Microbial Ecology       Indoor Microbial Ecology      (DNA Sequencing Focus)                Indoor Air 2011Workshop on Mic...
Outline•   Introduction•   Sequencing in microbial studies•   Sequencing technologies•   Current and future issues
microBEnet• /               http://microbe.net
microBEnet
MICROBES
A Field Guide to Microbes• What should be included  •   Catalog of types of organism  •   Functional diversity  •   Biogeo...
Microbial Ecology• Much more than just a field guide• Interactions of microbes with each other  with macroorganisms, and t...
I: Sequencing and Microbes• Sequencing is useful as a tool in studies  of microbial ecology for many reasons• It is compli...
Era I: rRNA Tree of Life  Bacteria                                             • Appearance of                            ...
Era II: rRNA in environment
Great Plate Count Anomaly Culturing    Microscope  Count         Count
Great Plate Count Anomaly Culturing      Microscope  Count      <<<< Count
Great Plate Count Anomaly                          DNA Culturing      Microscope  Count      <<<< Count
PCR & phylogenetic analysis of rRNA              DNA              extraction                             PCR              ...
Era II: rRNA in environmentThe Hidden Majority            Richness estimates             Hugenholtz 2002         Bohannan ...
Era III: Genome Sequencing                                   Genomes Online             Fleischmann et al. 1995 Science 26...
Lateral Gene TransferPerna et al. 2003
Era IV: Genomes in Environment                   shotgun                        sequenceMetagenomics
Weighted % of Clones                                                                                           0          ...
Metagenomics & Ecology
Sequencing Technology
Generation I: Manual Sanger
Generation II: Automation
Generation III: No clones
Generation IV: ????
Challenges and Outlook
What’s Coming?• Sequencing  •   Speed up; cost down  •   Mini-sequencers with massive capacity  •   Automation of sample p...
Beyond Sequencing• Array methods should not be ignored  • Bad gene array  • Phylochips• High throughput/low cost approache...
Challenge 1: Data overload• Major current issue is massive size of  sequence data sets• Creates many new challenges not wi...
Solutions?•   Throw away data (analogous to CERN)•   New algorithms to limit RAM needs•   Complete automation of algorithm...
Challenge 2: Short reads• Some specific challenges come from  short reads• Key step in analysis of mixed  communities is “...
Solutions• Longer reads• More full length reference data  • Reference is annotated  • Reads are used to count• New algorit...
Challenge 3: Real time• New sequencing and array technologies  allow almost real time data collection• Analysis generally ...
Solutions?• New automated high throughput  methods  • Must be updated continuously to deal with    new data types  • Need ...
Challenge 4: Reference Data• Microbial diversity woefully  undersampled• Greatly limits ability to  • Identify new organis...
Solution?• Systematic efforts to sample diversity• Some decent efforts in this regard in  terms of diversity of known Cate...
Spatial Diversity of Isolates
Genomic Diversity of Isolates               Bacteria                                                      Archaea         ...
Gene tree ≠ Genome tree               16s                                              WGT, 23SBadger et al. 2005 Int J Sy...
Phylogenetic Diversity• Phylogenetic  diversity poorly  sampled• GEBA project at DOE-  JGI correcting this
Metagenomic Diversity
Challenge 5: Knowledge• Data collection is of course not enough• Need to be able to turn the data into  knowledge• This is...
Solutions• More curators• Populate databases with experimental  information not more predictions• Bioinformatics expansion...
Acknowledgements• $$$  •   Sloan Foundation  •   DOE  •   NSF  •   GBMF  •   DARPA• People, places  • DOE JGI: Eddy Rubin,...
Eisen #microBEnet #IndoorAir2011
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Eisen #microBEnet #IndoorAir2011

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  • Send it out for sequencing, do an alignment with your gene and blast it (search for other organisms) with a similar sequence\n
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  • Eisen #microBEnet #IndoorAir2011

    1. 1. Microbial Ecology Indoor Microbial Ecology (DNA Sequencing Focus) Indoor Air 2011Workshop on Microbiomes of the Built Environment Jonathan A. Eisen, Ph.D. University of California, Davis DOE Joint Genome Institute Twitter: @phylogenomics
    2. 2. Outline• Introduction• Sequencing in microbial studies• Sequencing technologies• Current and future issues
    3. 3. microBEnet• / http://microbe.net
    4. 4. microBEnet
    5. 5. MICROBES
    6. 6. A Field Guide to Microbes• What should be included • Catalog of types of organism • Functional diversity • Biogeography (space and time) • Niche information • Means for identification• “Natural” locations• “Non natural (i.e., built) locations
    7. 7. Microbial Ecology• Much more than just a field guide• Interactions of microbes with each other with macroorganisms, and the environment• Mechanisms and rules of such interactions• Can be applied to any environment(s) including built ones
    8. 8. I: Sequencing and Microbes• Sequencing is useful as a tool in studies of microbial ecology for many reasons• It is complimentary to other means of study
    9. 9. Era I: rRNA Tree of Life Bacteria • Appearance of microbes not informative (enough) • rRNA Tree of Life Archaea identified two major groups of organisms w/o nuclei • rRNA powerful for many reasons, though not perfect EukaryotesBarton, Eisen et al. “Evolution”, CSHL Press. 2007.Based on tree from Pace 1997 Science 276:734-740
    10. 10. Era II: rRNA in environment
    11. 11. Great Plate Count Anomaly Culturing Microscope Count Count
    12. 12. Great Plate Count Anomaly Culturing Microscope Count <<<< Count
    13. 13. Great Plate Count Anomaly DNA Culturing Microscope Count <<<< Count
    14. 14. PCR & phylogenetic analysis of rRNA 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
    15. 15. Era II: rRNA in environmentThe Hidden Majority Richness estimates Hugenholtz 2002 Bohannan and Hughes 2003
    16. 16. Era III: Genome Sequencing Genomes Online Fleischmann et al. 1995 Science 269:496-512
    17. 17. Lateral Gene TransferPerna et al. 2003
    18. 18. Era IV: Genomes in Environment shotgun sequenceMetagenomics
    19. 19. Weighted % of Clones 0 0.1250 0.2500 0.3750 0.5000 Al ph a Be pro ta teo G p b am rot ac m eo te ba ria Ep ap ct si ro lo t e np eob ria D el rot ac ta e t pr ob eria ot ac C eo te ya b rEFG no ac iaEFTurRNARecARpoB b teHSP70 Fi act ria rm e Ac ic ria tin ut es ob a C cte hl r or ia ob C i FB C hl o Major Phylogenetic Group Sp rof Metagenomic Phylotyping Sargasso Phylotypes iro lex i Fu cha D304: 66. 2004 ei so et no ba es co ct cc er Euus ia ry -T a hVenter et al., Science C rcherm re na aeous rc t ha a eo ta
    20. 20. Metagenomics & Ecology
    21. 21. Sequencing Technology
    22. 22. Generation I: Manual Sanger
    23. 23. Generation II: Automation
    24. 24. Generation III: No clones
    25. 25. Generation IV: ????
    26. 26. Challenges and Outlook
    27. 27. What’s Coming?• Sequencing • Speed up; cost down • Mini-sequencers with massive capacity • Automation of sample processing • Portable and remote systems • Massive databases• Computational changes • Clusters vs. RAM • Cloud computing • GPU acceleration
    28. 28. Beyond Sequencing• Array methods should not be ignored • Bad gene array • Phylochips• High throughput/low cost approaches to characterizing other macromolecules • Proteomics • Metabolomics • Transcriptomics
    29. 29. Challenge 1: Data overload• Major current issue is massive size of sequence data sets• Creates many new challenges not widely anticipated • Data transfer and storage • RAM limits for some processes • Databases overstretched
    30. 30. Solutions?• Throw away data (analogous to CERN)• New algorithms to limit RAM needs• Complete automation of algorithms• Distributed data (e.g., Biotorrents)• Emphasis on standards and metadata
    31. 31. Challenge 2: Short reads• Some specific challenges come from short reads• Key step in analysis of mixed communities is “binning”• Binning methods perform poorly on short reads • nucleotide composition • blast hits • phylogenetic analysis
    32. 32. Solutions• Longer reads• More full length reference data • Reference is annotated • Reads are used to count• New algorithms • Phylogeny w/ short reads • Cobinning/combining data • New markers • Better HMM searches
    33. 33. Challenge 3: Real time• New sequencing and array technologies allow almost real time data collection• Analysis generally not done in real time • e.g., metagenome annotation can take weeks to months • e.g., phylogenetics bottleneck • systems not set up for rapid, open sharing of results
    34. 34. Solutions?• New automated high throughput methods • Must be updated continuously to deal with new data types • Need to be tested and verified• Rapid sharing of results • PLoS Currents 0.700 0.525 0.350 0.175 0 C eob ria Ba ac ria oi a s or es xi te ri le hl et te b e te de of no ct yc pr bac ya a om er o C ct te ct ot ro an ap Pl ta ph el D Al
    35. 35. Challenge 4: Reference Data• Microbial diversity woefully undersampled• Greatly limits ability to • Identify new organisms from DNA fragments • Determine if organisms are out of “place” in some way compared to natural diversity • Perform reliable attribution/matching • Understand EIDs • Know what is “normal”
    36. 36. Solution?• Systematic efforts to sample diversity• Some decent efforts in this regard in terms of diversity of known Category ABC pathogens• Much more needed
    37. 37. Spatial Diversity of Isolates
    38. 38. Genomic Diversity of Isolates Bacteria Archaea Eukaryotes Figure from Barton, Eisen et al. “Evolution”, CSHL Press. Based on tree from Pace NR, 2003.
    39. 39. Gene tree ≠ Genome tree 16s WGT, 23SBadger et al. 2005 Int J System Evol Microbiol 55: 1021-1026.
    40. 40. Phylogenetic Diversity• Phylogenetic diversity poorly sampled• GEBA project at DOE- JGI correcting this
    41. 41. Metagenomic Diversity
    42. 42. Challenge 5: Knowledge• Data collection is of course not enough• Need to be able to turn the data into knowledge• This is difficult to automate
    43. 43. Solutions• More curators• Populate databases with experimental information not more predictions• Bioinformatics expansion• Better linking with ecology, building science, etc.
    44. 44. Acknowledgements• $$$ • Sloan Foundation • DOE • NSF • GBMF • DARPA• People, places • DOE JGI: Eddy Rubin, Phil Hugenholtz et al. • UC Davis: Aaron Darling, Dongying Wu • Other: Jessica Green, Katie Pollard, Martin Wu, Tom Slezak, Jack Gilbert
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