H3ABioNet 16S rRNA Microbiome Intermediate Bioinformatics Training 2019

Aim

The H3ABioNet 16S rRNA Microbiome Intermediate Bioinformatics course will provide training to enable participants to gain the knowledge and skills to perform 16S rRNA microbiome data analyses using a variety of bioinformatics methods and tools.

Intended Audience

Anyone who will be working with 16S rRNA microbiome data and would like to learn more about the bioinformatics analyses.

Course curriculum

Module 1: Introduction to the Linux command line / intro to R
Module 2: Introduction to the microbiome and study design – why 16S?
Module 3: Sample collection, extraction and library prep for 16S NGS analyses
Module 4: 16S rRNA gene amplicon sequencing bioinformatics pipeline: the theory
Module 5: 16S analysis pipeline - QC, ASV picking, taxonomic classification and alignment
Module 6: Downstream analysis in R - using the packages phyloseq, NMF, vegan, metagenomeSeq (among others)
Potential bonus module: Shotgun sequencing

Course objectives

By the end of the course participants should be able to:

Describe the importance of the microbiome and why it should be studied
Understand how to design 16S rRNA microbiome studies
Be able to apply basic syntax and operations in R
Understand the different NGS data types (e.g. MiSeq reads) produced for a 16S rRNA microbiome study
Evaluate the quality of NGS sequence reads and samples
Understand the various bioinformatics tools used for 16S microbiome studies
Understand the various 16S rRNA bioinformatics pipelines being used to study the microbiome.
Apply the H3ABioNet’s 16S rRNA pipeline and understand how to execute this
Understand the use of workflow languages (Nextflow) and containerized images (Singularity) to automate analyses
Analyze 16S rRNA microbiome data and interpret results

Learning from this resource

This repository contains the trainer's presentations, tutorial scripts as well as instructions on how to set up Nextflow for automation of 16S rRNA pipeline. The trainers' presentation videos are available on H3ABioNet's Youtube channel on this playlist.

This resource assumes that you are running Linux with R and RStudio installed. Additionally, the latest version of the following R packages should be installed:

dada2
DECIPHER
phangorn
metagenomeSeq
vegan
ggplot2
NMF
gridExtra
dplyr
phyloseq

This training offers a step-by-step dada2 tutorial on how to analyze 16S rRNA microbiome data (covered in Module 5 and 6) as well as automation of the analysis using Nextflow workflow language and containerized images (Module 6 Session 1), before downstream statistical analysis in R.

To run the step-by-step tutorial, you require R and RStudio and the R packages listed above installed. The dada2 tutorial starts in Module 5 and continues in Module 6 on downstream analysis. The scripts and required data for the tutorials are also linked in the section below.

To run dada2 nextflow pipeline on a computer cluster, follow the instructions outlined on this document for software set up and running the pipeline. It involves setting up Singularity, R and RStudio, and Nextflow. For the training at ICIPE, please follow the instructions outlined here.

Links to resources referenced in modules

Module 1: Introduction to Linux and R

Module4: 16S rRNA sequencing bioinformatics pipeline theory

Module 5: 16S analysis pipeline

dada2 tutorial site
dada2 tutorial R script
Required tutorial data: http://web.cbio.uct.ac.za/~gerrit/downloads/dog_stool_full.tgz
RefSeq-RDP 16S database: https://zenodo.org/record/3266798/files/RefSeq-RDP16S_v3_May2018.fa.gz

Module 6: Downstream analysis in R

mbbu / intbt_2019

readme