Oxford Nanopore Sequencing, Hybrid Error Correction, and<i>de novo</i>Assembly of a Eukaryotic Genome

Goodwin, Sara; Gurtowski, James; Ethe-Sayers, Scott; Deshpande, Panchajanya; Schatz, Michael C.; McCombie, W. Richard

doi:10.1101/013490

Cited by 52 publications

(51 citation statements)

References 21 publications

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“…The MinION works on the principle of nanopore strand sequencing in real time Jain et al 2016) and results in sequence read lengths from several hundred bases to hundreds of thousands of bases. To date, most studies have used the MinION to sequence small genomes or to partially survey larger genomes to assess chromosomal structure and copy-number variations (Goodwin et al 2015;Loman et al 2015;Norris et al 2016;Wei and Williams 2016). The long read lengths combined with recent improvements in performance and the development of software to assemble genomes from long sequence reads ) make MinION sequencing a viable option for whole-genome sequencing of complex metazoan genomes.…”

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confidence: 99%

MinION-based long-read sequencing and assembly extends the Caenorhabditis elegans reference genome

et al. 2017

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Advances in long-read single molecule sequencing have opened new possibilities for ‘benchtop’ whole-genome sequencing. The Oxford Nanopore Technologies MinION is a portable device that uses nanopore technology that can directly sequence DNA molecules. MinION single molecule long sequence reads are well suited for de novo assembly of complex genomes as they facilitate the construction of highly contiguous physical genome maps obviating the need for labor-intensive physical genome mapping. Long sequence reads can also be used to delineate complex chromosomal rearrangements, such as those that occur in tumor cells, that can confound analysis using short reads. Here, we assessed MinION long-read-derived sequences for feasibility concerning: (1) the de novo assembly of a large complex genome, and (2) the elucidation of complex rearrangements. The genomes of two Caenorhabditis elegans strains, a wild-type strain and a strain containing two complex rearrangements, were sequenced with MinION. Up to 42-fold coverage was obtained from a single flow cell, and the best pooled data assembly produced a highly contiguous wild-type C. elegans genome containing 48 contigs (N50 contig length = 3.99 Mb) covering >99% of the 100,286,401-base reference genome. Further, the MinION-derived genome assembly expanded the C. elegans reference genome by >2 Mb due to a more accurate determination of repetitive sequence elements and assembled the complete genomes of two co-extracted bacteria. MinION long-read sequence data also facilitated the elucidation of complex rearrangements in a mutagenized strain. The sequence accuracy of the MinION long-read contigs (∼98%) was improved using Illumina-derived sequence data to polish the final genome assembly to 99.8% nucleotide accuracy when compared to the reference assembly.

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confidence: 99%

MinION-based long-read sequencing and assembly extends the Caenorhabditis elegans reference genome

et al. 2017

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“…After a nanopore specific option was added by the authors of BWA-MEM (Li & Durbin 2009), it soon attained ascendancy. Good results can also be obtained from BLAST, especially with tuned penalty parameters (Goodwin et al 2015). marginAlign is a nanopore-specific alignment tool that uses expectation maximisation to train an HMM and estimate Maximum Likelihood Estimation parameters in order to find higher confidence alignments (Jain et al 2015).…”

Section: Discussionmentioning

confidence: 99%

“…The assembler SPAdes has an option to utilise Nanopore or PacBio data for gap closure and repeat resolution in hybrid assemblies with Illumina data (Bankevich et al 2012). Nanocorr uses alignments of Illumina reads against nanopore reads to correct the longer reads (Goodwin et al 2015). MiSeq reads are aligned against the nanopore reads and an algorithm selects an optimal set of alignments spanning the read.…”

Section: Genome Assemblymentioning

confidence: 99%

A world of opportunities with nanopore sequencing

Leggett

Clark

2017

Preprint

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Oxford Nanopore Technologies' MinION sequencer was launched in pre-release form in 2014 and represents an exciting new sequencing paradigm. The device offers multi-kilobase reads and a streamed mode of operation that allows processing of reads as they are generated. Crucially, it is an extremely compact device that is powered from the USB port of a laptop computer, enabling it to be taken out of the lab and facilitating previously impossible in-field sequencing experiments to be contemplated. Many of the initial publications concerning the platform focussed on provision of tools to access and analyse the new sequence formats and then demonstrating the assembly of microbial genomes. More recently, as throughput and accuracy have increased, it has been possible to consider work involving more complex genomes and metagenomes. With the release of the high throughput GridION X5 and PromethION platforms the sequencing of large genomes will become more cost efficient, and enable the leveraging of extremely long (>100kb) reads for resolution of complex genomic structures. This review provides a brief overview of nanopore sequencing technology, describes the growing range of nanopore bioinformatics tools and highlights some of the most influential publications that have emerged over the last two years. Finally, we look to the future and the potential the platform has to disrupt work in human, microbiome and plant genomics.

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“…However, this technology is at an early stage of development with respect to the sequencing of large genomes and has a fairly high rate of error. Initial nanopore-based genome assemblies used a hybrid approach with short-read Illumina sequences to correct such errors in nanopore yeast-genome assemblies 33,34 . More recent progress has been rapid: for example, the 29× coverage of the Escherichia coli genome that was achieved with the long-read nanopore sequencing method MinION was sufficient to generate a single 4.9 Mb contig of 99.5% accuracy 35 .…”

Section: Review Insightmentioning

confidence: 99%

Genomic innovation for crop improvement

Bevan

Uauy

Wulff

et al. 2017

Nature

341

240

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Crop production needs to increase to secure future food supplies, while reducing its impact on ecosystems. Detailed characterization of plant genome structure and genetic diversity is crucial for meeting these challenges. Advances in genome sequencing and assembly are being used to access to the large and complex genomes of crops and their wild relatives. Sequencing of wild crop relatives is identifying a wide spectrum of genetic variation, permitting the association of genetic diversity with diverse agronomic phenotypes. In combination with improved and automated phenotyping assays and functional genomic studies, genomics is providing new foundations for crop-breeding systems. In the twentieth century, famines caused by political crises, mismanagement of food production or genocide killed an estimated 70 million people and were second only to war as the greatest manmade cause of death 1 . The father of the Green Revolution, Norman Borlaug, summarized the importance of crops: "Without food, people perish, social and political organizations disintegrate, and civilizations collapse. " For thousands of years, people have dedicated considerable resources to securing food supplies; for example, grain supplies to ancient Rome were secured through an extensive network of long-distance transport, and the distribution of grain was coordinated and subsidized by the cura annonae ('care for the grain supply'), an important figure who contributed to the maintenance of political unity and power.During the past 10,000 years, a period known as the Holocene, Earth's environment has been unusually stable. This probably facilitated the domestication of crops from wild species, resulting in steadily improved yields and adaptation to new agricultural areas. The production of food is now carried out on a vast scale, with 38% of Earth's surface dedicated to agriculture 2 . This increased production is having a pervasive influence on ecosystems worldwide: nitrogen production for agriculture accounts for 1.2% of global energy consumption 3 ; photosynthesis can no longer maintain stable levels of atmospheric carbon dioxide; and food production consumes around 70% of freshwater supplies. The modelling of crop responses to increases in temperature predicts that there will be a considerable reduction in the yield of rice, an important crop around the world 4 . Climate change could also alter the dynamics of crop pathogenic agents by altering the range of vectors and by compromising the immune response of crops 5 .Crop production must therefore adapt to more variable environments and the substantial impact it has on the environment needs to be reduced. Productivity must also increase at a much greater rate than in the past to meet the needs of Earth's growing population 6 . Genetic improvements in crop performance continue to be crucial for increasing crop productivity, but current rates of improvement are unable to meet the demands of sustainability and food security 7 . Plant genomics has a central role in the improvement of crops, includi...

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Oxford Nanopore Sequencing, Hybrid Error Correction, andde novoAssembly of a Eukaryotic Genome

Cited by 52 publications

References 21 publications

MinION-based long-read sequencing and assembly extends the Caenorhabditis elegans reference genome

MinION-based long-read sequencing and assembly extends the Caenorhabditis elegans reference genome

A world of opportunities with nanopore sequencing

Genomic innovation for crop improvement

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