1 About This Vignette

This vignette aims to be a short tutorial for the main functionalities of SIAMCAT. Examples of additional workflows or more detailed tutorials can be found in other vignettes (see the BioConductor page).

SIAMCAT is part of the suite of computational microbiome analysis tools hosted at EMBL by the groups of Peer Bork and Georg Zeller. Find out more at EMBL-microbiome tools.

2 Introduction

Associations between microbiome and host phenotypes are ideally described by quantitative models able to predict host status from microbiome composition. SIAMCAT can do so for data from hundreds of thousands of microbial taxa, gene families, or metabolic pathways over hundreds of samples. SIAMCAT produces graphical output for convenient assessment of the quality of the input data and statistical associations, for model diagnostics and inference revealing the most predictive microbial biomarkers.

3 Quick Start

For this vignette, we use an example dataset included in the SIAMCAT package. As example dataset we use the data from the publication of Zeller et al, which demonstrated the potential of microbial species in fecal samples to distinguish patients with colorectal cancer (CRC) from healthy controls.


data("feat_crc_zeller", package="SIAMCAT")
data("meta_crc_zeller", package="SIAMCAT")

First, SIAMCAT needs a feature matrix (can be either a matrix, a data.frame, or a phyloseq-otu_table), which contains values of different features (in rows) for different samples (in columns). For example, the feature matrix included here contains relative abundances for bacterial species calculated with the mOTU profiler for 141 samples:

feat.crc.zeller[1:3, 1:3]
##                                  CCIS27304052ST-3-0 CCIS15794887ST-4-0
## UNMAPPED                                   0.589839          0.7142157
## Methanoculleus marisnigri [h:1]            0.000000          0.0000000
## Methanococcoides burtonii [h:10]           0.000000          0.0000000
##                                  CCIS74726977ST-3-0
## UNMAPPED                                  0.7818674
## Methanoculleus marisnigri [h:1]           0.0000000
## Methanococcoides burtonii [h:10]          0.0000000
## [1] 1754  141

Please note that SIAMCAT is supposed to work with relative abundances. Other types of data (e.g. counts) will also work, but not all functions of the package will result in meaningful outputs.

Secondly, we also have metadata about the samples in another data.frame:

##                    Age BMI Gender AJCC_stage     FOBT Group
## CCIS27304052ST-3-0  52  20      F         -1 Negative   CTR
## CCIS15794887ST-4-0  37  18      F         -1 Negative   CTR
## CCIS74726977ST-3-0  66  24      M         -1 Negative   CTR
## CCIS16561622ST-4-0  54  26      M         -1 Negative   CTR
## CCIS79210440ST-3-0  65  30      M         -1 Positive   CTR
## CCIS82507866ST-3-0  57  24      M         -1 Negative   CTR

In order to tell SIAMCAT, which samples are cancer cases and which are healthy controls, we can construct a label object from the Group column in the metadata.

label.crc.zeller <- create.label(meta=meta.crc.zeller,
    label='Group', case='CRC')
## Label used as case:
##    CRC
## Label used as control:
##    CTR
## + finished create.label.from.metadata in 0.001 s

Now we have all the ingredients to create a SIAMCAT object. Please have a look at the vignette about input formats for more information about supported formats and other ways to create a SIAMCAT object.

sc.obj <- siamcat(feat=feat.crc.zeller,
## + starting
## +++ checking overlap between labels and features
## + Keeping labels of 141 sample(s).
## +++ checking sample number per class
## +++ checking overlap between samples and metadata
## + finished in 0.03 s

A few information about the SIAMCAT object can be accessed with the show function from phyloseq (SIAMCAT builds on the phyloseq data structure):

## siamcat-class object
## label()                Label object:         88 CTR and 53 CRC samples
## contains phyloseq-class experiment-level object @phyloseq:
## phyloseq@otu_table()   OTU Table:            [ 1754 taxa and 141 samples ]
## phyloseq@sam_data()    Sample Data:          [ 141 samples by 6 sample variables ]

Since we have quite a lot of microbial species in the dataset at the moment, we can perform unsupervised feature selection using the function filter.features.

sc.obj <- filter.features(sc.obj,
    filter.method = 'abundance',
    cutoff = 0.001)
## Features successfully filtered

4 Association Testing

Associations between microbial species and the label can be tested with the check.associations function. The function computes for each species the significance using a non-parametric Wilcoxon test and different effect sizes for the association (e.g. AUC or fold change).

sc.obj <- check.associations(
    sc.obj, = 'fc',
    alpha = 0.05,
    mult.corr = "fdr",
    detect.lim = 10 ^-6,
    plot.type = "",
    panels = c("fc", "prevalence", "auroc"))

The function produces a pdf file as output, since the plot is optimized for a landscape DIN-A4 layout, but can also used to plot on an active graphic device, e.g. in RStudio. The resulting plot then looks like that: Association Plot

5 Confounder Testing

As many biological and technical factors beyond the primary phenotype of interest can influence microbiome composition, simple association studies may suffer confounding by other variables, which can lead to spurious results. The check.confounders function provides the option to test the associated metadata variables for potential confounding influence. No information is stored in the SIAMCAT object, but the different analyses are visualized and saved to a combined pdf file for qualitative interpretation.

sc.obj <- check.confounders(
    fn.plot = 'confounder_plots.pdf', = NULL,
    feature.type = 'filtered'

The conditional entropy check primarily serves to remove nonsensical variables from subsequent checks. Conditional entropy quantifies the unique information contained in one variable (row) respective to another (column). Identical variables and derived variables which share the exact same information will have a value of zero. In this example, the label was derived from the Group variable which was determined from AJCC stage, so both are excluded.