ISEEM:Paper DiversityMethods Computational methods for microbial diversity review

Paper by Eisen et al for PLoS Computational Biology

Outline

Summary and introduction

Organized in part around the metagenomic diversity workflow See Here

A key point is to compare and contrast tools for analyzing rRNA with those for analyzing metagenomic data.

Generation of data

rRNA PCR

• Background
  • PCR amplification with conserved primers
  • Sequencing
    • Standard
    • 454

See Media:Margulies_etal_Nature_2005.pdf, Media:Mardis_et_al._Cell_2008.pdf

• • Indirect characterization
    • ARISA
    • T-RFLP
    • Array
      • Desantis et al 2007 on array vs. clones
• Informatics issues
  • Primer design

See "Review and re-analysis of domain-specific 16S primers."

• • Chimera checking
  • Assembly of sequences from paired ends
• rRNA databases
  • Greengenes
  • Silva

PCR of other genes

• Done sometimes to cover broad diversity
  • RecA/RadA best example
• Frequently done to focus on narrower groups or functional classes
  • Amanox
  • Nitrogen fixation

Metagenomic Sequencing

• Background
• Identifying rRNA genes
• Identifying other genes
  • Which genes should be used?
• Assembly
• Metagenomic databases
  • CAMERA
  • RAST
  • IMG/M

General analysis of gene families (rRNA and protein)

Sequence quality issues

• Chimera checking
  • Bellerephon

Alignment

• rRNA
  • STAP
  • NAST
  • Sean Eddy's infernal
• Protein families

Dividing into OTUs

• rRNA focused methods
  • DOTUR
  • FastGroup II to dereplicate

Phylogenetic trees

• Building trees
• Phylogenetic classification
  • Alignment independent method
  • PhyloPythia - doesn't use tree, SVM at each node
• Phylogenetic structure
• Phylogenetic based OTUs

Web servers that do multiple things

• Fast Group II
• NAST Server
• EZ TAXON

Species Diversity Measurements Focused on Single Communities

Richness estimators

• Reviews
  • Hughes et al. AEM Review
  • Lozupone and Knight FEMS Review
• Parametric
• Non parametric

Issues with different sequencing methods

• 454 Sequencing
  • See recent paper by R. Knight on methods

Comparing between samples

General approaches and Introduction

• Multiple ways to compare communities and to think about the comparisons
• Good discussion of this on p559 in Lozupone and Knight review
• I (Eisen) view things a little differently
• Would be useful to at least briefly link the microbial diversity/phylodiversity literature to the non-microbial ecology literature on this subject
  • Note parallel development of methods and tools to answer many of the same questions with different datasets. Emphasize need to unify.
• First key issue is whether to analyze communities one by one with some metric and then to compare the scores or to develop scores explicitly from comparing communities
• i.e. alpha vs. beta diversity
  • Within community metric could be
    • Richness
    • Diversity
• Lozupone and Knight outline a few other issues including
  • Qualitative vs. quantitative
    • Qualitative do not take into account species counts, just presence/absence
  • Species based or tree based

OTU and phylotype focused

• Presence/absence of OTUs
• Presence/absence of phylotypes (at all levels)
• SONS

Coverage and Statistical Significance

• See f-Libshuff
• See Original libshuff

Phylogenetic structure of communities

• See Tree Climber
• See AP Martin paper on phylogenetic based comparison where FST and P test introduced
• See AP Martin paper #1
• Unifrac
  • Online version paper here
  • Original paper here
• Weighted Unifrac

Metagenomic distances

• Rohwer Marine Viromes
• Edwards Rohwer et al Statistics paper
• Also see Rusch paper

Statistical issues

• Sampling
• Statistical concepts (e.g. experimental unit (species, community, environment, day, etc))
• Assessing reliability and repeatability of results

Case Studies and Science

Reviews on methods

• See New Review by Schloss
• See new Schloss paper
• http://www.nature.com/ismej/journal/v2/n7/abs/ismej200844a.html;jsessionid=12A1751090E8D55918D9AC086B76837F
• http://www.ncbi.nlm.nih.gov/pubmed/18435746?ordinalpos=18&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum

Metagenomics informatics

• Reviews
  • Chen and Pachter. PLoS Comp. Biol 2005
  • Schloss and Handelsman Statistical toolbox
• Binning
  • Phylopythia by McHardy et al.

General diversity things

• See Curtis et al. review
• See Global patterns in microbial diversity paper
• See Beta diversity paper by Knight
• See Hughes rarefaction paper

Biogeography

• See Taxa Area Paper
• Also see Other island paper
• See Freir paper
• See Green et al on microbial euks

Tables

Data Types

• • rRNA sequence
    • Pros
    • Cons
  • rRNA tRFLP
    • Pros
    • Cons
  • metagenomics
    • Pros
    • Cons

Diversity Methods

• alpha diversity
  • Phylogenetic diversity (PD)
    • Pros
    • Cons
    • Applications
    • References
• beta diversity