Paul J. McMurdie!Research Associate!Prof Susan Holmes Group!Statistics Department!Stanford University

Shiny-phyloseq: Web Application for Interactive Microbiome Analysis

with Provenance Tracking

• Intro to Microbiome Research!

• phyloseq - a microbiome BioC package!

• (RNA-Seq methods solve a microbiome problem)!

• Shiny-phyloseq: a shiny interface to phyloseq

Overview

What are microbes? Cell structure

(they don’t all look like this)

What are microbes?

http://en.wikipedia.org/wiki/Tree_of_life_(biology)

Bacteria

Archaea

Eukaryota

Ancestry of Life

• 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.

Cow Rumen

Human Microbiomes

Oceans, soils, waterwaysWastewater Treatment

Why study microbiomes?

Deep-Sea Hydrothermal Vent

Earth Microbiome Project:

Human Body Sites, HMP

>10 times more microbial cells than human cells !Entire human microbiome weighs less than 2 kg, at most

Fecal Transplants

Borody, et al (2011)!Nature Rev Gastroenterology &!Hepatology

(Clostridium difficile infection)

• Culture-based methods fail to detect most microbes!

• Microbes are easy to miss (except pathogens)!

• Most microbes are NOT pathogens (even the human-associated)

Bias for cultivable microbes, especially pathogens

• PCR, fast & cheap DNA sequencing, microarrays, etc!

• Discovery of culture-independent techniques - 16S-rRNA

Availability of tools limited to last 3 decades

Why is microbiome research new?

ribosome16S rRNA

ribosome!in action

How do we query microbiomes??

• Universal (e.g. 16S rRNA) Gene census!

• Shotgun Metagenome Sequencing!

• Transcriptomics (shotgun mRNA)!

• Proteomics (protein fragments)!

• Metabolomics (excreted chemicals)

Number of Microbial Species

Counted

How do we query microbiomes??

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

Microbiome data !heterogeneity and processing

microbiome!samples

amplify 16S rRNA!(barcoded)

demultiplex and !species clustering

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

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

graphics

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−0.4 −0.2 0.0 0.2 0.4NMDS1

NMDS

2

SampleType●●●●●●●●●●●●●●●●●●

FecesFreshwaterFreshwater (creek)MockOceanSediment (estuary)SkinSoilTongue

plot_ordination, NMDS, wUF

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

ockM

ockM

ockFecesFecesFecesFecesSedim

ent (estuary)TongueTongueO

ceanO

ceanO

ceanSedim

ent (estuary)Sedim

ent (estuary)

SampleType

OTU

1

100

10000

Abundance

plot_heatmap; bray−curtis, NMDS

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SeqTech●

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IlluminaPyro454Sanger

Enterotype● 1

23

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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76

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67

81

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Abundance●

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12562515625

SampleType●

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FecesFreshwaterFreshwater (creek)MockOceanSediment (estuary)SkinSoilTongue

Order● Bacteroidales

FlavobacterialesSphingobacteriales

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

0e+00

2e+05

4e+05

6e+05

Feces

Freshwater

Freshwater (creek)

Mock

Ocean

Sediment (estuary)

Skin

Soil

Tongue

SampleType

Abun

danc

e

FamilyBacteroidaceaeBalneolaceaeCryomorphaceaeCyclobacteriaceaeFlavobacteriaceaeFlexibacteraceaePorphyromonadaceaePrevotellaceaeRikenellaceaeSaprospiraceaeSphingobacteriaceae

plot_bar; Bacteroidetes−only

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S.obs S.chao1 S.ACE

2000

4000

6000

8000

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

http://joey711.github.io/waste-not-supplemental/

edgeR, DESeq(2), metagenomeSeq!perform better than popular alternatives!in differential abundance detection:!!McMurdie and Holmes (2014) PLoS Comp Biol!DOI: 10.1371/journal.pcbi.1003531

Side Note: BioC tools for microbiome

genes

samples

species

samples

species counts

gene counts

Acknowledgements

Susan Holmes

Wolfgang Huber

BioC and CRAN

Helpful advice and feedback re: DESeq(2)

Postdoc Advisor, Mentor, Co-author

Support, Feedback, Distribution of phyloseq and biom

RStudio Shiny, RStudio IDE

Hadley Wickham ggplot2, reshape2, plyr R packages

Holmes Group Helpful advice and feedback

Shiny-phyloseq

Live Demoinstall.packages(“shiny”)shiny::runGitHub(“shiny-phyloseq”, “joey711”)

How to Run:

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

End.!Questions?