Assembling Large Genomes with Single-Molecule Sequencing and Locality Sensitive Hashing

Berlin, Konstantin; Koren, Sergey; Chin, Chen-Shan; Drake, James; Landolin, Jane M.; Phillippy, Adam M.

doi:10.1101/008003

Cited by 189 publications

(350 citation statements)

References 88 publications

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“…De novo assembly of the genome sequencing data. De novo assembly of the long reads from SMRT Sequencing was performed using two assemblers: the Celera Assembler PBcR -MHAP pipeline 33 and Falcon 34 with different parameter settings. Quiver from SMRT Analysis v2.3.0 was used to polish base calling of contigs.…”

Section: Methodsmentioning

confidence: 99%

Improved maize reference genome with single-molecule technologies

Jiao

Peluso

Shi

et al. 2017

Nature

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1,107

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Complete and accurate reference genomes and annotations provide fundamental tools for characterization of genetic and functional variation 1 . These resources facilitate the determination of biological processes and support translation of research findings into improved and sustainable agricultural technologies. Many reference genomes for crop plants have been generated over the past decade, but these genomes are often fragmented and missing complex repeat regions 2 . Here we report the assembly and annotation of a reference genome of maize, a genetic and agricultural model species, using single-molecule real-time sequencing and high-resolution optical mapping. Relative to the previous reference genome 3 , our assembly features a 52-fold increase in contig length and notable improvements in the assembly of intergenic spaces and centromeres. Characterization of the repetitive portion of the genome revealed more than 130,000 intact transposable elements, allowing us to identify transposable element lineage expansions that are unique to maize. Gene annotations were updated using 111,000 full-length transcripts obtained by single-molecule real-time sequencing 4 . In addition, comparative optical mapping of two other inbred maize lines revealed a prevalence of deletions in regions of low gene density and maize lineage-specific genes.

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Section: Methodsmentioning

confidence: 99%

Improved maize reference genome with single-molecule technologies

Jiao

Peluso

Shi

et al. 2017

Nature

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1,107

1,153

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“…This suggests that contig break points occur at the start of repeats or that most assembly breaks are caused by other factors, such as within-genome heterozygosity or haplotype-specific structural variation. To test this, we also tried 'diploid-aware' assemblers Falcon (https://github.com/PacificBiosciences/ falcon) and MinHash Alignment Process (MHAP) 14 . These assemblies had similar metrics but were less contiguous overall (Extended Data Fig.…”

mentioning

confidence: 99%

Single-molecule sequencing of the desiccation-tolerant grass Oropetium thomaeum

VanBuren

Bryant

Edger

et al. 2015

Nature

290

271

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Plant genomes, and eukaryotic genomes in general, are typically repetitive, polyploid and heterozygous, which complicates genome assembly 1 . The short read lengths of early Sanger and current next-generation sequencing platforms hinder assembly through complex repeat regions, and many draft and reference genomes are fragmented, lacking skewed GC and repetitive intergenic sequences, which are gaining importance due to projects like the Encyclopedia of DNA Elements (ENCODE) 2 . Here we report the whole-genome sequencing and assembly of the desiccationtolerant grass Oropetium thomaeum. Using only single-molecule real-time sequencing, which generates long (>16 kilobases) reads with random errors, we assembled 99% (244 megabases) of the Oropetium genome into 625 contigs with an N50 length of 2.4 megabases. Oropetium is an example of a 'near-complete' draft genome which includes gapless coverage over gene space as well as intergenic sequences such as centromeres, telomeres, transposable elements and rRNA clusters that are typically unassembled in draft genomes. Oropetium has 28,466 protein-coding genes and 43% repeat sequences, yet with 30% more compact euchromatic regions it is the smallest known grass genome. The Oropetium genome demonstrates the utility of single-molecule real-time sequencing for assembling high-quality plant and other eukaryotic genomes, and serves as a valuable resource for the plant comparative genomics community.The genomes of Arabidopsis 3 , rice 4 , poplar, grape and Sorghum 5 were first sequenced using high-quality and reiterative Sanger-based approaches producing a series of 'gold standard' reference genomes. The advent of next-generation sequencing (NGS) technologies reduced costs of sequencing substantially, which has enabled sequencing of over 100 plant genomes 1 . The quality of plant genome assemblies depends on genome size, ploidy, heterozygosity and sequence coverage, but most NGS-based genomes have on the order of tens of thousands of short contigs distributed in thousands of scaffolds. The short read lengths of NGS, inherent biases and non-random sequencing errors have resulted in highly fragmented draft genome assemblies that are not complete, which means they are missing biologically meaningful sequences including entire genes, regulatory regions, transposable elements, centromeres, telomeres and haplotype-specific structural variations. It is becoming clear from ENCODE projects that complete genomes are needed to better understand the importance of the non-coding regions of genomes 2 .More than 40% of calories consumed by humans are derived from grasses, and the grass family (Poaceae) is arguably the most important plant family with regard to global food security 6 . The size and complexity of most grass genomes has challenged progress in gene discovery and comparative genomics, although draft genomes are now available for most agriculturally important grasses 1 . The largest genome assemblies, such as maize (2,300 megabases (Mb)) 7 , barley (5,100 Mb) 8 and wheat (hexaploid, 1...

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“…We assembled the PacBio reads from A. filiculoides and S. cucullata genomes using PBcR 61 , and the resulting drafts were then polished by Quiver 62 (A. filiculoides) or Pilon 63 (S. cucullata). Plastid genomes were separately assembled using Mitobim 64 and annotated in Geneious 65 with manual adjustments.…”

Section: Methodsmentioning

confidence: 99%

Azolla, a little fern with massive green potential

Pryer¹

2014

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Ferns are the closest sister group to all seed plants, yet little is known about their genomes other than that they are generally colossal. Here, we report on the genomes of Azolla filiculoides and Salvinia cucullata (Salviniales) and present evidence for episodic whole-genome duplication in ferns-one at the base of 'core leptosporangiates' and one specific to Azolla. One fernspecific gene that we identified, recently shown to confer high insect resistance, seems to have been derived from bacteria through horizontal gene transfer. Azolla coexists in a unique symbiosis with N 2 -fixing cyanobacteria, and we demonstrate a clear pattern of cospeciation between the two partners. Furthermore, the Azolla genome lacks genes that are common to arbuscular mycorrhizal and root nodule symbioses, and we identify several putative transporter genes specific to Azolla-cyanobacterial symbiosis. These genomic resources will help in exploring the biotechnological potential of Azolla and address fundamental questions in the evolution of plant life.

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Assembling Large Genomes with Single-Molecule Sequencing and Locality Sensitive Hashing

Cited by 189 publications

References 88 publications

Improved maize reference genome with single-molecule technologies

Improved maize reference genome with single-molecule technologies

Single-molecule sequencing of the desiccation-tolerant grass Oropetium thomaeum

Azolla, a little fern with massive green potential

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