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 [1], [2]

• • 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)

rRNA vs. 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

Alpha diversity

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