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Nat Biotechnol. 2018 Oct 15. doi: 10.1038/nbt.4266. [Epub ahead of print]

High-quality genome sequences of uncultured microbes by assembly of read clouds.

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Department of Computer Science, Stanford University, Stanford, California, USA.
Department of Medicine (Hematology, Blood and Marrow Transplantation) and Department of Genetics, Stanford University, Stanford, California, USA.
Department of Computer Science and Engineering, University of California San Diego, La Jolla, California, USA.
Department of Earth System Science, Stanford University, Stanford, California, USA.
Department of Pathology, Stanford University School of Medicine, Stanford, California, USA.


Although shotgun metagenomic sequencing of microbiome samples enables partial reconstruction of strain-level community structure, obtaining high-quality microbial genome drafts without isolation and culture remains difficult. Here, we present an application of read clouds, short-read sequences tagged with long-range information, to microbiome samples. We present Athena, a de novo assembler that uses read clouds to improve metagenomic assemblies. We applied this approach to sequence stool samples from two healthy individuals and compared it with existing short-read and synthetic long-read metagenomic sequencing techniques. Read-cloud metagenomic sequencing and Athena assembly produced the most comprehensive individual genome drafts with high contiguity (>200-kb N50, fewer than ten contigs), even for bacteria with relatively low (20×) raw short-read-sequence coverage. We also sequenced a complex marine-sediment sample and generated 24 intermediate-quality genome drafts (>70% complete, <10% contaminated), nine of which were complete (>90% complete, <5% contaminated). Our approach allows for culture-free generation of high-quality microbial genome drafts by using a single shotgun experiment.


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