Published on March 5, 2014
Making Sense of Microbes Metagenomic Analysis Workflow Jack Simpson 10 Februrary 2014 CSIRO COMPUTATIONAL INFORMATICS
Cells in the human body Microbial: 100 trillion 2 | Jack Simpson Human 10 trillion
Human Microbiome Project • Investigate impact on human health • ~ 240 healthy individuals • 18 microbial habitats (e.g. airways) • > 5000 samples • Taxonomic marker: 16S rRNA gene 3 | Jack Simpson
OTU counts Samples 4 | Jack Simpson
Metagenomics workflow • Three areas I found interesting over the summer • Background of the data • Working with count data • Finding associations 5 | Jack Simpson
Where does metagenomic data start? • Where did our counts and OTUs come from? • Count data is not raw data: many processing decisions • 16S rRNA gene resolution and primers • Multiple variable regions • Lab protocols and comparing projects • Biological data starts in the real world 6 | Jack Simpson
Working with count data 7 | Jack Simpson
Zeroes: Much Ado About Nothing…? • Does absence of evidence == evidence of absence? • What do we do with zeroes? • Remove or Pseudocounts? • When to remove/replace? • Merged at the class level: visualise and replace zeroes 8 | Jack Simpson
Heatmap of all counts 9 | Jack Simpson
Beware hidden complexity 10 | Jack Simpson
Heatmap of grouped counts 11 | Jack Simpson
Zoom in on the heatmap 12 | Jack Simpson
Processing the Data • OTU count data analysis • Dealt with zeroes • Visualised the data • Normalization and transformation: log or Aitchison’s CLR? • What do different transformations do to the data with different sample numbers? • See artefacts related to discretization and zeroes 13 | Jack Simpson
Gut log compositional and log raw data 14 | Jack Simpson
Gut log compositional & clr compositional 15 | Jack Simpson
Finding associations • Warning: compositional data! • Be careful with correlation • Fractions are not independent == negative correlation • What can be done? • Proportionality 16 | Presentation title | Presenter name
Summary • Metagenomic data background • Processing our data • Looking for associations the right way 17 | Jack Simpson
Thank-you! 18 | Jack Simpson
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