Finding Biology in the Human Microbiome

Finding Biology in the Human Microbiome

24 Pages · 2011 · 1011 KB · English

George Weinstock. What’s next for the Human Microbiome? George Weinstock. You are here. Metagenomics Unfolds. Setting Up Descriptive Phase. Hypothesis Testing. You are

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Finding Biology in the Human Microbiome George Weinstock What’s next for the Human Microbiome? George Weinstock You are here Metagenomics Unfolds Setting Up Descriptive Phase Hypothesis Testing You are here Metagenomics Unfolds Setting Up Descriptive Phase Hypothesis Testing Human Microbiome Research Thoughts •St. Louis (6 months ago) showed a wide -ranging field • Vancouver shows momentum and maturation continues • Analogy to Human Genome Project? • Probably not a “reference” microbiome •Distinguish a “healthy” from a “perturbed” microbiome? • Genomic, Transcriptomic, Proteomic, Metabolomic features • Ecological concepts apply to define disease? •Involvement of clinical concepts early in the project • Close interactions between clinicians and basic scientists • Massive amount of clinical samples in play; many clinical studies • Opportunities for new strategic and funding models early on? Sequencing Technology •HMP • 3000 reference genomes, euks and viruses too • Metagenomic 16S: 50 million reads at first data freeze • Metagenomic shotgun: >7 , ~10 11 reads at first data freeze •454 for 16S, 200 samples/run • Illumina for shotgun • Then: GAIIx @ 40 Gb/run • Now: HiSeq @ 500 Gb/run and increasing •Coming: PacBio, 454 Junior, Ion Torrent, MiSeq, SOLiD Early Pac Bio Experiences •What’s special? • Longer reads: assembly, novel organism/virus detection, 16S • Shorter run time: flexible throughput, match experiment better •Whole genome reference sequencing • Enterococcus faecalis example •16S sequencing • Whole 16S gene E. faecalis genome sequencing with Pac Bio Percent of Reference Bases Covered 93% (Ref = V583) Average Coverage Depth 48x Number of Gaps 50 Average Gap Length 4464 Alignment All Reads Avgread length 587 bp 12% > 1kb 3% > 2kb E. faecalis genome sequencing with Pac Bio Percent of Reference Bases Covered 93% (Ref = V583) Average Coverage Depth 48x Number of Gaps 50 Average Gap Length 4464 Percent of Reference Bases Covered75% Average Coverage Depth 1.6x Number of Gaps 380 Average Gap Length 2144 Alignment Reads >2kbAlignment All Reads Coverage Metrics Read Length & Accuracy Allora Assembly of E. faecalis •Velvet, Illumina: 162 contigs/scaffolds, N50 19kb • Allora, Pac Bio: 622 contigs/scaffolds, N50 7.8kb • Allora, Illumina + Pac Bio: 49 contigs/scaffolds, N50 243kb 16S sequencing on Pac Bio •Full length sequences possible due to read length • High error rate (85%) challenging: • Different ribotypes vs sequencing errors • High coverage

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