A Short Tutorial on Processing 16S Metagenomic Sequences Using mothur, Python, and R

Mitch Fernandez

June 17th, 2014

Metagenomics

https://www.sciencenews.org/article/year-review-your-body-mostly-microbes

PCR

http://calculatedimages.blogspot.com/2013/06/need-to-teach-pcr.html

16S rRNA Gene

http://www.clinchem.org/content/55/5/856.fullhttp://rna.ucsc.edu/rnacenter/ribosome_images.html

Pyrosequencing

http://www.nature.com/nmeth/journal/v8/n7/fig_tab/nmeth.1634_F1.htmlhttp://aem.asm.org/content/78/20/7480/F1.expansion.html

Workflow• Data Preparation

• Data Preprocessing

• Data Clustering

• Downstream Analysis

mothur

http://www.cbs.dtu.dk/researchgroups/metagenomics/metagenomics.php

Oligos

Map barcode to sample

Identify primer and adaptor

Shell Scripts

Loop through the

files

Modify mothur

template file

Call mothur and pass modified template

Clean up output

Denoising

Merging Files

Preprocessing pt. 1

Alignment

http://genome.cshlp.org/content/17/2/127/F2.expansion.html

Preprocessing pt. 2

Pre.clustering

http://www.nature.com/nmeth/journal/v9/n5/full/nmeth.1990.html

Preprocessing pt. 3

Chimeras

http://genome.cshlp.org/content/21/3/494/F1.expansion.html

Preprocessing pt. 4

Contaminants

http://www.reids-workouts.com/the-reason-you-are-not-building-muscle-part-2; http://acceleratingscience.com/proteomics/proteomic-analysis-of-mitochondria-unravels-the-pathophysiology-of-pre-eclampsia/; http://vickgaza.deviantart.com/art/Mountain-Yeti-360756260; http://www.funchap.com/pictures-of-dogs/; http://en.wikipedia.org/wiki/Chloroplast

Operational Taxonomic Units

OTU Clustering

http://rosalind.info/glossary/distance-matrix/https://peerj.com/articles/237/

Classifying OTUs

• Low Diversity

Richness and Diversity• High Diversity

Equal Richness

Text Parsing

• Automate Oligos file creation• Produce read count tables• Parse richness and diversity results• Parse error logs• Split data into groups

Normalization

Visualization

Metastats

  Former (n=24) Active (n=22)Difference in Relative Abundance

   OTU Reads Relative Abundance Variance Std. Error Reads Relative Abundance Variance Std. Error p-value q-valueSelenomonas_003 79 8.71E-04 1.00E-06 1.85E-04 216 2.03E-03 4.00E-06 4.01E-04 -1.16E-03 1.10E-02 2.33E-02Porphyromonas_001 607 8.30E-03 8.50E-05 1.89E-03 331 3.96E-03 7.00E-06 5.68E-04 4.34E-03 3.10E-02 6.38E-02Family_Burkholderiaceae_001 96 1.51E-03 5.00E-06 4.40E-04 67 5.04E-04 0.00E+00 1.48E-04 1.00E-03 3.50E-02 7.05E-02Neisseria_001 4,851 4.58E-02 4.68E-03 1.40E-02 1,353 2.00E-02 2.55E-04 3.41E-03 2.57E-02 3.80E-02 7.38E-02Campylobacter_001 121 1.10E-03 2.00E-06 2.76E-04 115 2.10E-03 4.00E-06 4.26E-04 -1.00E-03 5.29E-02 8.54E-02Rhizobium_001 20 3.42E-04 0.00E+00 1.37E-04 76 7.55E-04 1.00E-06 1.74E-04 -4.13E-04 5.99E-02 9.21E-02

Class_Gammaproteobacteria_002 158 2.03E-03 6.00E-06 5.06E-04 454 4.29E-03 2.90E-05 1.14E-03 -2.26E-03 7.09E-02 9.93E-02Catonella_001 274 2.13E-03 1.10E-05 6.81E-04 65 9.22E-04 1.00E-06 2.00E-04 1.21E-03 7.19E-02 1.00E-01Family_Carnobacteriaceae_001 621 6.83E-03 4.30E-05 1.34E-03 283 4.31E-03 1.20E-05 7.30E-04 2.52E-03 1.01E-01 1.25E-01Prevotella_001 3,064 3.58E-02 5.73E-04 4.89E-03 6,061 5.58E-02 2.50E-03 1.07E-02 -2.00E-02 1.06E-01 1.30E-01Paracoccus_001 24 2.76E-04 0.00E+00 1.28E-04 138 1.44E-03 1.40E-05 8.07E-04 -1.17E-03 1.06E-01 1.30E-01Actinomyces_001 903 7.32E-03 4.50E-05 1.37E-03 1,055 1.23E-02 1.76E-04 2.83E-03 -5.01E-03 1.07E-01 1.31E-01Prevotella_005 46 6.95E-04 2.00E-06 2.86E-04 328 3.09E-03 6.80E-05 1.76E-03 -2.40E-03 1.10E-01 1.34E-01Family_Rhodocyclaceae_001 14 1.92E-04 0.00E+00 1.36E-04 87 1.14E-03 8.00E-06 5.94E-04 -9.47E-04 1.25E-01 1.47E-01

Running the Workflow1. Gather your data

2. Prepare an Oligos file

3. Zip everything up and copy to the “work” folder

4. Run mothur.sh

5. Come back in a few hours/days

6. Run ReadCountTable.py on the taxonomy output

7. Do additional downstream processing

8. Publish results

What we need help with

Data managementPost-hoc OTU namingImproved scriptingIdentifying new toolsOther stuff I haven’t thought of

Thanks