Transcript Introducing DOTUR, a Computer Program for Defining

Introducing DOTUR, a Computer Program
for Defining Operational Taxonomic Units
and Estimating Species Richness
Patric D. Schloss and Jo Handelsman
Department of Plant Pathology, University of Wisconsin-Madison
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 2005
Presenter: Mingjie Wang
The Schloss Lab
http://schloss.micro.umass.edu/
What is in there
Statistical approaches for quantifying and comparing
the number and composition of lineages in microbial
communities are lacking. (species richness)
Species richness estimation
• Based on 16S rRNA gene sequences
• Grouped as Operational taxonomic units
(OTUs)/(Phylotypes)
• Defined by
Electrophoretic pattern
DNA sequence
Nucleotide sequence: 97%, 95%, 80%
OTU determined by
DNA sequence
Phlip L. Bond. et al. Bacterial Community Structures of Phosphate-Removing and NonPhosphate-Removing Activated Sludges from Sequencing Batch Reactors. Applied and
Environment Microbiology, 1995
Electrophoretic pattern
Restriction fragment length
polymorphism (RFLP) analysis of
16S rDNA of a six-member
bacterial
model
community
corresponding to HhaI digestion
Larry J. Forney et al. Characterization of Microbial Diversity by Determining Terminal Restriction
Fragment Length Polymorphisms of Genes Encoding 16S rRNA. Applied and Environment
Microbiology, 1997
General flowchart
ClustalW
Sequence Alignment
PHYLIP
Distance matrix generated
(input for DOTUR)
DOTUR
Sequence assignment at
every possible distance. etc.
Clustering algorithms
• Nearest neighbor (NN): Each of the sequences
within an OTU are at most X% distant from the
most similar sequence in the OTU
• Furthest neighbor (FN): All of the sequences
within an OTU are at most X% distant from all
of the other sequences within the OTU
• Average neighbor (AN): A middle ground
between the other two algorithms
DOTUR makes appropriate sequence
assignment
• NN: nearest neighbor assignment algorithm
• AN: average neighbor assignment algorithm
• FN: furthest neighbor assignment algorithm
• n1: no. of singletons;
• n2: no. of doubletons; etc.
Lineage-through-time plots by DOTUR
Application of DOTUR
• Construction of rarefaction and collector’s curves,
Shannon’s and Simpson’s diversity index, ACE, and
Chao1, Jackknife, and Bootstrap richness estimators
Rarefaction curves
Richness comparison between two Soil
samples using DOTUR
Scottish soil
Amazonian soil
Richness comparison between two soil
samples using DOTUR
• Result:
The number of observed OTUs from the Amazonian
soil falls within the 95 confidence interval (CI) of the
Scottish soil with 98 sequences sampled
• Conclusion:
The two samples have the same level of richness.
Application of DOTUR to the Sargasso
Sea metagenome sequence
16S rDNA
rpoB gene
Question
• What is the expected number of OTUs in a
microbial community?
• How to determine the minimum number of
sequences to estimate the overall OTUs?
Chao1 richness estimation
• DOTUR could give the full bias corrected Chao1 richness
estimates as described by Chao and modified by Colwell
(http://viceroy.eeb.uconn.edu/estimates)
Construction of collector’s curves
using Chao1 richness estimator
16S rDNA
rpoB gene
Summary
• DOTUR assigns sequences accurately and
consistently to OTUs for every distance level.
• DOTUR can be used to tell the relative
richness between two communities by
generating rarefaction curves.
• DOTUR can be used to compare different
phylogenetic anchors for measuring richness.
Summary (cont.)
• DOTUR can generate collector’s curves that
help determine the minimum number of
sequences to estimate the overall OTUs.