Microbiomes and DNA based studies of microbial diversity - talk by Jonathan Eisen at Singularity University
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Microbiomes and DNA based studies of microbial diversity - talk by Jonathan Eisen at Singularity University

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Talk by Jonathan Eisen 3/15/13

Talk by Jonathan Eisen 3/15/13

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  • Nice. Shame there aren't words to go with the slides but I saw enough to get the jist. I'll probably use a few of those references in my discussion for my thesis re: what is sequenced what isn't. Thanks. :)
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  • Thank you indeed
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Microbiomes and DNA based studies of microbial diversity - talk by Jonathan Eisen at Singularity University Presentation Transcript

  • 1. Microbiomes and DNA based Studies of DNA based Studies of Microbial Diversity Microbial Diversity Jonathan A. Eisen Jonathan A. Eisen University of California, Davis University of California, Davis 1Friday, March 15, 13
  • 2. Sequencing and Microbes • Four major “ERAs” in use of sequencing for microbial diversity studies • Each area represented by the Eras is being revolutionized by new sequencing methods 2Friday, March 15, 13
  • 3. Era I: rRNA Tree of Life Era I: rRNA Tree of Life 3Friday, March 15, 13
  • 4. Ernst Haeckel 1866 Plantae Protista Animalia 4 www.mblwhoilibrary.orgFriday, March 15, 13
  • 5. Whittaker – Five Kingdoms 1969 Monera Protista Plantae Fungi Animalia 5Friday, March 15, 13
  • 6. Woese 6Friday, March 15, 13
  • 7. WoeseWoese 1987 - rRNA Microbiological Reviews 51:221 7Friday, March 15, 13
  • 8. Tree of Life • Three main kinds of organisms  Bacteria  Archaea  Eukaryotes • Viruses not alive, but some call them microbes • Many misclassifications occurred before the use of molecular methods 8Friday, March 15, 13
  • 9. Era II: rRNA in the Environment Era II: rRNA in the Environment 9Friday, March 15, 13
  • 10. Great Plate Count Anomaly 10Friday, March 15, 13
  • 11. Great Plate Count Anomaly Culturing Microscopy 11Friday, March 15, 13
  • 12. Great Plate Count Anomaly Culturing Microscopy Count Count 12Friday, March 15, 13
  • 13. Great Plate Count Anomaly Culturing Microscopy Count <<<< Count 13Friday, March 15, 13
  • 14. Great Plate Count Anomaly Solution? Culturing Microscopy Count <<<< Count 14Friday, March 15, 13
  • 15. Great Plate Count Anomaly Solution? DNA Culturing Microscopy Count <<<< Count 15Friday, March 15, 13
  • 16. Analysis of uncultured microbes Collect from environment 16Friday, March 15, 13
  • 17. PCR and phylogenetic analysis of rRNA genes DNA extraction PCR Makes lots Sequence PCR of copies of rRNA genes the rRNA genes in sample rRNA1 5’ ...TACAGTATAGGT Phylogenetic tree Sequence alignment = Data matrix GGAGCTAGCGATC GATCGA... 3’ rRNA1 Yeast rRNA1 A C A C A C Yeast T A C A G T E. coli A G A C A G E. coli Humans Humans T A T A G T 17Friday, March 15, 13
  • 18. PCR and phylogenetic analysis of rRNA genes DNA extraction PCR Makes lots Sequence PCR of copies of rRNA genes the rRNA genes in sample rRNA1 5’ ...ACACACATAGGT Phylogenetic tree Sequence alignment = Data matrix GGAGCTAGCGATC GATCGA... 3’ rRNA1 rRNA2 rRNA1 A C A C A C rRNA2 T A C A G T rRNA2 5’ E. coli A G A C A G ...TACAGTATAGGT E. coli Humans Humans T A T A G T GGAGCTAGCGATC GATCGA... 3’ Yeast Yeast T A C A G T 18Friday, March 15, 13
  • 19. PCR and phylogenetic analysis of rRNA genes DNA extraction PCR Makes lots Sequence PCR of copies of rRNA genes the rRNA genes in sample rRNA1 5’...ACACACATAGGTGGAGC TAGCGATCGATCGA... 3’ Phylogenetic tree Sequence alignment = Data matrix rRNA2 rRNA1 rRNA2 rRNA1 A C A C A C 5’..TACAGTATAGGTGGAGCT rRNA4 AGCGACGATCGA... 3’rRNA3 rRNA2 T A C A G T rRNA3 rRNA3 C A C T G T 5’...ACGGCAAAATAGGTGGA E. coli Humans rRNA4 C A C A G T TTCTAGCGATATAGA... 3’ Yeast E. coli A G A C A G rRNA4 5’...ACGGCCCGATAGGTGG Humans T A T A G T ATTCTAGCGCCATAGA... 3’ Yeast T A C A G T 19Friday, March 15, 13
  • 20. PCR and phylogenetic analysis of rRNA genes PCR 20Friday, March 15, 13
  • 21. Major phyla of bacteria & archaea (as of 2002) No cultures Some cultures 21Friday, March 15, 13
  • 22. The Hidden Majority Richness estimates Hugenholtz 2002 Bohannan and Hughes 2003 22Friday, March 15, 13
  • 23. Example: Human biogeography Censored Censored 23Friday, March 15, 13
  • 24. Era III: Genome Sequencing Era III: Genome Sequencing 24Friday, March 15, 13
  • 25. 1st Genome Sequence Fleischmann et al. 1995 25Friday, March 15, 13
  • 26. Genomes Revolutionized Microbiology • Predictions of metabolic processes • Better vaccine and drug design • New insights into mechanisms of evolution • Genomes serve as template for functional studies • New enzymes and materials for engineering and synthetic biology 26Friday, March 15, 13
  • 27. Lateral Gene Transfer Perna et al. 2003 27Friday, March 15, 13
  • 28. Era IV: Genomes in the environment Era IV: Genomes in the Environment 28Friday, March 15, 13
  • 29. l gene own transducer of light stimuli [for example, the kinetics of its photochemical reaction cy- leDelong Lab ge- Htr (22, 23)]. Although sequence analysis of cle. The transport rhodopsins (bacteriorho- iden- proteorhodopsin shows moderate statistical dopsins and halorhodopsins) are character- roteo- support for a specific relationship with sen- ized by cyclic photochemical reaction se- fromopsinsferent.hereas philes r than rmine l, wea coli pres- rotein 3A). nes ofpopro-m was (Fig.at 520 band- erated odop-nce of dth is 29 rption March 15, 13 Friday,
  • 30. generated D ownloaded from w Delong Lab eorhodop-resence ofndwidth isabsorption. The red- nm in theated Schiffably to the on was de-s in a cellward trans- in proteor-nd only in (Fig. 4A).edium wasce of a 10re carbonyl19). Illumi-ical poten- right-side-nce of reti-light onsethat proteo- capable of Fig. 1. (A) Phylogenetic tree of bacterial 16S rRNA gene sequences, including that encoded on the physiolog- 130-kb bacterioplankton BAC clone (EBAC31A08) (16). (B) Phylogenetic analysis of proteorhodop- sin with archaeal (BR, HR, and SR prefixes) and Neurospora crassa (NOP1 prefix) rhodopsins (16).e activities Nomenclature: Name_Species.abbreviation_Genbank.gi (HR, halorhodopsin; SR, sensory rhodopsin; containing BR, bacteriorhodopsin). Halsod, Halorubrum sodomense; Halhal, Halobacterium salinarum (halo- proteorho- bium); Halval, Haloarcula vallismortis; Natpha, Natronomonas pharaonis; Halsp, Halobacterium sp;main to be Neucra, Neurospora crassa. 30 www.sciencemag.org Friday, March 15, 13 SCIENCE VOL 289 15 SEPTEMBER 2000 1903
  • 31. 31Friday, March 15, 13
  • 32. of the surface of a single cell3. This in amino-acid sequences were not restricted to the hydrophilicnt to produce substantial amounts ofefore, the high density of proteorho-rane indicated by our calculations otein has a signi®cant role in the MB 0m2 MB 40m5 Monterey Bay and shallow HOT BAC 40E8 HOT 0m1 MB 20m2 MB 40m12ed membranes MB 100m10 MB 20m12 BAC 31A8 MB 40m1 MB 100m5 MB 20m5 BAC 64A5 MB 100m7 10 –3 AU MB 0m1 MB 100m9 10 –1 s PAL E6 HOT 75m3e-treated membranes Antarctica and deep HOT PAL B1 PAL E7 PAL B2 PAL B8 HOT 75m1 PAL B7 PAL E1 HOT 75m4 PAL B5stituted membranes PAL B6 HOT 75m8 0.01 Figure 3 Phylogenetic analysis of the inferred amino-acid sequence of cloned proteorhodopsin genes. Distance analysis of 220 positions was used to calculate the tree at 500 nm of a Monterey Bay bacterioplankton by neighbour-joining using the PaupSearch program of the Wisconsin Package version tion of hydroxylamine; middle, after 0.2 M 10.0 (Genetics Computer Group; Madison, Wisconsin). H. salinarum bacteriorhodopsinC, with 500-nm illumination for 30 min; bottom, was used as an outgroup, and is not shown. Scale bar represents number of substitutionsn in 100 mM phosphate buffer, pH 7.0, followed per site. Bold names indicate the proteorhodopsins that were spectrally characterized in incubation for 1 h. this study. 32 Friday,nature.com March 15, 132001 Macmillan Magazines Ltd © 787
  • 33. clearly related to Burkholderia (fig. S2) and were found in the main scaffold set, coveredSargassoof scaffolds representing two dis- two groups Sea at depths ranging from 4ϫ to 36ϫ (indicated tinct strains closely related to the published with shading in table S3 with nine depicted in Fig. 1. MODIS-Aqua satellite image of ocean chlorophyll in the Sargasso Sea grid about the BATS site from 22 February 2003. The station locations are overlain with their respective identifications. Note the elevated levels of chlorophyll (green color shades) around station 3, which are not present around stations 11 and 13. Fig. 2. Gene conser- 33Friday, March 15, 13 closely vation among
  • 34. Functional Diversity of Proteorhodopsins? Venter et al., 34Friday, March 15, 13 2004
  • 35. t can be used as an- nomic group using the phylogenetic analysis marker genes, is roughly comparable to the ish eukaryotic mate- described for rRNA. For example, our data set 97% cutoff traditionally used for rRNA. Thus nverse relation was size of the pre-filters the fraction of se- table S5). This rela- sence of 18S rRNAstrong evidence that deed captured. es richness. Most ncultured organisms ies of rRNA genes eaction (PCR) with ed positions in those small subunit rRNA ersity of prokaryotic17). However, PCR-y biased, because not th the same “univer-hotgun sequence data tified 1164 distinct es or fragments ofd II assemblies andorcerer II reads (5).milarity cutoff to dis- s, we identified 148otypes in our samplehe RDP II database Fig. 6. Phylogenetic diversity of Sargasso Sea sequences using multiple phylogenetic markers. They cutoff, this number relative contribution of organisms from different major phylogenetic groups (phylotypes) was equence similarity is measured using multiple phylogenetic markers that have been used previously in phylogenetic e predictor of func- studies of prokaryotes: 16S rRNA, RecA, EF-Tu, EF-G, HSP70, and RNA polymerase B (RpoB). The equence divergence relative proportion of different phylotypes for each sequence (weighted by the depth of coverage ate with the biologi- of the contigs from which those sequences came) is shown. The phylotype distribution wasefining species (also determined as follows: (i) Sequences in the Sargasso data set corresponding to each of these genes were identified using HMM and BLAST searches. (ii) Phylogenetic analysis was performed for each sequence similarity phylogenetic marker identified in the Sargasso data separately compared with all members of that he accepted standard gene family in all complete genome sequences (only complete genomes were used to control for icrobes. All sampled the differential sampling of these markers in GenBank). (iii) The phylogenetic affinity of each to taxonomic groups sequence was assigned based on the classification of the nearest neighbor in the phylogenetic tree. 35 Friday, March 15, 13
  • 36. ARTICLES A human gut microbial gene catalogue established by metagenomic sequencing Junjie Qin1*, Ruiqiang Li1*, Jeroen Raes2,3, Manimozhiyan Arumugam2, Kristoffer Solvsten Burgdorf4, Chaysavanh Manichanh5, Trine Nielsen4, Nicolas Pons6, Florence Levenez6, Takuji Yamada2, Daniel R. Mende2, Junhua Li1,7, Junming Xu1, Shaochuan Li1, Dongfang Li1,8, Jianjun Cao1, Bo Wang1, Huiqing Liang1, Huisong Zheng1, Yinlong Xie1,7, Julien Tap6, Patricia Lepage6, Marcelo Bertalan9, Jean-Michel Batto6, Torben Hansen4, Denis Le Paslier10, Allan Linneberg11, H. Bjørn Nielsen9, Eric Pelletier10, Pierre Renault6, Thomas Sicheritz-Ponten9, Keith Turner12, Hongmei Zhu1, Chang Yu1, Shengting Li1, Min Jian1, Yan Zhou1, Yingrui Li1, Xiuqing Zhang1, Songgang Li1, Nan Qin1, Huanming Yang1, Jian Wang1, Søren Brunak9, Joel Dore6, Francisco Guarner5, ´ Karsten Kristiansen , Oluf Pedersen , Julian Parkhill , Jean Weissenbach , MetaHIT Consortium{, Peer Bork2, 13 4,14 12 10 S. Dusko Ehrlich6 & Jun Wang1,13 To understand the impact of gut microbes on human health and well-being it is crucial to assess their genetic potential. Here we describe the Illumina-based metagenomic sequencing, assembly and characterization of 3.3 million non-redundant microbial genes, derived from 576.7 gigabases of sequence, from faecal samples of 124 European individuals. The gene set, ,150 times larger than the human gene complement, contains an overwhelming majority of the prevalent (more frequent) microbial genes of the cohort and probably includes a large proportion of the prevalent human intestinal microbial genes. The genes are largely shared among individuals of the cohort. Over 99% of the genes are bacterial, indicating that the entire cohort harbours between 1,000 and 1,150 prevalent bacterial species and each individual at least 160 such species, which are also largely shared. We define and describe the minimal gut metagenome and the minimal gut bacterial genome in terms of functions present in all individuals and most bacteria, respectively. 36 8,16,17Friday, March 15, 13 that the microbes in our bodies collectively It has been estimated individuals from the United States or Japan . To get a broader
  • 37. ARTICLES 40 PC2 • • • 30 • •• Cluster (%) • • • • Ulcerative colitis • • • • • • • • 20 • • • • • • PC1 • 10 • • • • • • Healthy • • Crohn’s disease • P value: 0.031 0 1 • • • • • Figure 5 | Cluste were ranked by t Figure 4 | Bacterial species abundance differentiates IBD patients and length and copy n healthy individuals. Principal component analysis with health status as clusters with the instrumental variables, based on the abundance of 155 species with $1% groups of 100 clu genome coverage by the Illumina reads in at least 1 individual of the cohort, that contains 86% was carried out with 14 healthy individuals and 25 IBD patients (21 ulcerative colitis and 4 Crohn’s disease) from Spain (Supplementary Table 1). Two first components (PC1 and PC2) were plotted and represented 7.3% of whole were within th inertia. Individuals (represented by points) were clustered and centre of This suggests th gravity computed for each class; P-value of the link between health status and (Supplementary species abundance was assessed using a Monte-Carlo test (999 replicates). functions impo We found tw Almost all (99.96%) of the phylogenetically assigned genes belonged required in37 b allFriday, March 15, 13 to the Bacteria and Archaea, reflecting their predominance in the gut. for the gut. Am
  • 38. Woese Tree of Life ?????? adapted from Baldauf, et al., in Assembling the 38Friday, March 15, 13 Tree of Life, 2004
  • 39. PD: All From Wu et al. 2009 Nature 462, 1056-1060 39Friday, March 15, 13