EVE161 Lecture 1

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EVE161 Lecture 1

Microbial Phylogenomics course at UC Davis

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EVE161 Lecture 1

  1. 1. Lecture 1: EVE 161:
 Microbial Phylogenomics ! Lecture #1: Introduction ! UC Davis, Winter 2014 Instructor: Jonathan Eisen Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !1
  2. 2. Where we are going and where we have been • Previous lecture: ! • Current Lecture: ! 1. Introduction • Next Lecture: ! 2. Evolution of DNA sequencing Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !2
  3. 3. Lecture 1 Outline • Course details • Four eras of sequencing • Introduction to phylogeny Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !3
  4. 4. Main topics of the course • DNA sequence based studies of microbial diversity • Four Eras of sequencing ! The Tree of Life ! rRNA from environments ! Genome Sequencing ! Metagenomics Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !4
  5. 5. Textbook/Reading • Each lecture will have some associated background reading and 1+ primary literature papers • Whenever possible, the primary literature used will be “Open Access” material • There will also be news stories, blogs and other “media” to review / read Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !5
  6. 6. What you should learn from the course • History of sequence based studies of microbial diversity • Current practice in sequence based studies of microbial diversity • Broad view of what we know about microbial diversity • How to read and analyze a research paper Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !6
  7. 7. Grading • • • • • Attendance and class participation 20 % Weekly assignments 20 % Midterm 20 % Final presentation 20% Final exam 20% Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !7
  8. 8. Student project • Select 1-2 papers on one of the topics of the course (approval needed) • Review the paper and write up a summary of your assessment of the paper (more detail on this later) • Post your assessment on the course blog • Present a short summary of what you did to the class • Ask and answer questions about your and other people’s reviews on the course blog Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !8
  9. 9. Contact information • Jonathan Eisen, Professor – jaeisen@ucdavis.edu – Phone 752-3498 – Office Hours: TBD • Holly Ganz – hhganz@ucdavis.edu – Office Hours: TBD Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !9
  10. 10. Course Information • SmartSite • Also will be posting for the broader community at http:// microbe.net/eve161 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !10
  11. 11. Introduction to EVE161 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !11
  12. 12. Microbial Diversity Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !12
  13. 13. Microbial Diversity • Microbes are small • But diversity and numbers are very high • Appearance not a good indicator of type or function • Field observations of limited value Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !13
  14. 14. Diversity of Form Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !14
  15. 15. Diversity of Function The Bad The Good The Consumable The Burnable The Unusual The Planet Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !15
  16. 16. Phylogenetic Diversity Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !16
  17. 17. Phylogeny was central to Darwin’s Work on Natural Selection Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !17
  18. 18. Phylogeny • Phylogeny is a description of the evolutionary history of relationships among organisms (or their parts). • This is portrayed in a diagram called a phylogenetic tree. • Phylogenetic trees are used to depict the evolutionary history of populations, species and genes. • The Tree of Life refers to the concept that all living organisms are related to one another through shared ancestry. Ch. 25.1 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !18
  19. 19. Four Eras of Sequence & Microbial Diversity Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !19
  20. 20. Relevant Reading • Eisen JA. Environmental shotgun sequencing: its potential and challenges for studying the hidden world of microbes. PLOS Biology 5(3): e82. Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !20
  21. 21. Moore’s Law Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !21
  22. 22. Era I: rRNA Tree of Life Era I: rRNA Tree of Life Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !22
  23. 23. Era I: rRNA Tree of Life Era I: rRNA Tree of Life Lectures 3-4 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !23
  24. 24. Ernst Haeckel 1866 Plantae Protista Animalia Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !24
  25. 25. Whittaker – Five Kingdoms 1969 Monera Protista Plantae Fungi Animalia Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !25
  26. 26. Carl Woese http://mcb.illinois.edu/faculty/ profile/1204 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !26
  27. 27. Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !27
  28. 28. Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !28
  29. 29. Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !29
  30. 30. Woese and Fox 
 • Abstract: A phylogenetic analysis based upon ribosomal RNA sequence characterization reveals that living systems represent one of three aboriginal lines of descent: (i) the eubacteria, comprising all typical bacteria; (ii) the archaebacteria, containing methanogenic bacteria; and (iii) the urkaryotes, now represented in the cytoplasmic component of eukaryotic cells. Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !30
  31. 31. Propose “three aboriginal lines of descent” ! Eubacteria ! Archaebacteria ! Urkaryotes Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !31
  32. 32. Woese 1987 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !32
  33. 33. • Appearance of microbes not informative (enough) • rRNA Tree of Life identified two major groups of organisms 
 w/o nuclei • rRNA powerful for many reasons, though not perfect Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !33
  34. 34. 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 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !34
  35. 35. Tree of Life adapted from Baldauf, et al., in Assembling the Tree of Life, 2004 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !35
  36. 36. Most of the phylogenetic diversity of life is microbial adapted from Baldauf, et al., in Assembling the Tree of Life, 2004 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !36
  37. 37. Simplified, Rooted Tree of Life Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !37
  38. 38. Alternative rooted tree of life Archaea Archaea Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !38
  39. 39. Era II: rRNA in the Environment Era II: rRNA in the Environment Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !39
  40. 40. Era II: rRNA in the Environment Era II: rRNA in the Environment Lectures 5-9 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !40
  41. 41. Plant/Animal Field Studies Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !41
  42. 42. Microbial Field Studies Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !42
  43. 43. Culturing Microbes Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !43
  44. 44. Great Plate Count Anomaly Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !44
  45. 45. Great Plate Count Anomaly Culturing Microscopy Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !45
  46. 46. Great Plate Count Anomaly Culturing Count Microscopy Count Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !46
  47. 47. Great Plate Count Anomaly Culturing Count Microscopy <<<< Count Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !47
  48. 48. Great Plate Count Anomaly Problem because appearance not effective for “who is out there?” or “what are they doing?” Culturing Count Microscopy <<<< Count Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !48
  49. 49. Great Plate Count Anomaly Solution? Problem because appearance not effective for “who is out there?” or “what are they doing?” Culturing Count Microscopy <<<< Count Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !49
  50. 50. Great Plate Count Anomaly Solution? Problem because appearance not effective for “who is out there?” or “what are they doing?” DNA Culturing Count Microscopy <<<< Count Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !50
  51. 51. Analysis of uncultured microbes Collect from environment Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !51
  52. 52. PCR and phylogenetic analysis of rRNA genes DNA extraction PCR PCR Phylogenetic tree rRNA1 Sequence alignment = Data matrix Yeast A C A C A T A C A G T A G A C T A G rRNA1 5’ ...TACAGTATAGG TGGAGCTAGCGAT CGATCGA... 3’ C E. coli Humans rRNA1 Yeast E. coli Sequence rRNA genes Makes lots of copies of the rRNA genes in sample Humans A T A G T Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !52
  53. 53. PCR and phylogenetic analysis of rRNA genes DNA extraction PCR PCR Phylogenetic tree rRNA1 Sequence alignment = Data matrix rRNA2 Yeast A C A C A T A C A G T A G A C Humans T A T A G T Yeast T A C A G rRNA1 5’ ...ACACACATAG GTGGAGCTAGCGA TCGATCGA... 3’ C E. coli Humans rRNA1 rRNA2 E. coli Sequence rRNA genes Makes lots of copies of the rRNA genes in sample T A G rRNA2 5’ ...TACAGTATAGG TGGAGCTAGCGAT CGATCGA... 3’ Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !53
  54. 54. PCR and phylogenetic analysis of rRNA genes DNA extraction PCR PCR Phylogenetic tree rRNA1 Sequence alignment = Data matrix rRNA2 rRNA1 A C A C rRNA2 T A C A G T C A C T G T rRNA4 C A C A G T E. coli A G A C T A T A G T Yeast Yeast C Humans Humans E. coli A rRNA3 rRNA4 rRNA3 Sequence rRNA genes Makes lots of copies of the rRNA genes in sample T A C A G rRNA1 5’...ACACACATAGGTGGAGC TAGCGATCGATCGA... 3’ rRNA2 5’..TACAGTATAGGTGGAGCT AGCGACGATCGA... 3’ T A G rRNA3 5’...ACGGCAAAATAGGTGGA TTCTAGCGATATAGA... 3’ rRNA4 5’...ACGGCCCGATAGGTGG ATTCTAGCGCCATAGA... 3’ Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !54
  55. 55. PCR and phylogenetic analysis of rRNA genes PCR Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !55
  56. 56. Major phyla of bacteria & archaea (as of 2002) No cultures Some cultures Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !56
  57. 57. The Hidden Majority Hugenholtz 2002 Richness estimates Bohannan and Hughes 2003 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !57
  58. 58. Human microbiome case study Censored Censored Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !58
  59. 59. Built Environment Case Study Microbial Biogeography of Public Restroom Surfaces Gilberto E. Flores1, Scott T. Bates1, Dan Knights2, Christian L. Lauber1, Jesse Stombaugh3, Rob Knight3,4, Noah Fierer1,5* Bacteria of Public Restrooms 1 Cooperative Institute for Research in Environmental Science, University of Colorado, Boulder, Colorado, United States of America, 2 Department of Computer Science, University of Colorado, Boulder, Colorado, United States of America, 3 Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado, United States of America, 4 Howard Hughes Medical Institute, University of Colorado, Boulder, Colorado, United States of America, 5 Department of Ecology and Evolutionary Biology, University of Colorado, Boulder, Colorado, United States of America Abstract We spend the majority of our lives indoors where we are constantly exposed to bacteria residing on surfaces. However, the diversity of these surface-associated communities is largely unknown. We explored the biogeographical patterns exhibited by bacteria across ten surfaces within each of twelve public restrooms. Using high-throughput barcoded pyrosequencing of the 16 S rRNA gene, we identified 19 bacterial phyla across all surfaces. Most sequences belonged to four phyla: www.nature.com/ismej Actinobacteria, Bacteriodetes, Firmicutes and Proteobacteria. The communities clustered into three general categories: those found on surfaces associated with toilets, those on the restroom floor, and those found on surfaces routinely touched with Figure hands. On toilet surfaces, gut-associated taxa wereof discriminating suggesting fecal contamination of theseblue indicates low 3. Cartoon illustrations of the relative abundance more prevalent, taxa on public restroom surfaces. Light surfaces. Floor abundance while dark blue indicates high abundance of taxa. (A) Although skin-associated taxa (Propionibacteriaceae, Corynebacteriaceae, surfaces were the most diverse of all communities and contained several taxa commonly found in soils. Skin-associated Staphylococcaceae and Streptococcaceae) were abundant on all surfaces, they were relatively more abundant on surfaces routinely touched with bacteria, especially the Propionibacteriaceae, dominated surfaces routinely touched Prevotellaceae and Bacteroidaceae) were most hands. (B) Gut-associated taxa (Clostridiales, Clostridiales group XI, Ruminococcaceae, Lachnospiraceae, with our hands. Certain taxa were more common surfaces. (C) Although soil-associated taxa (Rhodobacteraceae, Rhizobiales, Microbacteriaceae and Nocardioidaceae) were in low abundant on toilet in female than in male restrooms as vagina-associated Lactobacillaceae were widely distributed in female restrooms, likely from urine were relatively more abundant SourceTracker algorithm confirmed Figure not drawn to scale. abundance on all restroom surfaces, they contamination. Use of the on the floor of the restrooms we surveyed.many of our taxonomic doi:10.1371/journal.pone.0028132.g003 was the primary source of bacteria on restroom surfaces. Overall, these results demonstrate that observations as human skin restroom surfaces host relatively diverse microbial communities dominated by human-associated bacteria with Bacteria of P clear 1 1 1,2 1,2 1,2 linkages between communities on or in different body sites and those communities found on restroom surfaces. More Steven W Kembel , Evan Jones , Jeff Kline , Dale Northcutt , Jason Stenson , Results of SourceTracker microbes are commonly found the stall in), they were work is relevant to the public women field as we show that human-associatedanalysis support the taxonomic likely dispersed manually after health used 1 1 1,2 1,3 generally, this Bohannan , G Z Brown and Jessica L Green Ann time, the M Womack , Brendan JM 100 SOURCES on Coupling 1 Bathroomthe toilet.restroom surfaces suggesting thatwith those of the could readily be transmitted between individuals by the touching biogeography. Bythese observations bacterial pathogens patterns highlighted above, indicating that human skin was the Biology and the Built Environment Center, Institute of Ecology and Evolution, Department of distribution of gut-associated bacteriademonstrate that we can of high-throughput analyses of bacterial communities to determine indicate that routine use use primary source of bacteria on all public restroom surfaces Soil un to take of surfaces in 2 swabbing differentsurfaces. Furthermore, we and fecal-associated bacteria Biology, University of Oregon, Eugene, OR, USA; Energy Studies in Buildings Laboratory, toilets results in the bacteria onurine- surfaces, an approach which examined, used to track pathogen transmission and teston or sources of dispersal of indoor could be while the human gut was an important source the Water 80 of outside Department of Architecture, University of Oregon, Eugene, OR, USA and 3Santa Fe Institute, public restrooms, researchers While these results are not unexpected, around the toilet, and urine was an important source in women’s throughout the of hygiene practices. restroom. efficacy Mouth Santa om plants Fe, NM, USA restrooms (Figure 4, Table S4). Contrary to expectations (see they do highlight the importance of hand-hygiene when using determined that microbes vary in Urine 60 SourceTracker 6(11): e28132. ours after where theypublic restrooms since these Knights D, Lauber CL,also be potential(2011) above), soil was not identified by theSurfaces. PLoS ONE algorithm as come from Flores GE, Bates ST, surfaces could Stombaugh J, et al. Citation: dependMicrobial Biogeography of Public Restroom Gut being a major source of bacteria on any of the surfaces, including vehicles for the transmission of human pathogens. Unfortunately, doi:10.1371/journal.pone.0028132 ing on the surface (chart). have documented that college students (who are ere shut floors (Figure 4). Although the floor samples contained family-level previous studies R. Liles, Auburn University, United States of America Buildings are complex ecosystems that house trillions of microorganisms interactingSkin each with Editor: Mark 40 taxa that are common in soil, the SourceTracker algorithm likely the most frequent users of the studied restrooms) are not ortion of other, with humans and with their environment. Understanding the ecological and evolutionary Received September 12, 2011; Accepted November 1, 2011; Published November 23, 2011 processes that determine the diversity and composition of the built environment microbiome—thepant in indoor microbial of hand-washers [42,43]. probably underestimates the relative importance of sources, like always the most diligent e human Copyright: ß 2011 Flores et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits community of microorganisms that live indoors—is important for understanding the relationship 20 unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The ISME Journal (2012), 1–11 & 2012 International Society for Microbial Ecology All rights reserved 1751-7362/12 ORIGINAL ARTICLE between building design, biodiversity and human health. In this study, we used high-throughputecology research, Peccia Funding: This was sequencing of the bacterial 16S rRNA gene to quantify relationships between building attributes and thinks that the field workand the Howard with funding from the Alfred P. Sloan had no role andstudy design, data collection and analysis, in part bytothe National has supported Hughes Medical Institute. The funders Foundation in their Indoor Environment program, and decision publish, or Institutes of Health airborne bacterial communities at 0 health-care facility. We quantified airborne bacterial community a preparation structure and environmental conditions in patient rooms exposed to mechanical or windowyet to gel. And the of the manuscript. wh i c h Sloan Competing Interests: The authors have declared that no competing interests exist. ventilation and in outdoor air. The phylogenetic diversity of airborne bacterial communities was 26 JanuOlsiewski lower indoors than outdoors, and mechanically ventilated rooms contained less diverse microbialFoundation’sE-mail: noah.fierer@colorado.edu * communities than did window-ventilated rooms. Bacterial communities in indoor environmentsshares some of his conJournal, contained many taxa that are absent or rare outdoors, including taxa closely related to potential hanically human pathogens. Building attributes, specifically the source of ventilation air, airflow rates, relativecern. “Everybody’s gencommunities and revealed a greater diversity of bacteria on Introduction humidity and temperature, were correlated with the diversity and composition of indoor bacterialerating vast amounts of had lower indoor surfaces than captured using cultivation-based techniques communities. The relative abundance of bacteria closely related to human pathogens was higher More data sets y than ones with openthan outdoors, and higher in rooms withquantify those con- lower relative humidity. looking across than ever, individuals across the globe spend a large [10–13]. Most of the organisms identified in these studies are they move around. But to lower airflow rates and data,” she says, but indoors winportion of their The observed relationship between building design and airborne bacterial can be difficult because groups choose lives indoors, yet relatively little is known about the ility of fresh air translated tributions, Peccia’s team has had to develop diversity suggests that dif- indoor environments. Of the studies that related to human commensalsrestroom surfaces. Communities are Figure 2. Relationship between bacterial communities associated with ten public suggesting that the organisms wer microbial diversity of of the unweighted UniFrac distance we can manage indoor environments, altering through building design and operation the community growing the surfaces but rather were PCoA rtions of microbes associ- new methods to collect airborne bacteria and our timeanalytical tools. With have examined microorganisms associated with indoormatrix. Each point represents touching) on indirectly (e.g. shedding of skindeposited(a Sloan support, clusters distinct from surfaces touchedenviron- not actively a single sample. Note that the floor (triangles) and toilet of microbial species that potentially colonize the human microbiome during ferent indoors. form with hands. directly (i.e. or cells) by The ISME Journal extract their DNA, 26 January 2012; doi:10.1038/ismej.2011.211 ments, most have relied upon cultivation-based techniques to doi:10.1371/journal.pone.0028132.g002 an body, and consequently, advance online publication,as the microbes are much though, a data archive and integrated analythumans. Despite these efforts, we still have an incomplete Subject Category: microbial population and community ecology detect organisms residing on a variety of household surfaces [1–5]. understanding of bacterial communities associated with indoor pathogens. Although this less abundant in builtthan on surfaces. ical tools dispersal; Keywords: aeromicrobiology; bacteria; air environment microbiome; community ecology; are in the works. Not surprisingly, these studies have identified surfaces in kitchens g on February 9, 2012 Do or Do in or ou t St all in Fa Sta uc et ll ou So han t ap d dis les pe ns To T e ile oile r tf lus t sea hh t a To ndle ile tf lo Si or nk flo or ck to pre- Average contribution (%) Architectural design influences the diversity and structure of the built environment microbiome high diversity of floor communities is likely due to the frequency of because limitations of traditional 16abundances of s environments related differences in the relative S rRNA gene environmental filtering In one recent study, they used air filters hat having natural airflow To foster collaborations between micro- with the bottombacterial which would track in a diversity sequencing techniques have made replicate sampling and restrooms as being hot spots of of shoes, contamination. contact some surfaces (Figure 1B, Table S2). Most notably cloning and Because several of microorganisms from a variety ofto survive on pathogenic bacteria are known sources including soil, which is characterizations of the communities prohibitive. Green says answering that to sample airborne particles and microbes biologists, architects, and building scientists, and in-depth were clearly more abundant on certain surfaces surfaces for extended to be a time [6–8], these studies are of known microbial With the advent of high-throughput sequencing techniques, we Introduction microbiome—includes the foundation and comclinical data; she’s hoping in a classroom during 4 days during which human pathogensalso sponsored a symposiuminperiods of highly-diverse of human habitat [27,39]. Indeed, restrooms than male restrooms (Figure 1B). Some obvious importance preventing the spread disease. bacteria commonly associated with soil (e.g. Rhodobacteraceae, investigate are the most common, and often most abu family indoor microbial communities at an can now mensals interacting with ital to participate in a study 90% of theirwere present and 4 days during with each other and haveof the built environment widely recognized that the majority of on average, depth andthe vagina understand reproductive age on the microbiome their However, it is Rhizobiales, Microbacteriaceae and Nocardioidaceae) were, unprecedented found in begin to of healthy the relationship now Humans spend up to students lives indoors environment (Eames et al., 2009). There been (Klepeis et al., 2001). Consequently,roomway we the Slides few attempts to comprehensively survey the built Taught by Jonathanon floor surfaces (Figure 2014 S2). for UC Davis EVE161Indoor Air conference in Austin, abundant cultivated [9]Winter 3C, between humans,and are relatively built abundant in male urine Course microorganisms cannot be readily Eisen and thus, the Table more microbes and the less environment. !59 dence of hospital-acquired which the was vacant. They measured at the 2011 design and operate the indoor environment has a overall diversityInterestingly, some of the toilet flush handles harbored bacterial of microorganisms associated with indoor analysis of female urine samples collected as par
  60. 60. Era III: Genome Sequencing Era III: Genome Sequencing Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !60
  61. 61. Era III: Genome Sequencing Era III: Genome Sequencing Lectures 10-14 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !61
  62. 62. 1st Genome Sequence Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Fleischmann et al. 1995 !62
  63. 63. 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 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !63
  64. 64. Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !64
  65. 65. Metabolic Predictions Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !65
  66. 66. Lateral Gene Transfer Perna et al. 2003 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !66
  67. 67. Network of Life Bacteria Archaea Eukaryotes Figure from Barton, Eisen et al. “Evolution”, CSHL Press. Based on tree from Pace NR, 2003. Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !67
  68. 68. GEBA Case Study • Phylogenetic diversity poorly sampled • GEBA project at DOE-JGI correcting this Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !68
  69. 69. Era IV: Genomes in the environment Era IV: Genomes in the Environment Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !69
  70. 70. Era IV: Genomes in the environment Era IV: Genomes in the Environment Lectures 15-19 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !70
  71. 71. Era IV: Metagenomics DNA extraction Sequence all genes PCR 5’ ...TACAGTATAGGTG GAGCTAGCGATCGAT CGA... 3’ Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014
  72. 72. Delong GENOMIC FRAGMENTS FROM PLANKTONIC MARINE ARCHAEA Lab 593 ments isolated from fosmid clones with various restriction endonucle10 kb, the F-factor-based vector the fosmid subfragments. Partial of restriction enzyme to 1 ⇥g of mixture. The reaction mixture was removed at 10, 40, and 60 min. dding 1 ⇥l of 0.5 M EDTA to the e. The partially digested DNA was s described above except using a 1he sizes of the separated fragments n standards. The distances of the d SP6 promoter sites on the excised pmol of T7- or SP6-specific oligol) and hybridizing with Southern artial sequences reported in Table the following accession numbers: U40243, U40244, and U40245. The and EF2 have been submitted to and U41261. FIG. 1. Flowchart depicting the construction and screening of an environmental library from a mixed picoplankton sample. MW, molecular weight; PFGE, pulsed-field gel electrophoresis. Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 D ownloa de d from jb.a sm .org a t U N IV O fosmid and pBAC clones digested probed with labeled T7 and SP6 eled subclones and PCR fragments otgun sequencing described above. e estimates from the partial digesof the fosmids and their subclones. and DeSoete distance (9) analyses n using GDE 2.2 and Treetool 1.0, (RDP) (23). DeSoete least squares ng pairwise evolutionary distances, to account for empirical base fretained from the RDP, version 4.0 rRNA sequences were performed the RDP. For distance analyses of lutionary distances were estimated d tree topology was inferred by the n addition and global branch swapprotein sequences, the Phylip proaddition and ordinary parsimony !72
  73. 73. D ownloa de d from w generated eorhodopresence of ndwidth is absorption . The rednm in the ated Schiff ably to the Delong Lab on was des in a cell ward transin proteornd only in (Fig. 4A). edium was ce of a 10 re carbonyl 19). Illumiical potenright-sidence of retilight onset hat proteocapable of physiologe activities containing proteorhomain to be Fig. 1. (A) Phylogenetic tree of bacterial 16S rRNA gene sequences, including that encoded on the 130-kb bacterioplankton BAC clone (EBAC31A08) (16). (B) Phylogenetic analysis of proteorhodopsin with archaeal (BR, HR, and SR prefixes) and Neurospora crassa (NOP1 prefix) rhodopsins (16). Nomenclature: Name_Species.abbreviation_Genbank.gi (HR, halorhodopsin; SR, sensory rhodopsin; BR, bacteriorhodopsin). Halsod, Halorubrum sodomense; Halhal, Halobacterium salinarum (halobium); Halval, Haloarcula vallismortis; Natpha, Natronomonas pharaonis; Halsp, Halobacterium sp; Neucra, Neurospora crassa. Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !73
  74. 74. Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !74
  75. 75. Shotgun metagenomics shotgun sequence Metagenomics Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !75
  76. 76. articles Community structure and metabolism through reconstruction of microbial genomes from the environment Gene W. Tyson1, Jarrod Chapman3,4, Philip Hugenholtz1, Eric E. Allen1, Rachna J. Ram1, Paul M. Richardson4, Victor V. Solovyev4, Edward M. Rubin4, Daniel S. Rokhsar3,4 & Jillian F. Banfield1,2 1 Department of Environmental Science, Policy and Management, 2Department of Earth and Planetary Sciences, and 3Department of Physics, University of California, Berkeley, California 94720, USA 4 Joint Genome Institute, Walnut Creek, California 94598, USA RESEARCH ARTICLE ........................................................................................................................................................................................................................... Microbial communities are vital in the functioning of all ecosystems; however, most microorganisms are uncultivated, and their roles in natural systems are unclear. Here, using random shotgun sequencing of DNA from a natural acidophilic biofilm, we report reconstruction of near-complete genomes of Leptospirillum group II and Ferroplasma type II, and partial recovery of three other genomes. This was possible because the biofilm was dominated by a small number of species populations and the frequency of genomic rearrangements and gene insertions or deletions was relatively low. Because each sequence read came from a different individual, we could determine that single-nucleotide polymorphisms are the predominant form of heterogeneity at the strain level. The Leptospirillum group II genome had remarkably few nucleotide polymorphisms, despite the existence of low-abundance variants. The Ferroplasma type II genome seems to be a composite from three ancestral strains that have undergone homologous recombination to form a large population of mosaic genomes. Analysis of the gene complement for each organism revealed the pathways for carbon and nitrogen fixation and energy generation, and provided insights into survival strategies in an extreme J. Craig Venter,1* Karin Remington,1 John F. Heidelberg,3 environment. 2 2 3 Environmental Genome Shotgun Sequencing of the Sargasso Sea Aaron L. Halpern, Doug Rusch, Jonathan A. Eisen, Dongying Wu,3 Ian Paulsen,3 Karen E. Nelson,3 William Nelson,3 The study of microbial evolution and ecology has been revolutio- fluorescence3in situ hybridization Anthony H. Knap,6 biofilms Derrick E. Fouts, Samuel Levy,2 (FISH) revealed that all nized by DNA sequencing and analysis1–3. However, isolates have contained mixtures of bacteria (Leptospirillum, Sulfobacillus and, in Michael W. Lomas,6 Ken Nealson,5 Owen White,3 and other been the main source of sequence data, and only a small fraction of a few cases, Acidimicrobium) and1archaea (Ferroplasma 6 Jeremy Peterson,3 Thermoplasmatales). The genome of one microorganisms have been cultivated4–6. Consequently, focus has members of theJeff Hoffman, Rachel Parsons, of these shifted towards the analysis of uncultivated microorganisms via archaea, Ferroplasma acidarmanus fer1, isolated fromRogers,4 Holly Baden-Tillson,1 Cynthia Pfannkoch,1 Yu-Hui the Richmond 5 cloning of conserved genes and genome fragments directly from mine, has been sequenced previously (http://www.jgi.doe.gov/JGI_ Hamilton O. Smith1 the environment7–9. To date, only a small fraction of genes have been microbial/html/ferroplasma/ferro_homepage.html). Slides for UC Davis EVE161 Course biofilm (Fig.Jonathan Eisen Winter 2014 was recovered from individual environments, limiting the analysis of A pink Taught by 1a) typical of AMD communities chlorococcus, tha photosynthetic bio Surface water were collected ab from three sites o February 2003. A lected aboard the S station S” in May are indicated on F S1; sampling prot one expedition to was extracted from genomic libraries w 2 to 6 kb were m prepared plasmid both ends to!76 provi
  77. 77. Binning challenge A B C D E F G T U V W X Y Z Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !77
  78. 78. Binning challenge A B C D E F G Best binning method: reference genomes Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 T U V W X Y Z !78
  79. 79. Case Study - Human Microbiome Metagenomics 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, 13 4,14 12 10 Karsten Kristiansen , Oluf Pedersen , Julian Parkhill , Jean Weissenbach , MetaHIT Consortium{, Peer Bork2, 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. Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !79
  80. 80. ARTICLES 40 PC2 • • • • 30 • •• • • Ulcerative colitis • • • • • • • • • • • • • • PC1 • • • • Healthy • • 20 10 • • Cluster (%) • • Crohn’s disease • • 0 P value: 0.031 • • • 1 • Figure 4 | Bacterial species abundance differentiates IBD patients and healthy individuals. Principal component analysis with health status as instrumental variables, based on the abundance of 155 species with $1% genome coverage by the Illumina reads in at least 1 individual of the cohort, 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 inertia. Individuals (represented by points) were clustered and centre of gravity computed for each class; P-value of the link between health status and species abundance was assessed using a Monte-Carlo test (999 replicates). Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Almost all (99.96%) of the phylogenetically assigned Winter 2014 genes belonged Figure 5 | Cluste were ranked by t length and copy n clusters with the groups of 100 clu that contains 86% were within th This suggests th (Supplementar functions impo We found tw !80

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