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

Individual microbes in metagenomic samples are sequenced by assembly of barcoded short reads 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...

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Published inNature biotechnology Vol. 36; no. 11; pp. 1067 - 1075
Main Authors Bishara, Alex, Moss, Eli L, Kolmogorov, Mikhail, Parada, Alma E, Weng, Ziming, Sidow, Arend, Dekas, Anne E, Batzoglou, Serafim, Bhatt, Ami S
Format Journal Article
LanguageEnglish
Published New York Nature Publishing Group US 01.12.2018
Nature Publishing Group
Subjects
45
45/22
45/23
631/208/212/2142
631/208/212/2302
631/326/171
631/326/2565
631/326/41
Agriculture
Analysis
Assembly
Bacteria
Bioinformatics
Biomedical Engineering/Biotechnology
Biomedicine
Biotechnology
Clouds
Community structure
Gene sequencing
Genomes
Genomics
Life Sciences
Microbial genetics
Microbiomes
Microorganisms
Scientific software
Sediment samplers
Sediments (Geology)
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Abstract Individual microbes in metagenomic samples are sequenced by assembly of barcoded short reads 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.
AbstractList 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.
Although shotgun metagenomic sequencing of microbiome samples enables partial reconstruction of the strain-level community structure, it remains difficult to obtain high-quality microbial genome drafts without isolation and culture. Here we present a novel 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 apply this approach to sequence stool samples from two healthy individuals, and compare it to existing short-read and synthetic long-read metagenomic sequencing techniques. Read cloud metagenomic sequencing and Athena assembly produce the most complete individual genome drafts with high contiguity (>200 kbp N50, <10 contigs), even for bacteria that have relatively low (20x) raw short-read sequence coverage. We also sequence a complex marine sediment sample and generate 24 intermediate-quality genome drafts (>70% complete, <10% contaminated), nine of which are complete (>90% complete, <5% contaminated). Thus, our approach allows culture-free generation of high-quality microbial genome drafts using a single shotgun experiment.
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 ( [greater than] 200-kb N50, fewer than ten contigs), even for bacteria with relatively low (20x) raw short-read-sequence coverage. We also sequenced a complex marine-sediment sample and generated 24 intermediate-quality genome drafts ( [greater than] 70% complete, [less than] 10% contaminated), nine of which were complete ( [greater than] 90% complete, [less than] 5% contaminated). Our approach allows for culture-free generation of high-quality microbial genome drafts by using a single shotgun experiment.
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.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.
Individual microbes in metagenomic samples are sequenced by assembly of barcoded short reads 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.
Audience Academic
Author Parada, Alma E
Weng, Ziming
Sidow, Arend
Bishara, Alex
Kolmogorov, Mikhail
Bhatt, Ami S
Moss, Eli L
Batzoglou, Serafim
Dekas, Anne E
AuthorAffiliation 3 Department of Computer Science and Engineering, University of California San Diego, La Jolla, California, USA
2 Department of Medicine (Hematology, Blood and Marrow Transplantation) and Department of Genetics, Stanford University, Stanford, California, USA
4 Department of Earth System Science, Stanford University, Stanford, CA, USA
1 Department of Computer Science, Stanford University, Stanford, California, USA
5 Department of Pathology, Stanford University School of Medicine, Stanford, California, USA
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– name: 4 Department of Earth System Science, Stanford University, Stanford, CA, USA
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  organization: Department of Medicine (Hematology, Blood and Marrow Transplantation) and Department of Genetics, Stanford University
BackLink https://www.ncbi.nlm.nih.gov/pubmed/30320765$$D View this record in MEDLINE/PubMed
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COPYRIGHT 2018 Nature Publishing Group
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A.B., E.L.M., A.S.B. and S.B. conceived of the study. Z.W. prepared read cloud libraries. E.L.M. extracted DNA and prepared SLR sequencing libraries. E.L.M. performed PCR validation, and Sanger sequencing. A.B. and S.B. conceived of the assembly approach. A.B. implemented the Athena assembler. M.K. modified the Flye assembler for use with Athena. A.E.P. and A.E.D. collected marine sediment sample, extracted DNA from the marine sediment sample and helped in analysis of these samples. A.B, A.S.B. and E.L.M. carried out all analyses, wrote the manuscript, and generated figures. All authors commented on the manuscript.
Author contributions
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Snippet Individual microbes in metagenomic samples are sequenced by assembly of barcoded short reads Although shotgun metagenomic sequencing of microbiome samples...
Although shotgun metagenomic sequencing of microbiome samples enables partial reconstruction of strain-level community structure, obtaining high-quality...
Although shotgun metagenomic sequencing of microbiome samples enables partial reconstruction of the strain-level community structure, it remains difficult to...
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SubjectTerms 45
45/22
45/23
631/208/212/2142
631/208/212/2302
631/326/171
631/326/2565
631/326/41
Agriculture
Analysis
Assembly
Bacteria
Bioinformatics
Biomedical Engineering/Biotechnology
Biomedicine
Biotechnology
Clouds
Community structure
Gene sequencing
Genomes
Genomics
Life Sciences
Microbial genetics
Microbiomes
Microorganisms
Scientific software
Sediment samplers
Sediments (Geology)
Title High-quality genome sequences of uncultured microbes by assembly of read clouds
URI https://link.springer.com/article/10.1038/nbt.4266
https://www.ncbi.nlm.nih.gov/pubmed/30320765
https://www.proquest.com/docview/2157638598
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https://pubmed.ncbi.nlm.nih.gov/PMC6465186
Volume 36
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