Analysis of microbial communities with QIIME

Justin Kuczynski February 2012

Because Microbial communities are important

Microbial communities are important

Microbial communities are important

Microbial communities are important

photos: stockphoto.it, eurasnet.info, elkhorn.unl.edu

One option: 16S / SSU sequence based profiling

Focus on the ribosomal SSU / 16S gene Target and amplify the 16S/18S ribosomal RNA as a ‘fingerprint’ for different taxonomic groups.

Why 16S/18S?

- gene is ubiquitous - contains conserved and variable regions

SSU / 16S experimental workflow Extract DNA and amplify marker gene with barcoded primers Pool amplicons and sequence

Sequences are not directly interpretable line 12,000 of 1.2 M

Sequences are not directly interpretable And beware: ▫ chimeric PCR products ▫ sequencing error ▫ uneven sequencing depth ▫ short read effects line 12,000 of 1.2 M

From sequences to results

From sequences to results E. Costello et al., 2009

That is QIIME’s purpose

Extract DNA and amplify marker gene with barcoded primers Pool amplicons and sequence > GCACCTGAGGAC AGGCATGAGGAA… > GCACCTGAGGAC AGGGGAGGAGGA… > TCACATGAACCT AGGCAGGACGAA… > CTACCGGAGGAC AGGCATGAGGAT… > TCACATGAACCT AGGCAGGAGGAA… > GCACCTGAGGAC ACGCAGGACGAC… > CTACCGGAGGAC AGGCAGGAGGAA… > CTACCGGAGGAC ACACAGGAGGAA… > GAACCTTCACAT AGGCAGGAGGAT… > TCACATGAACCT AGGGGCAAGGAA… > GCACCTGAGGAC AGGCAGGAGGAA… Assign reads to communities Assign millions of sequences from thousands of communities to OTUs www.qiime.org

Visualize and compare community relationships

QIIME analysis in more detail

QIIME analysis in more detail

QIIME analysis in more detail beta_diversity_through_plots.py

QIIME analysis example beta_diversity_through_plots.py

QIIME analysis example

QIIME analysis example

QIIME analysis example

Demo of beta diversity analysis results

Demo of beta diversity analysis results

QIIME tech support • www.qiime.org

• Overview Tutorial • QIIME forum

Running QIIME Laptop EC2 Cluster

images: pcdownload.asia, ostatic.com

Acknowledgements FUNDING Crohn’s and Colitis Foundation of America, the Bill and Melinda Gates Foundation, NASA, the HHMI and the NIH

[email protected]

www.qiime.org

RESEARCH Antonio González Bharath Prithiviraj Catherine Lozupone Chris Lauber Dan Knights Daniel McDonald Donna Berg-Lyons Doug Wendel Greg Caporaso Jens Reeder Jerry Kennedy Jess Metcalf Jesse Stombaugh Jesse Zaneveld José Clemente Justin Kuczynski Laura Parfrey Meg Pirrung Rob Knight Tony Walters Ulla Westermann Will Van Trueren