Talk by Jonathan Eisen and Indoor Air 2011 meeting

1. Microbial Ecology
Indoor Microbial Ecology
(DNA Sequencing Focus)
Indoor Air 2011
Workshop on Microbiomes of the Built Environment
Jonathan A. Eisen, Ph.D.
University of California, Davis
DOE Joint Genome Institute
Twitter: @phylogenomics

2. Outline
• Introduction
• Sequencing in microbial studies
• Sequencing technologies
• Current and future issues

3. microBEnet
• /
http://microbe.net

4. microBEnet

6. MICROBES

7. 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

8. 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

9. 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

10. 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
Eukaryotes
Barton, Eisen et al. “Evolution”, CSHL Press. 2007.
Based on tree from Pace 1997 Science 276:734-740

11. Era II: rRNA in environment

12. Great Plate Count Anomaly
Culturing Microscope
Count Count

13. Great Plate Count Anomaly
Culturing Microscope
Count <<<< Count

14. Great Plate Count Anomaly
DNA
Culturing Microscope
Count <<<< Count

15. 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

16. Era II: rRNA in environment
The Hidden Majority Richness estimates
Hugenholtz 2002 Bohannan and Hughes 2003

17. Era III: Genome Sequencing
Genomes Online
Fleischmann et al. 1995 Science 269:496-512

18. Lateral Gene Transfer
Perna et al. 2003

19. Era IV: Genomes in Environment
shotgun
sequence
Metagenomics

20. Weighted % of Clones
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21. Metagenomics & Ecology

22. Sequencing Technology

23. Generation I: Manual Sanger

24. Generation II: Automation

25. Generation III: No clones

26. Generation IV: ????

27. Challenges and Outlook

28. 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

29. Beyond Sequencing
• Array methods should not be ignored
• Bad gene array
• Phylochips
• High throughput/low cost approaches to
characterizing other macromolecules
• Proteomics
• Metabolomics
• Transcriptomics

30. 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

31. 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

32. 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

33. 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

34. 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

35. 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
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36. 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”

37. Solution?
• Systematic efforts to sample diversity
• Some decent efforts in this regard in
terms of diversity of known Category
ABC pathogens
• Much more needed

38. Spatial Diversity of Isolates

39. Genomic Diversity of Isolates
Bacteria
Archaea
Eukaryotes
Figure from Barton, Eisen et al.
“Evolution”, CSHL Press.
Based on tree from Pace NR, 2003.

40. Gene tree ≠ Genome tree
16s WGT, 23S
Badger et al. 2005 Int J System Evol Microbiol 55: 1021-1026.

41. Phylogenetic Diversity
• Phylogenetic
diversity poorly
sampled
• GEBA project at DOE-
JGI correcting this

42. Metagenomic Diversity

43. 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

44. Solutions
• More curators
• Populate databases with experimental
information not more predictions
• Bioinformatics expansion
• Better linking with ecology, building
science, etc.

45. 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