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


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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Figure 1: Overview of the read-cloud shotgun sequencing and assembly approach.
Figure 2: Composition of stool microbiome communities from two healthy human participants.
Figure 3: Combined genome-draft results of the read-cloud, SLR, and short-read approaches applied to stool samples from healthy humans.
Figure 4: Completeness of genome bins produced by read-cloud, SLR, and short-read sequencing for various taxa present in stool samples from healthy humans.
Figure 5: Comparisons of representative read-cloud genome drafts to reference genomes, and corresponding short-read and SLR drafts.
Figure 6: Comparison of marine-sediment genome drafts generated by read-cloud sequencing with standard short-read versus Athena assembly.

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The authors thank E. Tkachenko for assistance in preparing TruSeq libraries and M. Snyder and members of the laboratory of A.S.B. for helpful feedback. The authors also thank H. Xu at Illumina for sharing read-cloud sequencing data of ATCC 20 for the mock metagenome. This work was supported by NCI K08 CA184420, the Amy Strelzer Manasevit Award from the National Marrow Donor Program, and a Damon Runyon Clinical Investigator Award to A.S.B. E.L.M. was supported by National Science Foundation Graduate Research Fellowship DGE-114747. A.B. was supported by the Stanford Genome Training Program (SGTP; NIH/NHGRI) and a Training Grant of the Joint Initiative for Metrology in Biology (JIMB; NIST). A.E.D. and the marine-sample collection and extraction were supported by National Science Foundation grant OCE-1634297. A.E.P. was supported by a Center for Dark Energy Biosphere Investigations Postdoctoral Fellowship. Access to shared computer resources was supported in part by NIH P30 CA124435 via the Stanford Cancer Institute Shared Resource Genetics Bioinformatics Service Center.

Author information

Authors and Affiliations



A.B., E.L.M., A.S.B. and S.B. conceived 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 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 the marine-sediment sample, extracted DNA from the marine-sediment sample and assisted 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.

Corresponding authors

Correspondence to Serafim Batzoglou or Ami S Bhatt.

Ethics declarations

Competing interests

S.B. is an employee of and owns stock in Illumina. Shotgun sequencing products developed, marketed and/or sold by Illumina were used in this work.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5, Supplementary Tables 1, 3, 4, and Supplementary Notes 1–8 (PDF 5546 kb)

Life Sciences Reporting Summary (PDF 160 kb)

Supplementary Table 2

Per-species MetaQUAST statistics for ATCC 20 (XLSX 40 kb)

Supplementary Table 5

Assignment of metagenomic contigs to bins for healthy gut samples (TXT 1592 kb)

Supplementary Table 6

Per-taxon genome bin statistics for healthy gut samples (CSV 10 kb)

Supplementary Table 7

Comparisons of healthy gut genome bins and available references (XLSX 9 kb)

Supplementary Table 8

Assignment of metagenomic contigs to bins for marine sediment sample (TXT 6609 kb)

Supplementary Table 9

Genome bin statistics for marine sample (CSV 5 kb)

Supplementary Table 10

High-copy repeat statistics for P. copri (XLSX 7 kb)

Supplementary Table 11

Per-species MetaQUAST statistics for in silico downsampled ATCC 20 (XLSX 37 kb)

Supplementary Table 12

Per-taxon genome bin statistics for in silico downsampled human gut sample (CSV 3 kb)

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Bishara, A., Moss, E., Kolmogorov, M. et al. High-quality genome sequences of uncultured microbes by assembly of read clouds. Nat Biotechnol 36, 1067–1075 (2018).

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