Paul J. McMurdie Second Genome, South San Francisco, CA, USA !Formerly: Statistics Department Stanford University

Lecture 01: Introduction - Statistical analysis of Microbiome Count Data

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Goals for the day.

• Unit 1 - morning - Interactive/Exploratory Analyses - Alpha/Beta Diversity - Distances - Transformations - Ordination (Dimensional Reduction Techniques) - Perm. Multivariate ANOVA testing (adonis) • Lunch • Unit II - afternoon - Multiple Testing, Mixture Models • We can’t cover everything in one day! - An Introduction to Statistical Learning: - http://www-bcf.usc.edu/~gareth/ISL/

Link to NIOO Statistics-Day Materials

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• A population of a single species/strain is a culture, extremely rare outside of lab, some infections

• A microbiome is a mixed population of different microbial species (microbial ecosystem)

What is a microbiome?The totality of microbes in a defined environment, especially their genomes and interactions with each other and surrounding environment.

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• Universal Gene census (amplicon)

• Shotgun Metagenome Sequencing

• Transcriptomics (shotgun mRNA)

• Proteomics (protein fragments)

• Metabolomics (excreted chemicals)

Number of Species Counted

Methods for characterizing microbiomes

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Data Analysis Tools of the Trade: R - RStudio - Rmarkdown

Data analysis should be: • Modular - Save time, protect mistakes, share • Repeatable - Confirmable by others • Flexible - Analysis not predefined • There is no “write paper” button

• Interactive - Answers create new questions !Recommendation: R - RStudio - Rmarkdown (and Bioconductor for this crowd) Lab exercises today will be Rmarkdown based, and I will encourage you to save your work this way

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markdown (code + console) +

figures

phyloseq + ggplot2 + etc.

# Main title!This is an [R Markdown](my.link.com) document of my recent analysis.!## Subsection: some codeHere is some import code, etc.```{r}library("phyloseq")library("ggplot2")physeq = import_biom(“datafile.biom”)plot_richness(physeq)```

source.Rmd

Complete HTML5

knitr::knit2html()

microbiome data

Our Goal with Collaborators: Reproducible analysis workflow with R-markdown

Better Reproducibility

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Paul J. McMurdie !Statistics Department & CEHG Stanford University !with contributions from Prof Susan Holmes

Microbiome data heterogeneity and processing

http://joey711.github.io/phyloseq/

phyloseq

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phyloseq

http://joey711.github.io/phyloseq/

phyloseq Home Page

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apepackage

OTU Abundanceotu_table

Sample Variablessample_data

Taxonomy TabletaxonomyTable

Phylogenetic Treephylo

otu_table sample_data tax_table phy_tree

otu_table sample_data tax_table

read.treeread.nexusread_tree

as as as

import

phyloseqconstructor:

Biostringspackage

Reference Seq.XStringSet

DNAStringSet RNAStringSet

AAStringSet

phyloseq

Experiment Data

otu_table,sam_data,tax_table,phy_treerefseq

Accessors:get_taxaget_samplesget_variablensamplesntaxarank_namessample_namessample_sumssample_variablestaxa_namestaxa_sums

Processors:filter_taxamerge_phyloseqmerge_samplesmerge_taxaprune_samplesprune_taxasubset_taxasubset_samplestip_glomtax_glom

matrix matrixdata.frame

optional

refseq

data

data structure & APIphyloseq

http://joey711.github.io/phyloseq/

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phyloseq

Preprocessing

Import

Direct Plots

plot_network plot_heatmap plot_ordination

distance ordinate

Summary / ExploratoryGraphics

filter_taxafilterfun_samplegenefilter_sampleprune_taxaprune_samplessubset_taxasubset_samplestransform_sample_counts

import_biomimport_mothurimport_pyrotaggerimport_qiimeimport_RDP

plot_tree

plot_richness

plot_bar

bootstrappermutation testsregressiondiscriminant analysismultiple testinggap statisticclusteringprocrustes

Inference, Testing

sample data

OTU cluster output

Input

raw

phyloseqprocessed

work flowphyloseq

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graphics

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plot_ordination, NMDS, wUF

FreshwaterFreshwater (creek)FreshwaterFreshwater (creek)Freshwater (creek)SoilSoilSoilSkinSkinSkinM

ockM

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ockFecesFecesFecesFecesSedim

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plot_heatmap; bray−curtis, NMDS

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plot_network; Enterotype data, bray−curtis, max.dist=0.25

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CytophagaEmticicia

Sphingobacterium

Segetibacter

Haliscomenobacter

Pedobacter

Bacteroides

Alistipes

Bacteroides

Cytophaga

Porphyromonas

Prevotella

Parabacteroides

Algoriphagus

Odoribacter

CandidatusAquirestis

Capnocytophaga

Porphyromonas

Spirosoma

Prevotella

Balneola

Prevotella

Hymenobacter

Prevotella

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FecesFreshwaterFreshwater (creek)MockOceanSediment (estuary)SkinSoilTongue

Order● Bacteroidales

FlavobacterialesSphingobacteriales

plot_tree; Bacteroidetes−only. Merged samples, tip_glom=0.1

0e+00

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Feces

Freshwater

Freshwater (creek)

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Ocean

Sediment (estuary)

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Soil

Tongue

SampleType

Abun

danc

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FamilyBacteroidaceaeBalneolaceaeCryomorphaceaeCyclobacteriaceaeFlavobacteriaceaeFlexibacteraceaePorphyromonadaceaePrevotellaceaeRikenellaceaeSaprospiraceaeSphingobacteriaceae

plot_bar; Bacteroidetes−only

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S.obs S.chao1 S.ACE

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FALSE TRUE FALSE TRUE FALSE TRUEHuman Associated Samples

Num

ber o

f OTU

s

SampleTypeFecesFreshwaterFreshwater (creek)MockOceanSediment (estuary)SkinSoilTongue

plot_ordination()

plot_network()

plot_bar()

plot_heatmap()

plot_tree()

plot_richness()

phyloseq

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graphics

plot_ordination()

−1.0

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SampleTypeFecesFreshwaterFreshwater (creek)MockOceanSediment (estuary)SkinSoilTongue

Samples Only; type="samples"

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type

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samplestaxa

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Bacteroidia Flavobacteria Sphingobacteria

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Class

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Taxa Only; type="taxa"

samples taxa

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ClassFlavobacteriaSphingobacteriaBacteroidiasamples

SampleTypeFecesFreshwaterFreshwater (creek)MockOceanSediment (estuary)SkinSoilTonguetaxa

Split Plot; type="split"

type="scree"

split

taxa-only

biplot

samples-only

phyloseq

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http://joey711.github.io/phyloseq/tutorials-index

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