Summary and Setup

This is a new lesson built with The Carpentries Workbench.

Author Information

Developed by: Dr. Ashley Dungan and Dr. Gayle Philip School of Biosciences, University of Melbourne; Melbourne Bioinformatics

Created/Reviewed: April 2026

Overview

This page outlines the steps, beginning with raw data from the sequencing facility, that were followed to produce the 16S rRNA metabarocoding files used in R for data analysis. All intermediate .qza and .qzv files are provided. This data processing was completed in QIIME2 v2026.1 by Ashley Dungan in March 2026.

Anticipated workshop duration when delivered to a group of participants is 4 hours.

For queries relating to this workshop, contact Melbourne Bioinformatics ().

Learning Objectives

At the end of this introductory workshop, you will:

  • Take raw data from a sequencing facility and end with publication quality graphics and statistics
  • Answer the question What is the influence of captivity on gut microbiota of the fat-tailed dunnart?

Slides and workshop instructions

Click here for slides presented during this workshop.

Data

Data from the Walter and Eliza Hall Institute (WEHI) came as paired-end, demultiplexed (.fastq) files with primers and overhang sequences still attached. Samples were sequenced on a single NextSeq run.

Downloading data

  • No additional data needs to be downloaded for this workshop - it is all located on the Nectar Instance. FASTQs are located in the directory raw_data and a metadata (dunnart_metadata.tsv) file has also been provided.

  • If you wish to analyse the data independently at a later stage, it can be downloaded from here. This link contains both the FASTQs and associated metadata file.

  • If you are running this tutorial independently, you can also access the classifier that has been trained specifically for this data from here.

Prequisites

This workshop is designed for participants with command-line knowledge. You will need to be able to ssh into a remote machine, navigate the directory structure and scp files from a remote computer to your local computer.

Software Setup

Discussion

Details

For an in-person workshop, you will need access to Nectar instances. Login information will be provided to you prior to the workshop start date. You can find more information about Nectar instances here.

You will need to use a Google Chrome or Mozilla Firefox web browser to view files in QIIME2 View.

Use PuTTY

Use Terminal.app

Use Terminal

Initial Set up on Nectar

Byobu-screen

To ensure that commands continue to run should you get disconnected from your Nectar Instance, we’ll run a byobu-screen session.

Starting a byobu-screen session

On Nectar, to start a byobu-screen session called workshop, type

BASH 

byobu-screen -S workshop

Reconnecting to a byobu-screen session

If you get disconnected from your Nectar Instance, follow the instructions here to resume your session.

Data for this workshop is stored in a central location (/mnt/shared_data/) on the Nectar file system that we will be using. We will use symbolic links (ln -s) to point to it. Symbolic links (or symlinks) are just “virtual” files or folders (they only take up a very little space) that point to a physical file or folder located elsewhere in the file system. Sequencing data can be large, and rather than unnecessarily having multiple copies of the data which can quickly take up a lot of space, we will simply point to the files needed in the shared_data folder.

BASH 

cd
mkdir raw_data; cp -rs /mnt/shared_data/raw_data/* raw_data 
ln -s /mnt/shared_data/dunnart_metadata.tsv dunnart_metadata.tsv
ln -s /mnt/shared_data/silva_138.2_16s_v4_classifier.qza silva_138.2_16s_v4_classifier.qza