16S rRNA gene amplicon sequencing data analysis workshop (4/19/2016)

16S rRNA gene amplicon sequencing data analysis workshop

April 19, Tuesday, 1:30-5:00pm

By Despoina Lymperopoulou

Amplicon analysis is a multi-step process. There are decisions to be made in analysis and implementation. This tutorial contains the basic procedures using Mothur to analyze 16S rRNA gene sequences obtained by high throughput sequencing technology, and specifically Illumina sequencing. The flowchart and example provided in the tutorial is especially useful for Beginners. We will go through an example touching on a couple of the issues in amplicon analysis. We will thenbriefly touch on ways to explore the data using R.

Before the workshop, you need to:

1.Download and unzip Mothur software (v1.35.1) according to your OS.

https://github.com/mothur/mothur/releases/tag/v1.35.1

2. Download and install “FastQC” – make sure that you have Java installed too. If you have (Java) trouble, consider this as an optional installation. http://www.bioinformatics.babraham.ac.uk/projects/download.html#fastqc

3. Download the “Amplicon_Workshop_CGRL” through

https://drive.google.com/folderview?id=0BygjJgVFY1nUXzZ4amZNd244SG8&usp=sharing

– it contains two subfolders. One subfolder is called “Workshop_files” and includes the fastq (sequencing) files, the stability.files (mapping file), and reference files (SILVA). These reference files can be downloaded here for your future analyses http://www.mothur.org/wiki/Silva_reference_files. The second subfolder contains the output files of mothur from the analysis that we are going to run. I have already run it and I give you the files in case you get stuck somewhere in the tutorial today, so that we can all proceed with the analysis.

4. Download and install:

R: http://cran.rstudio.com

RStudio: http://www.rstudio.com/products/rstudio/download/

Optional

5. Install the following packages in R: “calibrate”, “shape”, “direct labels”, “knitr”, “clustsig”, “ellipse, “plyr”, “vegan”, “ggplot2”, and “phyloseq”. Except for “phyloseq”, all packages can simply be installed by running:install.packages(“package_name”). Please follow the directions to successfully install “phyloseq”:http://joey711.github.io/phyloseq/install