Phased diploid genome assembly with single-molecule real-time sequencing

Chin, Chen-Shan; Peluso, Paul; Sedlazeck, Fritz J.; Nattestad, Maria; Concepcion, Gregory T.; Clum, Alicia; Dunn, Christopher; O’Malley, Ronan; Figueroa‐Balderas, Rosa; Morales‐Cruz, Abraham; Cramer, Grant R.; Delledonne, Massimo; Luo, Chongyuan; Ecker, Joseph R.; Cantu, Dario; Rank, David R.; Schatz, Michael C.

doi:10.1038/nmeth.4035

Cited by 1,719 publications

(1,709 citation statements)

References 42 publications

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“…We used the diploid aware PacBio assembler FALCON 0.3.0 (ref. 32), which has recently been used to successfully assemble highly heterozygous genomes 33 , to assemble the F. cylindrus genome. The cut off for long reads was 2,000 bp.…”

Section: Methodsmentioning

confidence: 99%

Evolutionary genomics of the cold-adapted diatom Fragilariopsis cylindrus

Möck

Otillar

Strauss

et al. 2017

Nature

322

303

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The Southern Ocean houses a diverse and productive community of organisms 1,2 . Unicellular eukaryotic diatoms are the main primary producers in this environment, where photosynthesis is limited by low concentrations of dissolved iron and large seasonal fluctuations in light, temperature and the extent of sea ice 3-7 . How diatoms have adapted to this extreme environment is largely unknown. Here we present insights into the genome evolution of a cold-adapted diatom from the Southern Ocean, Fragilariopsis cylindrus 8,9 , based on a comparison with temperate diatoms. We find that approximately 24.7 per cent of the diploid F. cylindrus genome consists of genetic loci with alleles that are highly divergent (15.1 megabases of the total genome size of 61.1 megabases). These divergent alleles were differentially expressed across environmental conditions, including darkness, low iron, freezing, elevated temperature and increased CO 2 . Alleles with the largest ratio of non-synonymous to synonymous nucleotide substitutions also show the most pronounced condition-dependent expression, suggesting a correlation between diversifying selection and allelic differentiation.

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Section: Methodsmentioning

confidence: 99%

Evolutionary genomics of the cold-adapted diatom Fragilariopsis cylindrus

Möck

Otillar

Strauss

et al. 2017

Nature

322

303

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“…However, for the same reasons that a de novo assembly can carry more information than a simple catalog of differences with a reference sequence, these "phased haploid" assemblies carry less information than true diploid assemblies that separately display homologous loci. In a few cases, these diploid de novo assemblies have been demonstrated for small and midsized genomes (Jones et al 2004;Chin et al 2016). There are two extant instances of diploid de novo assemblies of human genomes-one obtained by Sanger sequencing of multiple libraries (Levy et al 2007), and one from thousands of separate clone pools, each representing a small, low-throughput partition of the genome (Cao et al 2015).…”

mentioning

confidence: 99%

Direct determination of diploid genome sequences

et al. 2017

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Determining the genome sequence of an organism is challenging, yet fundamental to understanding its biology. Over the past decade, thousands of human genomes have been sequenced, contributing deeply to biomedical research. In the vast majority of cases, these have been analyzed by aligning sequence reads to a single reference genome, biasing the resulting analyses, and in general, failing to capture sequences novel to a given genome. Some de novo assemblies have been constructed free of reference bias, but nearly all were constructed by merging homologous loci into single "consensus" sequences, generally absent from nature. These assemblies do not correctly represent the diploid biology of an individual. In exactly two cases, true diploid de novo assemblies have been made, at great expense. One was generated using Sanger sequencing, and one using thousands of clone pools. Here, we demonstrate a straightforward and low-cost method for creating true diploid de novo assemblies. We make a single library from ∼1 ng of high molecular weight DNA, using the 10x Genomics microfluidic platform to partition the genome. We applied this technique to seven human samples, generating low-cost HiSeq X data, then assembled these using a new "pushbutton" algorithm, Supernova. Each computation took 2 d on a single server. Each yielded contigs longer than 100 kb, phase blocks longer than 2.5 Mb, and scaffolds longer than 15 Mb. Our method provides a scalable capability for determining the actual diploid genome sequence in a sample, opening the door to new approaches in genomic biology and medicine.

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“…Similarly, long reads performed using the PacBio platform coupled to the fast alignment and consensus for assembly (FALCON)-Unzip genome assembler have been used to successfully define large-scale haplotypespecific assemblies of Arabidopsis trios and Vitis vinifera cv. Cabernet Sauvignon, which is a highly heterozygous F 1 hybrid 32 .…”

Section: Methods For Linking Sequences In Genome Assemblymentioning

confidence: 99%

“…This has proved useful for the production of higher-quality plant-genome assemblies and has been applied to the de novo assembly of smaller plant genomes such as Arabidopsis and the grape vine Vitis vinifera cv. Cabernet Sauvignon 32 .…”

Section: Review Insightmentioning

confidence: 99%

Genomic innovation for crop improvement

Bevan

Uauy

Wulff

et al. 2017

Nature

331

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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Phased diploid genome assembly with single-molecule real-time sequencing

Cited by 1,719 publications

References 42 publications

Evolutionary genomics of the cold-adapted diatom Fragilariopsis cylindrus

Evolutionary genomics of the cold-adapted diatom Fragilariopsis cylindrus

Direct determination of diploid genome sequences

Genomic innovation for crop improvement

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