The power of single molecule real-time sequencing technology in the de novo assembly of a eukaryotic genome

Sakai, Hiroaki; Naito, Kunihiko; Ogiso‐Tanaka, Eri; Takahashi, Yu; Iseki, Kazuo; Muto, Chiaki; Satou, Kazuhito; Teruya, Kuniko; Shiroma, Akino; Shimoji, Makiko; Hirano, Takashi; Itoh, Takeshi; Kaga, Akito; Tomooka, Norihiko

doi:10.1038/srep16780

Cited by 71 publications

(58 citation statements)

References 62 publications

(80 reference statements)

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“…The homozygous variants amounted to 1,532 SNPs, 20,479 deletions and 6,549 insertions showing that the assembly had 99.99% base accuracy. The insertion-deletion (in-del) errors had outnumbered the substitution errors, similar to the results observed in PacBio-based Vigna angularis 20 and Oropetium thomaeum 21 genome assemblies, and were replaced with the Illumina sequence bases. Mitochondrial and chloroplast derived sequences were identified to be 1.15 Mb from 51 contigs and were removed.…”

Section: Resultssupporting

confidence: 59%

“…However, a better resolution of such repeats was obtained in Oropetium thomaeum 21 genome assembly, possibly owing to the 15-kb lower end insert size selection, explaining the importance of longer read lengths in obtaining near-perfect assemblies. The potential of PacBio sequence data in long, eukaryotic genomes has been further showcased in the draft genomes of Gorilla gorilla 45 (scaffold N50 of 23.1 Mb), V. angularis 20 (scaffold N50 of 3.0 Mb), O. thomaeum 21 (contig N50 of 2.4 Mb) and Lates calcarifer 46 (scaffold N50 of 1.19 Mb). A rapid increase in PacBio sequencing for similar large-scale assemblies can be expected in the near future.…”

Section: Discussionmentioning

confidence: 99%

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Genome sequence and analysis of the Japanese morning glory Ipomoea nil

et al. 2016

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Ipomoea is the largest genus in the family Convolvulaceae. Ipomoea nil (Japanese morning glory) has been utilized as a model plant to study the genetic basis of floricultural traits, with over 1,500 mutant lines. In the present study, we have utilized second- and third-generation-sequencing platforms, and have reported a draft genome of I. nil with a scaffold N50 of 2.88 Mb (contig N50 of 1.87 Mb), covering 98% of the 750 Mb genome. Scaffolds covering 91.42% of the assembly are anchored to 15 pseudo-chromosomes. The draft genome has enabled the identification and cataloguing of the Tpn1 family transposons, known as the major mutagen of I. nil, and analysing the dwarf gene, CONTRACTED, located on the genetic map published in 1956. Comparative genomics has suggested that a whole genome duplication in Convolvulaceae, distinct from the recent Solanaceae event, has occurred after the divergence of the two sister families.

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Section: Resultssupporting

confidence: 59%

Section: Discussionmentioning

confidence: 99%

Genome sequence and analysis of the Japanese morning glory Ipomoea nil

et al. 2016

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“…However, the genome coverage rates of the assembly were low both in quinoa (73%) and P. veris (63%). Recently, near-complete genome assembly of a diploid species, Vigna angularis (azuki bean), was achieved using 27.6 Gbp of the PacBio long reads, which corresponds to 51× coverage of the genome size 68 . The authors constructed 2,529 scaffolds (N50 = 3.0 Mbp), covering 97.1% of the V. angularis genome.…”

Section: Resultsmentioning

confidence: 99%

“…In our analysis of quinoa, we attained a genome coverage of 30×. Given the complexity of the quinoa genome resulting from ploidy and large genome size, a much greater coverage than 51× of the genome size is needed to use the method reported by Sakai et al 68 Attaining a greater genome coverage is our next objective for refining our quinoa assembly. …”

Section: Resultsmentioning

confidence: 99%

Draft genome sequence of an inbred line ofChenopodium quinoa, an allotetraploid crop with great environmental adaptability and outstanding nutritional properties

Yasui

Hirakawa

Oikawa

et al. 2016

DNA Res

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Chenopodium quinoa Willd. (quinoa) originated from the Andean region of South America, and is a pseudocereal crop of the Amaranthaceae family. Quinoa is emerging as an important crop with the potential to contribute to food security worldwide and is considered to be an optimal food source for astronauts, due to its outstanding nutritional profile and ability to tolerate stressful environments. Furthermore, plant pathologists use quinoa as a representative diagnostic host to identify virus species. However, molecular analysis of quinoa is limited by its genetic heterogeneity due to outcrossing and its genome complexity derived from allotetraploidy. To overcome these obstacles, we established the inbred and standard quinoa accession Kd that enables rigorous molecular analysis, and presented the draft genome sequence of Kd, using an optimized combination of high-throughput next generation sequencing on the Illumina Hiseq 2500 and PacBio RS II sequencers. The de novo genome assembly contained 25 k scaffolds consisting of 1 Gbp with N50 length of 86 kbp. Based on these data, we constructed the free-access Quinoa Genome DataBase (QGDB). Thus, these findings provide insights into the mechanisms underlying agronomically important traits of quinoa and the effect of allotetraploidy on genome evolution.

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“…It has been used for the sequencing of bacterial genomes such as the plant pathogen Xanthomonas oryzae [4]. PacBio reads have also been used for the sequencing of complex plant nuclear genomes, such as that of the Adzuki bean, Vigna angularis [5], demonstrating the advantage of this technology for resolving repetitive regions during sequence assembly.…”

Section: Introductionmentioning

confidence: 99%

Organelle_PBA, a pipeline for assembling chloroplast and mitochondrial genomes from PacBio DNA sequencing data

et al. 2017

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BackgroundThe development of long-read sequencing technologies, such as single-molecule real-time (SMRT) sequencing by PacBio, has produced a revolution in the sequencing of small genomes. Sequencing organelle genomes using PacBio long-read data is a cost effective, straightforward approach. Nevertheless, the availability of simple-to-use software to perform the assembly from raw reads is limited at present.ResultsWe present Organelle-PBA, a Perl program designed specifically for the assembly of chloroplast and mitochondrial genomes. For chloroplast genomes, the program selects the chloroplast reads from a whole genome sequencing pool, maps the reads to a reference sequence from a closely related species, and then performs read correction and de novo assembly using Sprai. Organelle-PBA completes the assembly process with the additional step of scaffolding by SSPACE-LongRead. The program then detects the chloroplast inverted repeats and reassembles and re-orients the assembly based on the organelle origin of the reference. We have evaluated the performance of the software using PacBio reads from different species, read coverage, and reference genomes. Finally, we present the assembly of two novel chloroplast genomes from the species Picea glauca (Pinaceae) and Sinningia speciosa (Gesneriaceae).ConclusionOrganelle-PBA is an easy-to-use Perl-based software pipeline that was written specifically to assemble mitochondrial and chloroplast genomes from whole genome PacBio reads. The program is available at https://github.com/aubombarely/Organelle_PBA.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3412-9) contains supplementary material, which is available to authorized users.

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The power of single molecule real-time sequencing technology in the de novo assembly of a eukaryotic genome

Cited by 71 publications

References 62 publications

Genome sequence and analysis of the Japanese morning glory Ipomoea nil

Genome sequence and analysis of the Japanese morning glory Ipomoea nil

Draft genome sequence of an inbred line ofChenopodium quinoa, an allotetraploid crop with great environmental adaptability and outstanding nutritional properties

Organelle_PBA, a pipeline for assembling chloroplast and mitochondrial genomes from PacBio DNA sequencing data

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