An evaluation of the PacBio RS platform for sequencing and de novo assembly of a chloroplast genome.
ABSTRACT: BACKGROUND: 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. RESULTS: Following 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. CONCLUSIONS: 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.
Project description:<h4>Background</h4>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.<h4>Results</h4>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.<h4>Conclusions</h4>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.
Project description:BACKGROUND: The availability of diverse second- and third-generation sequencing technologies enables the rapid determination of the sequences of bacterial genomes. However, identifying the sequencing technology most suitable for producing a finished genome with multiple chromosomes remains a challenge. We evaluated the abilities of the following three second-generation sequencers: Roche 454 GS Junior (GS Jr), Life Technologies Ion PGM (Ion PGM), and Illumina MiSeq (MiSeq) and a third-generation sequencer, the Pacific Biosciences RS sequencer (PacBio), by sequencing and assembling the genome of Vibrio parahaemolyticus, which consists of a 5-Mb genome comprising two circular chromosomes. RESULTS: We sequenced the genome of V. parahaemolyticus with GS Jr, Ion PGM, MiSeq, and PacBio and performed de novo assembly with several genome assemblers. Although GS Jr generated the longest mean read length of 418 bp among the second-generation sequencers, the maximum contig length of the best assembly from GS Jr was 165 kbp, and the number of contigs was 309. Single runs of Ion PGM and MiSeq produced data of considerably greater sequencing coverage, 279× and 1,927×, respectively. The optimized result for Ion PGM contained 61 contigs assembled from reads of 77× coverage, and the longest contig was 895 kbp in size. Those for MiSeq were 34 contigs, 58×?coverage, and 733 kbp, respectively. These results suggest that higher coverage depth is unnecessary for a better assembly result. We observed that multiple rRNA coding regions were fragmented in the assemblies from the second-generation sequencers, whereas PacBio generated two exceptionally long contigs of 3,288,561 and 1,875,537 bps, each of which was from a single chromosome, with 73× coverage and mean read length 3,119 bp, allowing us to determine the absolute positions of all rRNA operons. CONCLUSIONS: PacBio outperformed the other sequencers in terms of the length of contigs and reconstructed the greatest portion of the genome, achieving a genome assembly of "finished grade" because of its long reads. It showed the potential to assemble more complex genomes with multiple chromosomes containing more repetitive sequences.
Project description:Although PacBio third-generation sequencers have improved the read lengths of genome sequencing which facilitates the assembly of complete genomes, no study has reported success in using PacBio data alone to completely sequence a two-chromosome bacterial genome from a single library in a single run. Previous studies using earlier versions of sequencing chemistries have at most been able to finish bacterial genomes containing only one chromosome with de novo assembly. In this study, we compared the robustness of PacBio RS II, using one SMRT cell and the latest P6-C4 chemistry, with Illumina HiSeq 1500 in sequencing the genome of Burkholderia pseudomallei, a bacterium which contains two large circular chromosomes, very high G+C content of 68-69%, highly repetitive regions and substantial genomic diversity, and represents one of the largest and most complex bacterial genomes sequenced, using a reference genome generated by hybrid assembly using PacBio and Illumina datasets with subsequent manual validation. Results showed that PacBio data with de novo assembly, but not Illumina, was able to completely sequence the B. pseudomallei genome without any gaps or mis-assemblies. The two large contigs of the PacBio assembly aligned unambiguously to the reference genome, sharing >99.9% nucleotide identities. Conversely, Illumina data assembled using three different assemblers resulted in fragmented assemblies (201-366 contigs), sharing only 92.2-100% and 92.0-100% nucleotide identities to chromosomes I and II reference sequences, respectively, with no indication that the B. pseudomallei genome consisted of two chromosomes with four copies of ribosomal operons. Among all assemblies, the PacBio assembly recovered the highest number of core and virulence proteins, and housekeeping genes based on whole-genome multilocus sequence typing (wgMLST). Most notably, assembly solely based on PacBio outperformed even hybrid assembly using both PacBio and Illumina datasets. Hybrid approach generated only 74 contigs, while the PacBio data alone with de novo assembly achieved complete closure of the two-chromosome B. pseudomallei genome without additional costly bench work and further sequencing. PacBio RS II using P6-C4 chemistry is highly robust and cost-effective and should be the platform of choice in sequencing bacterial genomes, particularly for those that are well-known to be difficult-to-sequence.
Project description:The chloroplast genome is important for plant development and plant evolution. Nelumbo nucifera is one member of relict plants surviving from the late Cretaceous. Recently, a new sequencing platform PacBio RS II, known as 'SMRT (Single Molecule, Real-Time) sequencing', has been developed. Using the SMRT sequencing to investigate the chloroplast genome of N. nucifera will help to elucidate the plastid evolution of basal eudicots.The sizes of the de novo assembled complete chloroplast genome of N. nucifera were 163,307 bp, 163,747 bp and 163,600 bp with average depths of coverage of 7×, 712× and 105× sequenced by Sanger, Illumina MiSeq and PacBio RS II, respectively. The precise chloroplast genome of N. nucifera was obtained from PacBio RS II data proofread by Illumina MiSeq reads, with a quadripartite structure containing a large single copy region (91,846 bp) and a small single copy region (19,626 bp) separated by two inverted repeat regions (26,064 bp). The genome contains 113 different genes, including four distinct rRNAs, 30 distinct tRNAs and 79 distinct peptide-coding genes. A phylogenetic analysis of 133 taxa from 56 orders indicated that Nelumbo with an age of 177 million years is a sister clade to Platanus, which belongs to the basal eudicots. Basal eudicots began to emerge during the early Jurassic with estimated divergence times at 197 million years using MCMCTree. IR expansions/contractions within the basal eudicots seem to have occurred independently.Because of long reads and lack of bias in coverage of AT-rich regions, PacBio RS II showed a great promise for highly accurate 'finished' genomes, especially for a de novo assembly of genomes. N. nucifera is one member of basal eudicots, however, evolutionary analyses of IR structural variations of N. nucifera and other basal eudicots suggested that IR expansions/contractions occurred independently in these basal eudicots or were caused by independent insertions and deletions. The precise chloroplast genome of N. nucifera will present new information for structural variation of chloroplast genomes and provide new insight into the evolution of basal eudicots at the primary sequence and structural level.
Project description:BACKGROUND: With the price of next generation sequencing steadily decreasing, bacterial genome assembly is now accessible to a wide range of researchers. It is therefore necessary to understand the best methods for generating a genome assembly, specifically, which combination of sequencing and bioinformatics strategies result in the most accurate assemblies. Here, we sequence three E. coli strains on the Illumina MiSeq, Life Technologies Ion Torrent PGM, and Pacific Biosciences RS. We then perform genome assemblies on all three datasets alone or in combination to determine the best methods for the assembly of bacterial genomes. RESULTS: Three E. coli strains - BL21(DE3), Bal225, and DH5α - were sequenced to a depth of 100× on the MiSeq and Ion Torrent machines and to at least 125× on the PacBio RS. Four assembly methods were examined and compared. The previously published BL21(DE3) genome [GenBank:AM946981.2], allowed us to evaluate the accuracy of each of the BL21(DE3) assemblies. BL21(DE3) PacBio-only assemblies resulted in a 90% reduction in contigs versus short read only assemblies, while N50 numbers increased by over 7-fold. Strikingly, the number of SNPs in PacBio-only assemblies were less than half that seen with short read assemblies (~20 SNPs vs. ~50 SNPs) and indels also saw dramatic reductions (~2 indel >5 bp in PacBio-only assemblies vs. ~12 for short-read only assemblies). Assemblies that used a mixture of PacBio and short read data generally fell in between these two extremes. Use of PacBio sequencing reads also allowed us to call covalent base modifications for the three strains. Each of the strains used here had a known covalent base modification genotype, which was confirmed by PacBio sequencing. CONCLUSION: Using data generated solely from the Pacific Biosciences RS, we were able to generate the most complete and accurate de novo assemblies of E. coli strains. We found that the addition of other sequencing technology data offered no improvements over use of PacBio data alone. In addition, the sequencing data from the PacBio RS allowed for sensitive and specific calling of covalent base modifications.
Project description:BACKGROUND:Chloroplasts are organelles that conduct photosynthesis in plant and algal cells. The information chloroplast genome contained is widely used in agriculture and studies of evolution and ecology. Correctly assembling chloroplast genomes can be challenging because the chloroplast genome contains a pair of long inverted repeats (10-30?kb). Typically, it is simply assumed that the gross structure of the chloroplast genome matches the most commonly observed structure of two single-copy regions separated by a pair of inverted repeats. The advent of long-read sequencing technologies should remove the need to make this assumption by providing sufficient information to completely span the inverted repeat regions. Yet, long-reads tend to have higher error rates than short-reads, and relatively little is known about the best way to combine long- and short-reads to obtain the most accurate chloroplast genome assemblies. Using Eucalyptus pauciflora, the snow gum, as a test case, we evaluated the effect of multiple parameters, such as different coverage of long-(Oxford nanopore) and short-(Illumina) reads, different long-read lengths, different assembly pipelines, with a view to determining the most accurate and efficient approach to chloroplast genome assembly. RESULTS:Hybrid assemblies combining at least 20x coverage of both long-reads and short-reads generated a single contig spanning the entire chloroplast genome with few or no detectable errors. Short-read-only assemblies generated three contigs (the long single copy, short single copy and inverted repeat regions) of the chloroplast genome. These contigs contained few single-base errors but tended to exclude several bases at the beginning or end of each contig. Long-read-only assemblies tended to create multiple contigs with a much higher single-base error rate. The chloroplast genome of Eucalyptus pauciflora is 159,942?bp, contains 131 genes of known function. CONCLUSIONS:Our results suggest that very accurate assemblies of chloroplast genomes can be achieved using a combination of at least 20x coverage of long- and short-reads respectively, provided that the long-reads contain at least ~5x coverage of reads longer than the inverted repeat region. We show that further increases in coverage give little or no improvement in accuracy, and that hybrid assemblies are more accurate than long-read-only or short-read-only assemblies.
Project description:PacBio long reads sequencing presents several potential advantages for DNA assembly, including being able to provide more complete gene profiling of metagenomic samples. However, lower single-pass accuracy can make gene discovery and assembly for low-abundance organisms difficult. To evaluate the application and performance of PacBio long reads and Illumina HiSeq short reads in metagenomic analyses, we directly compared various assemblies involving PacBio and Illumina sequencing reads based on two anaerobic digestion microbiome samples from a biogas fermenter. Using a PacBio platform, 1.58 million long reads (19.6 Gb) were produced with an average length of 7,604 bp. Using an Illumina HiSeq platform, 151.2 million read pairs (45.4 Gb) were produced. Hybrid assemblies using PacBio long reads and HiSeq contigs produced improvements in assembly statistics, including an increase in the average contig length, contig N50 size, and number of large contigs. Interestingly, depth-based hybrid assemblies generated a higher percentage of complete genes (98.86%) compared to those based on HiSeq contigs only (40.29%), because the PacBio reads were long enough to cover many repeating short elements and capture multiple genes in a single read. Additionally, the incorporation of PacBio long reads led to considerable advantages regarding reducing contig numbers and increasing the completeness of the genome reconstruction, which was poorly assembled and binned when using HiSeq data alone. From this comparison of PacBio long reads with Illumina HiSeq short reads related to complex microbiome samples, we conclude that PacBio long reads can produce longer contigs, more complete genes, and better genome binning, thereby offering more information about metagenomic samples.
Project description:The chloroplast genome harbors plenty of valuable information for phylogenetic research. Illumina short-read data is generally used for de novo assembly of whole plastomes. PacBio or Oxford Nanopore long reads are additionally employed in hybrid approaches to enable assembly across the highly similar inverted repeats of a chloroplast genome. Unlike for PacBio, plastome assemblies based solely on Nanopore reads are rarely found, due to their high error rate and non-random error profile. However, the actual quality decline connected to their use has rarely been quantified. Furthermore, no study has employed reference-based assembly using Nanopore reads, which is common with Illumina data. Using Leucanthemum Mill. as an example, we compared the sequence quality of seven chloroplast genome assemblies of the same species, using combinations of two sequencing platforms and three analysis pipelines. In addition, we assessed the factors which might influence Nanopore assembly quality during sequence generation and bioinformatic processing. The consensus sequence derived from de novo assembly of Nanopore data had a sequence identity of 99.59% compared to Illumina short-read de novo assembly. Most of the errors detected were indels (81.5%), and a large majority of them is part of homopolymer regions. The quality of reference-based assembly is heavily dependent upon the choice of a close-enough reference. When using a reference with 0.83% sequence divergence from the studied species, mapping of Nanopore reads results in a consensus comparable to that from Nanopore de novo assembly, and of only slightly inferior quality compared to a reference-based assembly with Illumina data. For optimal de novo assembly of Nanopore data, appropriate filtering of contaminants and chimeric sequences, as well as employing moderate read coverage, is essential. Based on these results, we conclude that Nanopore long reads are a suitable alternative to Illumina short reads in plastome phylogenomics. Few errors remain in the finalized assembly, which can be easily masked in phylogenetic analyses without loss in analytical accuracy. The easily applicable and cost-effective technology might warrant more attention by researchers dealing with plant chloroplast genomes.
Project description:BACKGROUND: With high quantity and quality data production and low cost, next generation sequencing has the potential to provide new opportunities for plant phylogeographic studies on single and multiple species. Here we present an approach for in silicio chloroplast DNA assembly and single nucleotide polymorphism detection from short-read shotgun sequencing. The approach is simple and effective and can be implemented using standard bioinformatic tools. RESULTS: The chloroplast genome of Toona ciliata (Meliaceae), 159,514 base pairs long, was assembled from shotgun sequencing on the Illumina platform using de novo assembly of contigs. To evaluate its practicality, value and quality, we compared the short read assembly with an assembly completed using 454 data obtained after chloroplast DNA isolation. Sanger sequence verifications indicated that the Illumina dataset outperformed the longer read 454 data. Pooling of several individuals during preparation of the shotgun library enabled detection of informative chloroplast SNP markers. Following validation, we used the identified SNPs for a preliminary phylogeographic study of T. ciliata in Australia and to confirm low diversity across the distribution. CONCLUSIONS: Our approach provides a simple method for construction of whole chloroplast genomes from shotgun sequencing of whole genomic DNA using short-read data and no available closely related reference genome (e.g. from the same species or genus). The high coverage of Illumina sequence data also renders this method appropriate for multiplexing and SNP discovery and therefore a useful approach for landscape level studies of evolutionary ecology.
Project description:OBJECTIVE:Passalora sequoiae (family Mycosphaerellaceae) causes a twig blight on Leyland cypress that requires numerous fungicide applications annually to minimize economic losses for ornamental plant nursery and Christmas tree producers. The objective was to generate a high-quality draft assembly of the genome of P. sequoiae as a resource for primer development to investigate genotype diversity. DATA DESCRIPTION:We report here the genome sequence of P. sequoiae 9LC2 that was isolated from Leyland cypress 'Leighton Green' in 2017 in southern Mississippi, USA. The draft genome was obtained using Pacific Biosciences (PacBio) SMRT and Illumina HiSeq 2500 sequencing. Illumina reads were mapped to PacBio assembled contigs to determine base call consistency. Based on a total of 44 contigs with 722 kilobase (kb) average length (range 9.4 kb to 3.4 Mb), the whole genome size was estimated at 31,768,716 bp. Mapping of Illumina reads to PacBio contigs resulted in a 1000?×?coverage and were used to confirm accuracy of the consensus sequences.