18-21 August 2009 METAGENOMIC WORKSHOP James R. Cole, Ph.D. Ribosomal Database Project Center for...
-
Upload
armando-chinnock -
Category
Documents
-
view
216 -
download
1
Transcript of 18-21 August 2009 METAGENOMIC WORKSHOP James R. Cole, Ph.D. Ribosomal Database Project Center for...
![Page 1: 18-21 August 2009 METAGENOMIC WORKSHOP James R. Cole, Ph.D. Ribosomal Database Project Center for Microbial Ecology Michigan State University .](https://reader038.fdocuments.net/reader038/viewer/2022103112/551c36d65503460d398b473e/html5/thumbnails/1.jpg)
18-21 August 2009
METAGENOMIC WORKSHOP
James R. Cole, Ph.D.Ribosomal Database ProjectCenter for Microbial Ecology
Michigan State University
http://rdp.cme.msu.edu
![Page 2: 18-21 August 2009 METAGENOMIC WORKSHOP James R. Cole, Ph.D. Ribosomal Database Project Center for Microbial Ecology Michigan State University .](https://reader038.fdocuments.net/reader038/viewer/2022103112/551c36d65503460d398b473e/html5/thumbnails/2.jpg)
18-21 August 2009
Some Bacterial Names
• Bacillus subtilis (Ehrenberg 1835) Cohn 1872
• Rhizobium meliloti Dangeard 1926• Sinorhizomium meliloti (Dangeard 1926) De Lajudie
et al. 1994• Ensifer meliloti (Dangeard 1926) Young 2003
• Actinobacteria Stackenbrandt et al. 1997
![Page 3: 18-21 August 2009 METAGENOMIC WORKSHOP James R. Cole, Ph.D. Ribosomal Database Project Center for Microbial Ecology Michigan State University .](https://reader038.fdocuments.net/reader038/viewer/2022103112/551c36d65503460d398b473e/html5/thumbnails/3.jpg)
18-21 August 2009
Prokaryotic Nomenclature
• Bacterial and Archaeal Names are covered by an official set of rules:
– “The International Code of Nomenclature of Prokaryotes”
– Covers ranks from Class to Subspecies.
– Ranks of Kingdom and Phylum (Domain) are not covered by the code.
![Page 4: 18-21 August 2009 METAGENOMIC WORKSHOP James R. Cole, Ph.D. Ribosomal Database Project Center for Microbial Ecology Michigan State University .](https://reader038.fdocuments.net/reader038/viewer/2022103112/551c36d65503460d398b473e/html5/thumbnails/4.jpg)
18-21 August 2009
Synonymy
New Combination (taxonomic change)Rhizobium meliloti -> Ensifer meliloti
Homotypic Synonym (same type strain)Aeromonas caviae = Aeromonas punctata
Heterotypic Synonym (different type strain)Wautersia eutropha => Cupriavidis necator
9612 Species Names But Only 8062 Species?
![Page 5: 18-21 August 2009 METAGENOMIC WORKSHOP James R. Cole, Ph.D. Ribosomal Database Project Center for Microbial Ecology Michigan State University .](https://reader038.fdocuments.net/reader038/viewer/2022103112/551c36d65503460d398b473e/html5/thumbnails/5.jpg)
18-21 August 2009
![Page 6: 18-21 August 2009 METAGENOMIC WORKSHOP James R. Cole, Ph.D. Ribosomal Database Project Center for Microbial Ecology Michigan State University .](https://reader038.fdocuments.net/reader038/viewer/2022103112/551c36d65503460d398b473e/html5/thumbnails/6.jpg)
18-21 August 2009
![Page 7: 18-21 August 2009 METAGENOMIC WORKSHOP James R. Cole, Ph.D. Ribosomal Database Project Center for Microbial Ecology Michigan State University .](https://reader038.fdocuments.net/reader038/viewer/2022103112/551c36d65503460d398b473e/html5/thumbnails/7.jpg)
18-21 August 2009
![Page 8: 18-21 August 2009 METAGENOMIC WORKSHOP James R. Cole, Ph.D. Ribosomal Database Project Center for Microbial Ecology Michigan State University .](https://reader038.fdocuments.net/reader038/viewer/2022103112/551c36d65503460d398b473e/html5/thumbnails/8.jpg)
18-21 August 2009
Prokaryotic Taxonomy
• There is no formal Prokaryotic taxonomyTaxonomy is a matter of opinion!
• Changes in opinion may require changes in nomenclature Rhizobium meliloti -> Ensifer meliloti
• Every taxon must be circumscribed•
![Page 9: 18-21 August 2009 METAGENOMIC WORKSHOP James R. Cole, Ph.D. Ribosomal Database Project Center for Microbial Ecology Michigan State University .](https://reader038.fdocuments.net/reader038/viewer/2022103112/551c36d65503460d398b473e/html5/thumbnails/9.jpg)
18-21 August 2009
![Page 10: 18-21 August 2009 METAGENOMIC WORKSHOP James R. Cole, Ph.D. Ribosomal Database Project Center for Microbial Ecology Michigan State University .](https://reader038.fdocuments.net/reader038/viewer/2022103112/551c36d65503460d398b473e/html5/thumbnails/10.jpg)
18-21 August 2009
![Page 11: 18-21 August 2009 METAGENOMIC WORKSHOP James R. Cole, Ph.D. Ribosomal Database Project Center for Microbial Ecology Michigan State University .](https://reader038.fdocuments.net/reader038/viewer/2022103112/551c36d65503460d398b473e/html5/thumbnails/11.jpg)
18-21 August 2009
![Page 12: 18-21 August 2009 METAGENOMIC WORKSHOP James R. Cole, Ph.D. Ribosomal Database Project Center for Microbial Ecology Michigan State University .](https://reader038.fdocuments.net/reader038/viewer/2022103112/551c36d65503460d398b473e/html5/thumbnails/12.jpg)
18-21 August 2009
Bacterial Species
• One strain of a species is designated as the “type strain”. Other closely related strains are of the same species.
• There is no completely objective method for species delimitation.
• The current accepted standard is 70% DNA-DNA hybridization (DDH)
![Page 13: 18-21 August 2009 METAGENOMIC WORKSHOP James R. Cole, Ph.D. Ribosomal Database Project Center for Microbial Ecology Michigan State University .](https://reader038.fdocuments.net/reader038/viewer/2022103112/551c36d65503460d398b473e/html5/thumbnails/13.jpg)
18-21 August 2009
Stackebrandt, E. and J. Ebers. (2006) Taxonomic parameters revisited: tarnished gold standards. Microbiology Today :152-155.
DDH vs 16S Similarity
![Page 14: 18-21 August 2009 METAGENOMIC WORKSHOP James R. Cole, Ph.D. Ribosomal Database Project Center for Microbial Ecology Michigan State University .](https://reader038.fdocuments.net/reader038/viewer/2022103112/551c36d65503460d398b473e/html5/thumbnails/14.jpg)
18-21 August 2009
Goris, J., Konstantinidis, K.T., Klappenbach, J.A., Coenye, T., Vandamme, P., Tiedje, J.M. (2007) DNA–DNA hybridization values and their relationship to whole-genome sequence similarities. IJSEM 57:81–91. doi:10.1099/ijs.0.64483-0
DDH vs Genome Similarity
![Page 15: 18-21 August 2009 METAGENOMIC WORKSHOP James R. Cole, Ph.D. Ribosomal Database Project Center for Microbial Ecology Michigan State University .](https://reader038.fdocuments.net/reader038/viewer/2022103112/551c36d65503460d398b473e/html5/thumbnails/15.jpg)
18-21 August 2009
Bacterial Species Definition
• The species concept in bacteria is not well defined.
• As an ad-hoc measure, 70% DHH has several limitations.
• Less than 98.5% 16S similarity indicates different species, but greater than 98.5% does not indicate the same species.
• ANI may be a good candidate to replace DHH as an ad-hoc species definition.
![Page 16: 18-21 August 2009 METAGENOMIC WORKSHOP James R. Cole, Ph.D. Ribosomal Database Project Center for Microbial Ecology Michigan State University .](https://reader038.fdocuments.net/reader038/viewer/2022103112/551c36d65503460d398b473e/html5/thumbnails/16.jpg)
18-21 August 2009
Nomenclatural References
• The International Code of Nomenclature of Prokaryotes: http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=icnb
• List of Prokaryotic Names with Standing in Nomenclature http://www.bacterio.cict.fr/index.html
• Bacterial Nomenclature Up-to-Date http://www.dsmz.de/microorganisms/bacterial_nomenclature.php
![Page 17: 18-21 August 2009 METAGENOMIC WORKSHOP James R. Cole, Ph.D. Ribosomal Database Project Center for Microbial Ecology Michigan State University .](https://reader038.fdocuments.net/reader038/viewer/2022103112/551c36d65503460d398b473e/html5/thumbnails/17.jpg)
18-21 August 2009
Taxonomy References
• NCBI Taxonomyhttp://www.ncbi.nlm.nih.gov/Taxonomy/
• TOBAhttp://www.taxonomicoutline.org/
• Bergey’s Taxonomyhttp://www.bergeys.org/outlines.html
![Page 18: 18-21 August 2009 METAGENOMIC WORKSHOP James R. Cole, Ph.D. Ribosomal Database Project Center for Microbial Ecology Michigan State University .](https://reader038.fdocuments.net/reader038/viewer/2022103112/551c36d65503460d398b473e/html5/thumbnails/18.jpg)
![Page 19: 18-21 August 2009 METAGENOMIC WORKSHOP James R. Cole, Ph.D. Ribosomal Database Project Center for Microbial Ecology Michigan State University .](https://reader038.fdocuments.net/reader038/viewer/2022103112/551c36d65503460d398b473e/html5/thumbnails/19.jpg)
18-21 August 2009
Phylogenetic Inference
Use simplest method that works.
![Page 20: 18-21 August 2009 METAGENOMIC WORKSHOP James R. Cole, Ph.D. Ribosomal Database Project Center for Microbial Ecology Michigan State University .](https://reader038.fdocuments.net/reader038/viewer/2022103112/551c36d65503460d398b473e/html5/thumbnails/20.jpg)
18-21 August 2009
Why rRNA?
• Universal
• No ortholog - paralog problem
• No horizontal gene transfer
• Easy to amplify and sequence
• Large database
![Page 21: 18-21 August 2009 METAGENOMIC WORKSHOP James R. Cole, Ph.D. Ribosomal Database Project Center for Microbial Ecology Michigan State University .](https://reader038.fdocuments.net/reader038/viewer/2022103112/551c36d65503460d398b473e/html5/thumbnails/21.jpg)
18-21 August 2009
Jukes & Cantor
To: A G C T --------------------------------- A | 1-3a a a a From: G | a 1-3a a a C | a a 1-3a a T | a a a 1-3a
p = 3/4 ( 1 - e- 4/3 u t )
ut = - 3/4 loge ( 1 - 4/3 p )
a = u dt
(ut = evolutionary distance)
![Page 22: 18-21 August 2009 METAGENOMIC WORKSHOP James R. Cole, Ph.D. Ribosomal Database Project Center for Microbial Ecology Michigan State University .](https://reader038.fdocuments.net/reader038/viewer/2022103112/551c36d65503460d398b473e/html5/thumbnails/22.jpg)
18-21 August 2009
Substitution Models
• Jukes & Cantor (0 parameters)– All rates equal
• Kimura (2 parameters)– Transition & transversion different
• HKY (6 parameters)– Plus different frequency of four nucleotides
• Generalized time reversible– 8 parameters
![Page 23: 18-21 August 2009 METAGENOMIC WORKSHOP James R. Cole, Ph.D. Ribosomal Database Project Center for Microbial Ecology Michigan State University .](https://reader038.fdocuments.net/reader038/viewer/2022103112/551c36d65503460d398b473e/html5/thumbnails/23.jpg)
18-21 August 2009
Alignment Mask
• Use to remove unstable regions from phylogenetic analysis– Inserts and deletes make homolog assigment difficult
• Rapidly changing regions add noise• Should be reported in publication (but often are
not)• Alignment masks should be provided with
alignment• Can be created by using ad-hoc rules
– Eg 50% residues in column
![Page 24: 18-21 August 2009 METAGENOMIC WORKSHOP James R. Cole, Ph.D. Ribosomal Database Project Center for Microbial Ecology Michigan State University .](https://reader038.fdocuments.net/reader038/viewer/2022103112/551c36d65503460d398b473e/html5/thumbnails/24.jpg)
18-21 August 2009
Phylogenetic Methods
• UPGMA– Assumes clock
• Neighbor Joining– No clock, distance based
• Parsimony– Slow, used in other fields
• Maximum Likelihood– Slow, but fast converging, allows different rates
• Bayesian– Can be faster then maximum likelihood
![Page 25: 18-21 August 2009 METAGENOMIC WORKSHOP James R. Cole, Ph.D. Ribosomal Database Project Center for Microbial Ecology Michigan State University .](https://reader038.fdocuments.net/reader038/viewer/2022103112/551c36d65503460d398b473e/html5/thumbnails/25.jpg)
18-21 August 2009
Confidence Estimation
• Bootstrapping alignment columns– Estimates confidence for individual branch
order– Sensitive to “unstable” sequenes
• Kishino-Hasegawa-Templeton test– Compares whole trees– Asks one is significantly better– Uses Maximum Likelihood
![Page 26: 18-21 August 2009 METAGENOMIC WORKSHOP James R. Cole, Ph.D. Ribosomal Database Project Center for Microbial Ecology Michigan State University .](https://reader038.fdocuments.net/reader038/viewer/2022103112/551c36d65503460d398b473e/html5/thumbnails/26.jpg)
18-21 August 2009
![Page 27: 18-21 August 2009 METAGENOMIC WORKSHOP James R. Cole, Ph.D. Ribosomal Database Project Center for Microbial Ecology Michigan State University .](https://reader038.fdocuments.net/reader038/viewer/2022103112/551c36d65503460d398b473e/html5/thumbnails/27.jpg)
18-21 August 2009
![Page 28: 18-21 August 2009 METAGENOMIC WORKSHOP James R. Cole, Ph.D. Ribosomal Database Project Center for Microbial Ecology Michigan State University .](https://reader038.fdocuments.net/reader038/viewer/2022103112/551c36d65503460d398b473e/html5/thumbnails/28.jpg)
18-21 August 2009
![Page 29: 18-21 August 2009 METAGENOMIC WORKSHOP James R. Cole, Ph.D. Ribosomal Database Project Center for Microbial Ecology Michigan State University .](https://reader038.fdocuments.net/reader038/viewer/2022103112/551c36d65503460d398b473e/html5/thumbnails/29.jpg)
18-21 August 2009
![Page 30: 18-21 August 2009 METAGENOMIC WORKSHOP James R. Cole, Ph.D. Ribosomal Database Project Center for Microbial Ecology Michigan State University .](https://reader038.fdocuments.net/reader038/viewer/2022103112/551c36d65503460d398b473e/html5/thumbnails/30.jpg)
18-21 August 2009
![Page 31: 18-21 August 2009 METAGENOMIC WORKSHOP James R. Cole, Ph.D. Ribosomal Database Project Center for Microbial Ecology Michigan State University .](https://reader038.fdocuments.net/reader038/viewer/2022103112/551c36d65503460d398b473e/html5/thumbnails/31.jpg)
18-21 August 2009
![Page 32: 18-21 August 2009 METAGENOMIC WORKSHOP James R. Cole, Ph.D. Ribosomal Database Project Center for Microbial Ecology Michigan State University .](https://reader038.fdocuments.net/reader038/viewer/2022103112/551c36d65503460d398b473e/html5/thumbnails/32.jpg)
18-21 August 2009
Phylogeny Programs
• Phylogeny Program List:http://evolution.genetics.washington.edu/phylip/software.html
• Phyliphttp://evolution.genetics.washington.edu/phylip
• MEGAhttp://www.megasoftware.net/
• Clearcuthttp://bioinformatics.hungry.com/clearcut/
• Weighborhttp://www.t6.lanl.gov/billb/weighbor/index.html
• Mr. Bayes• http://mrbayes.csit.fsu.edu/
![Page 33: 18-21 August 2009 METAGENOMIC WORKSHOP James R. Cole, Ph.D. Ribosomal Database Project Center for Microbial Ecology Michigan State University .](https://reader038.fdocuments.net/reader038/viewer/2022103112/551c36d65503460d398b473e/html5/thumbnails/33.jpg)
![Page 34: 18-21 August 2009 METAGENOMIC WORKSHOP James R. Cole, Ph.D. Ribosomal Database Project Center for Microbial Ecology Michigan State University .](https://reader038.fdocuments.net/reader038/viewer/2022103112/551c36d65503460d398b473e/html5/thumbnails/34.jpg)
18-21 August 2009
The Ribosomal Database Project
Sequences and tools
![Page 35: 18-21 August 2009 METAGENOMIC WORKSHOP James R. Cole, Ph.D. Ribosomal Database Project Center for Microbial Ecology Michigan State University .](https://reader038.fdocuments.net/reader038/viewer/2022103112/551c36d65503460d398b473e/html5/thumbnails/35.jpg)
18-21 August 2009
Aligned and annotated sequencesAligned and annotated sequences
tutorials and helptutorials and helpupload and align
your own 16Ssequences inyour private
myRDP space and use the new
analysis Pipeline
upload and alignyour own 16Ssequences inyour private
myRDP space and use the new
analysis Pipelineinteractive online toolsinteractive online tools
powerful search andselection features
powerful search andselection features
view by publication or genomeview by publication or genome
![Page 36: 18-21 August 2009 METAGENOMIC WORKSHOP James R. Cole, Ph.D. Ribosomal Database Project Center for Microbial Ecology Michigan State University .](https://reader038.fdocuments.net/reader038/viewer/2022103112/551c36d65503460d398b473e/html5/thumbnails/36.jpg)
18-21 August 2009
RDP-II Screenshotsfast search algorithm,limit searches to sequences spanning specific regions,
change depth and edit distance
fast search algorithm,limit searches to sequences spanning specific regions,
change depth and edit distance
place sequences into bacterial taxonomy,works well with partial or full-length sequences,
bootstrap confidence estimate,prior alignment not required
place sequences into bacterial taxonomy,works well with partial or full-length sequences,
bootstrap confidence estimate,prior alignment not required
finds nearest neighbor,more accurate than BLAST,
uses “q-gram” matching method
finds nearest neighbor,more accurate than BLAST,
uses “q-gram” matching method
![Page 37: 18-21 August 2009 METAGENOMIC WORKSHOP James R. Cole, Ph.D. Ribosomal Database Project Center for Microbial Ecology Michigan State University .](https://reader038.fdocuments.net/reader038/viewer/2022103112/551c36d65503460d398b473e/html5/thumbnails/37.jpg)
18-21 August 2009
examine sequences by publication
examine sequences by publication
Browse and select from taxonomic hierarchy
Browse and select from taxonomic hierarchy
![Page 38: 18-21 August 2009 METAGENOMIC WORKSHOP James R. Cole, Ph.D. Ribosomal Database Project Center for Microbial Ecology Michigan State University .](https://reader038.fdocuments.net/reader038/viewer/2022103112/551c36d65503460d398b473e/html5/thumbnails/38.jpg)
18-21 August 2009
rRNA sequence from genome projects
rRNA sequence from genome projects
Provides links to other genome-related information
Provides links to other genome-related information
![Page 39: 18-21 August 2009 METAGENOMIC WORKSHOP James R. Cole, Ph.D. Ribosomal Database Project Center for Microbial Ecology Michigan State University .](https://reader038.fdocuments.net/reader038/viewer/2022103112/551c36d65503460d398b473e/html5/thumbnails/39.jpg)
18-21 August 2009
Ribosomal Database Project-IImyRDP Personal/Collaborative Workspace
upload and align your own 16S sequences in your
myRDP space
upload and align your own 16S sequences in your
myRDP space
your gateway to thehigh-throughput pipeline
your gateway to thehigh-throughput pipeline
share specific data with research buddies
share specific data with research buddies
![Page 40: 18-21 August 2009 METAGENOMIC WORKSHOP James R. Cole, Ph.D. Ribosomal Database Project Center for Microbial Ecology Michigan State University .](https://reader038.fdocuments.net/reader038/viewer/2022103112/551c36d65503460d398b473e/html5/thumbnails/40.jpg)
18-21 August 2009
Ribosomal Database Project-IImyRDP High-Throughput Pipeline
sequencer debugging:status and quality
summary for each sequence
sequencer debugging:status and quality
summary for each sequence
Library-level information
Library-level information
![Page 41: 18-21 August 2009 METAGENOMIC WORKSHOP James R. Cole, Ph.D. Ribosomal Database Project Center for Microbial Ecology Michigan State University .](https://reader038.fdocuments.net/reader038/viewer/2022103112/551c36d65503460d398b473e/html5/thumbnails/41.jpg)
18-21 August 2009
Ribosomal Database Project-IImyRDP Single-Read Pipeline
![Page 42: 18-21 August 2009 METAGENOMIC WORKSHOP James R. Cole, Ph.D. Ribosomal Database Project Center for Microbial Ecology Michigan State University .](https://reader038.fdocuments.net/reader038/viewer/2022103112/551c36d65503460d398b473e/html5/thumbnails/42.jpg)
18-21 August 2009
Interactive assignments customized on a per-student basis
Interactive assignments customized on a per-student basis
Short video tutorials cover all major features
Short video tutorials cover all major features
![Page 43: 18-21 August 2009 METAGENOMIC WORKSHOP James R. Cole, Ph.D. Ribosomal Database Project Center for Microbial Ecology Michigan State University .](https://reader038.fdocuments.net/reader038/viewer/2022103112/551c36d65503460d398b473e/html5/thumbnails/43.jpg)
18-21 August 2009
RDP 10 Alignment
• INFERNAL Stochastic Context-Free Grammar Aligner (Eddy)
• INFERNAL (Eddy)– Fast
– Replaces RNAcad (Brown)
• Incorporates secondary structure information
• Probabilistic model
• New training set
http://www.rna.icmb.utexas.edu
![Page 44: 18-21 August 2009 METAGENOMIC WORKSHOP James R. Cole, Ph.D. Ribosomal Database Project Center for Microbial Ecology Michigan State University .](https://reader038.fdocuments.net/reader038/viewer/2022103112/551c36d65503460d398b473e/html5/thumbnails/44.jpg)