which are difficult to resolve with other sequencing platforms. in this investigation, the performance of the pacbio rs sequencing platform for the sequencing and de novo assembly of the chloroplast genome of a member of the rosaceae, potentilla micrantha, was evaluated. to our knowledge this is the first report of a chloroplast genome sequenced using pacbio rs data. since data generated using the illumina hiseq platform are considered to be of very high quality, the relative performance of the pacbio sequence data was evaluated in relation to a de novo assembly of the same genome performed with data generated from a single illumina library sequenced on a single lane of illumina hiseq the performance of the data generated from the pacbio rs platform is discussed.  data output from illumina hiseq and pacbio rs platforms following extraction of reads containing only chloroplast genome sequence data and prior to error-correction, pacbio rs reads with a mean length of. bp were recovered, totalling, nucleotides. post hgap error-correction, pacbio rs reads were recovered with a mean read length of. bp totalling, bp. following trimming,, paired illumina reads with a mean length of bp were recovered containing a total of, nucleotides. assembly of the chloroplast genome sequence pacbio rs a total of overlapping contigs were obtained from the celera assembly of the chloroplast reads of the hgap-corrected pacbio dataset, which were merged into a single contiguous sequence using minimus and seqman. the pacbio contig contained a total of nucleotides. the two irs in the pacbio dataset differed at three nucleotide positions which allowed the two irs to be resolved across nucleotides. the remaining bp section of the inverted repeat was identical in both irs and thus the total length for the p. micrantha chloroplast genome was bp. illumina hiseq2000 the chloroplast reads extracted from the illumina dataset were assembled into a total of seven contigs containing nucleotides, including a single bp inverted repeat. since the chloroplasts of angiosperms contain a large sequence repeated once in reverse polarity, the sequence was resolved manually based on read depth within the region and comparison to the ir of the fragaria chloroplast genome to identify ir borders, in line with the methodology used to defined the chloroplast genomes of other plant species, to give a total length of bp. contigs had a minimum length of bp, a maximum length of and a mean and n length of and respectively. the gaps in the illumina assembly had a minimum length of bp, a maximum length of and a mean length of bp. the average gc content of the gaps in the assembly was %, compared to an average gc content of the chloroplast consensus sequence of %. a summary of the data generated and the assemblies produced from the pacbio rs data in comparison to the data generated from the illumina hiseq platform is given table  summary statistics for the assembly of the p. micrantha chloroplast genome using pacbio rs and illumina hiseq sequencing data. 1trimmed illumina reads. 2error-corrected pacbio reads and raw illumina reads. 3in comparison to the chloroplast consensus sequence. depth of coverage and gc bias both the pacbio and illumina reads covered the majority of the p. micrantha chloroplast genome, with 100% and % of the genome covered by pacbio and illumina respectively following alignment of all reads from each dataset to the assembled chloroplast consensus sequence using blat. the high percentage coverage of both datasets following the blat alignment supports the use of closely related chloroplast genomes to extract chloroplast-containing reads from the raw datasets generated from both the illumina and pacbio platforms, and suggests this process did not bias the data towards longer pacbio reads. low read coverage in certain regions of the illumina assembly meant that the seven contigs resolved covered just % of the chloroplast consensus sequence, whilst the pacbio data were significantly more evenly distributed and were assembled into a single contig which formed the basis of the chloroplast consensus sequence presented here. blat aligned a total of reads containing a total of, bp from the pacbio rs dataset and, reads containing, bp from the illumina dataset. thus, the average depth of coverage of the p. micrantha chloroplast genome represented by the error-corrected pacbio rs data was 320×, whilst the average depth of coverage of the illumina reads was ×. figure  shows the base per base coverage of the reads aligned by blat for both the pacbio and illumina datasets across the p. micrantha chloroplast genome, showing a more uniform coverage of genome by the pacbio rs dataset. to determine whether a gc bias existed in the two sequencing datasets, the pearson correlation coefficient was computed between mean coverage and percentage gc content in contiguous non-overlapping windows of nucleotides. for the purposes of the calculation, data from the two inverted repeat regions was excluded. the calculated pearson correlation coefficients were and respectively for the pacbio and illumina datasets. thus, a much stronger positive dependency between the mean coverage against percentage gc content was observed in the illumina dataset than in the pacbio data. error rates the mean pre-assembly error rate in the pacbio rs reads in comparison to the p. micrantha chloroplast consensus sequence was %, whilst the mean error rate in the illumina reads was % compared to the chloroplast consensus sequence. post-assembly, the two assemblies were generally in concordance however, nucleotide sites could not be discriminated unambiguously in the illumina assembly. performing error-correction prior to assembly using coral on the illumina reads did not help resolve the ambiguities at these sites. however, inspection of coverage and base calling at those sites in the pacbio rs data showed a clear single nucleotide consensus and thus all nucleotides were resolved unambiguously in the chloroplast consensus sequence. chloroplast genome assemblies at different depths of sequence coverage to determine the effect of depth of sequence coverage on the assembly of the p. micrantha genome using pacbio rs data, a titration of sequence depths was performed with data sampled at 10×, 20×, 35×, 50×, 100×, 150× and 200× depth of coverage, following which the genome was assembled de novo from each dataset using the procedure described for the full datasets. of the seven assemblies performed, five returned a single contig spanning the chloroplast genome, whilst the assembly performed at 20× returned four contigs spanning % of the genome and the assembly at 10× returned contigs spanning % of the chloroplast genome. for comparison, illumina data were sampled at the same seven depths of coverage as the pacbio data and assemblies were performed, however, none returned more complete assemblies than that performed with 9111× depth of coverage. structural organisation of the potentilla micrantha chloroplast genome the assembled chloroplast genome of potentilla micrantha was bp in length. the inverted repeats were bp in length each, whilst the large single copy and small single copy regions were bp and bp in length respectively. the p. micrantha chloroplast contains genes, of which are duplicated in the irs, giving a total of genes of known function. of these genes, were trna coding genes, of which seven were located in the ir. a comparison with the f. vesca chloroplast genome sequence, the closest relative to p. micrantha for which a fully-sequenced chloroplast genome is available, revealed that the gene number and order within the genomes was identical between the two species. data relating to this project have been submitted to the ena sequence read archive of the embl database under the project accession number prjeb discussion here we present the first report of the sequencing and de novo assembly of a chloroplast genome using the pacbio rs sequencing platform in which we recovered a single contig containing bp that covered the entire p. micrantha chloroplast genome. to enable an evaluation of the relative performance of the pacbio rs sequencing platform for sequencing and de novo assembly of the p. micrantha chloroplast genome, we compared the results obtained to an assembly performed with a single library from the illumina hiseq platform. since the data from the two platforms were assembled by necessity using different assembly programs and assembly parameters, the results obtained clearly cannot be compared on a like-for-like basis, and the experimental design did not provide for the ‘optimal’ results that could be obtained for the assembly of a chloroplast genome using the illumina hiseq platform. nevertheless, illumina data are recognised as being of immense utility to sequencing and de novo assembly of draft genome sequences, and thus, whilst the comparison is not intended to be a reflection of the performance of the hiseq platform per se, the resultant illumina assembly provided a useful yardstick with which to judge the relative merits and short-comings of the pacbio rs sequencing platform. short-read sequencing platforms, including the illumina hiseq derive sequencing reads from template dna that has undergone pre-sequencing amplification by pcr. this amplification step results in sequencing bias, and thus poor or no sequencing coverage in certain regions of the genome, and a strong positive correlation between %gc content and read coverage. this lack of coverage is evident even when average depths of sequence coverage are high. such bias leads to regions of no sequence coverage within sequencing datasets and thus assemblies that contain multiple small gaps, leading to a large number of contigs and scaffolds even in modest sized genomes such as those of bacteria and chloroplast genomes. in this investigation, the p. micrantha chloroplast genome was sequenced on the hiseq platform to an average depth of × from a single illumina truseq library, but despite this depth of coverage, there remained a total of nucleotides of the genome which were not assembled from the illumina data and thus seven contigs were recovered from the genome assembly. the gap regions contained a much lower average gc content than the entire chloroplast genome, in line with other studies that have reported a lower gc content in low coverage and gapped regions in illumina assemblies and reinforcing evidence of a strong positive dependency between coverage and gc content observed in the illumina data set. in contrast, despite a lower depth of sequence coverage achieved following error-correction, data from the pacbio rs platform were assembled into a single contig spanning the entire p. micrantha chloroplast genome. coverage of pacbio reads across the entire chloroplast consensus sequence was relatively even, demonstrating that data from this platform does not suffer from % gc and other context-specific biases affecting data produced by short-read ‘second-generation’ sequencing platforms. our data were also in accord with the recently reported findings of tang et al who recovered two contigs spanning the mitochondrial genome of tomato in an assembly using 122 × of pacbio data, in contrast to scaffolds covering the same genome using 4098× of illumina data, suggesting longer read length and less genome coverage bias can result in significantly longer contigs in de novo plastid genome assemblies. it is possible that if multiple illumina libraries, including mate-pair libraries and overlapping fragment libraries, were sequenced, then a single scaffold covering the chloroplast genome would have been recovered. however, due to the inherent biases in the pcr amplification performed prior to sequencing, it is likely that the scaffold would still have contained gaps associated with the regions of poor and no coverage as was found in this investigation and in other studies of chloroplast assembly using second generation sequencing platforms. indeed, assemblies performed following a titration of sequence depths for both pacbio and illumina datasets demonstrated that the high depth of coverage of the illumina dataset did not confound the assembly process, and no assembly at a lower depth of coverage performed better than the assembly utilising the entire illumina dataset. pacbio assemblies at depths of coverage of 35× and above, recovered a single contig spanning the chloroplast genome, suggesting that de novo non-hybrid assemblies with pacbio data could be possible at relatively low depths of sequencing coverage. error-rates from single read data generated from the pacbio rs platform have been reported to be relatively high, in the region of – %. however, since sequencing errors are introduced randomly into the reads generated and are thus largely non-context specific, they are likely to have minimal effect on the final assembled sequence if sufficient depth of coverage is achieved and error-correction is performed prior to assembly. since data generated from the illumina hiseq platform has been established as the ‘gold standard’ for second-generation sequencing technologies, we evaluated the error-rate in the assembly of the pacbio rs data by comparison to illumina data and where both assemblies resolved the same result for a nucleotide, we took this as an indication that the base had been called correctly in both assemblies. in this investigation, error rates of % were observed in the pacbio rs data following processing and error correction using hgap when compared to the chloroplast consensus sequence. illumina sequencing data has been shown to contain non-random distribution of errors, with 3% of all error positions accounting for % of all substitution errors in one study and no universal motif responsible for the occurrence of these error-prone positions. this type of error was observed at nucleotide sites in the contigs derived from the illumina assembly of the p. micrantha chloroplast genome in this investigation which despite high sequence coverage, returned ambiguous base calls following assembly. in all cases however, these ambiguous nucleotides were unambiguously called in the assembly derived from pacbio rs data as one of the alternative bases in the illumina assembly. the pacbio and illumina assemblies were concordant at all other bases within the assemblies, indicating that post-error correction and assembly pacbio data are potentially as robust as data derived from other sequencing platforms if sufficient depth of coverage is achieved to permit reliable error-correction. indeed, recent reports suggest that with the latest chemistry and the most recent version of the hgap algorithm, accuracy rates in pacbio rs datasets post-error-correction as high as % could be achieved. it is important to highlight here however that the analyses performed for creating the consensus sequence favour the pacbio assembly since it contains more nucleotides than the illumina assembly. thus where no illumina data were available for comparison, the pacbio data may contain a low percentage of errors that could not be verified in this study. in previous studies, pacbio rs data have been reported to contain maximum read lengths of up to nucleotides and median lengths of nucleotides. such read lengths have been shown to significantly improve the quality of sequence assemblies when used for hybrid assemblies. in this investigation, the maximum and mean un-corrected read lengths were and nucleotides respectively, with an average read length following error-correction of. the data generated using the pacbio rs platform covered a greater proportion of the chloroplast genome and was able to resolve the small percentage of ambiguities that were present in the illumina assembly. thus the data from the chloroplast assembly reported here supports previous findings that pacbio rs data can produce high quality sequence assemblies covering a greater proportion of the genome than can be achieved by illumina sequencing alone. pacbio rs data is significantly less expensive to generate than data from traditional sanger sequencing, and reports indicate that for targeted exon sequencing, for use in genomic profiling of tumor biopsies, pacbio rs sequence data was in 100% concordance with traditional sanger sequencing. additionally, other researchers demonstrated the utility of pacbio rs data for snp validation in medical re-sequencing projects, where sanger sequencing has traditionally been employed. however, the additional read length of pacbio rs data comes at the cost of a higher cost per base than ‘second generation’ short read technologies, and higher single molecule error-rates necessitates the need for a greater depth of sequence coverage to be achieved to permit consensus accuracies of an acceptable level for de novo sequence assembly with currently available software. additionally, since the pacbio sequencing platform performs real-time sequencing from single molecules, a greater quantity of dna is required than second generation sequencing platforms, which could be a limiting factor for sequencing from organisms from which dna is hard to obtain or which are difficult to culture. despite the advantages to the use of pacbio rs sequencing data, and recent significant increases in throughput, the cost per base for de novo sequencing and assembly of larger genomes, such as those of plants are still significantly more expensive than data derived from the illumina hiseq platform. thus de novo assemblies of the genomes of minority species at the time of writing may be best served through the combination of pacbio data with data from other platforms. koren et al. demonstrated that the addition of a modest amount of illumina error-corrected pacbio data to supplement sequencing data from multiple libraries resulted in a 32% increase in n sizes in the parrot genome sequence assembly and other researchers have demonstrated the utility of pacbio sequence data for gap filling and genome finishing. the data presented here support the findings of those previous studies and illustrate the power and utility of pacbio rs sequencing data for sequencing and de novo assembly, as well as demonstrating that despite high initial single read error rates, following error-correction and assembly, the data produced by the platform are robust and reliable.  as part of an on-going effort to sequence the nuclear genome of p. micrantha, we are employing pacbio sequence data in combination with illumina small insert and mate pair sequencing libraries and initial data suggest that, as with the chloroplast data presented here and by other authors, pacbio rs sequencing data show great promise in scaffolding, gap filling and genome sequence finishing. in addition, if the current trend in increased throughput and reliability continue, it is reasonable to speculate that the technology may be able to deliver affordable high quality finished genomes for a variety of eukaryotic organisms. 
following error-correction, a total of pacbio rs reads were recovered with a mean read length of bp totalling, nucleotides and representing an average depth of coverage of 320× the chloroplast genome. 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. 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. 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 dataset covered the entire bp of the chloroplast genome in a single contig compared to seven contigs 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 ambiguities in the illumina data to be resolved. second 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. this is the first report to our knowledge of a chloroplast genome assembled de novo using pacbio sequence data. the additional read length also permitted small differences in the two inverted repeat regions to be assigned unambiguously.
 second 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.  following error-correction, a total of pacbio rs reads were recovered with a mean read length of bp totalling, nucleotides and representing an average depth of coverage of 320× the chloroplast genome. the dataset covered the entire bp of the chloroplast genome in a single contig compared to seven contigs 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 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.  this 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. third-generation sequencingngengenomicsassemblyannotationoxford nanoporepacific biosciencesroche 454 