You will need to bring your own PC

We will be using the ARDC Nectar Research Cloud to perform RNAseq analyses

Requirements

terminal application installed
or alternativly Visual Studio Code installed
please see instructions below if don’t have either of these

Accessing nectar using a ssh (Secure Shell) command

To access Nectar you will need the following


1	username	workshop
2	password	Sagc_2024
3	IP address	given on the day

Your individual IP address will be given on the day. Each participant will have their own VM (virtual machine). Do not log in with the same IP address as someone else!*

your ssh command should look like this, but with a different IP address

ssh workshop@172.243.264.45

press enter, and you should see be prompted with some text

The authenticity of host '118.138.235.141 (118.138.235.141)' can't be established.
ECDSA key fingerprint is SHA256:dgHPXk3BkFPTErqpjAYkB5qAnHLYeUZE7qg3YKcWVQw.
Are you sure you want to continue connecting (yes/no/[fingerprint])?

type in ‘yes’
enter your password if prompted

you should see your username in green text, e.g.

workshop@workshop

This means you have logged into the virtual machine.

you can now navigate to the workshop materials, which have been loaded to your instance.

cd ~/workshop/
ls -l

Install and set up a terminal application (if needed)

Linux terminals

If you use Linux, then likely no explanation is needed. Open your preferred terminal program

Mac operating systems have a terminal program, called Terminal. Just look for it in your Applications folder, or hit Command + Space and type ‘terminal’. You may find that other, 3rd party terminal programs are .

Use ssh in the Terminal app. For example, if your IP is 172.243.264.45 and user name is user, you would enter:

ssh user@172.243.264.45

Windows

The latest builds of Windows 10 and 11 come with SSH server and client. To see if your windows installation has SSH:

start Command Prompt (cmd) or Powershell
type in ‘ssh’

If SSH is available, you should see something like

usage: ssh [-46AaCfGgKkMNnqsTtVvXxYy] [-B bind_interface]
           [-b bind_address] [-c cipher_spec] [-D [bind_address:]port]
           [-E log_file] [-e escape_char] [-F configfile] [-I pkcs11]
           [-i identity_file] [-J [user@]host[:port]] [-L address]
           [-l login_name] [-m mac_spec] [-O ctl_cmd] [-o option] [-p port]
           [-Q query_option] [-R address] [-S ctl_path] [-W host:port]
           [-w local_tun[:remote_tun]] destination [command]

If SSH is not available, please install PuTTY.

Open PuTTY and connect using the IP address of your VM as the host name, and set port to 22.

When prompted, type in your username and password.

Environment Setup

**NO NEED TO CONTINUE** if accessing this content on the nectar instance

On the day of the workshop this will have already been done, and will be available when you log into the Nectar instance using ssh
however if you wish to use your own computing environment or from scratch you will have to install the dependencies

install nextflow
install conda
install nf-core tools
install singularity
install GO

you can fistly check whether these have been installed with the following commands

nextflow -version
conda -version
nf-core -version
singularity -version

install nextflow

mkdir ~/bin && cd ~/bin
curl -s https://get.nextflow.io | bash
chmod +x nextflow

adding ~/bin to PATH # in ~/.bashrc

echo 'export PATH=~/bin:${PATH}' >> ~/.bashrc && \
    source ~/.bashrc

test installation

nextflow -version

Setting the nextflow singularity cachedir

mkdir -p ~/SingularityCacheDir

#export NXF_SINGULARITY_CACHEDIR=~/SingularityCacheDir
echo 'export NXF_SINGULARITY_CACHEDIR=~/SingularityCacheDir' >> ~/.bashrc && \
    source ~/.bashrc

# check cache dir
echo $NXF_SINGULARITY_CACHEDIR

install Singularity

install the following dependencies: (Ubuntu)

sudo apt-get update && sudo apt-get install -y \
    build-essential \
    uuid-dev \
    libgpgme-dev \
    squashfs-tools \
    libseccomp-dev \
    wget \
    pkg-config \
    git \
    cryptsetup-bin

if you are using an HPC environment without superuser (su) access, then you will have to ask IT/administrator to install it Sahmri HPC, Phoenix (adelaide Uni) or Deepthought (Flinders Uni) will already have it installed.

install GO
GO is a dependency of Singularity

export VERSION=1.23.2 OS=linux ARCH=amd64 && \
    wget https://dl.google.com/go/go$VERSION.$OS-$ARCH.tar.gz && \
    sudo tar -C /usr/local -xzvf go$VERSION.$OS-$ARCH.tar.gz && \
    rm go$VERSION.$OS-$ARCH.tar.gz

# set up your environment for Go.

echo 'export GOPATH=${HOME}/go' >> ~/.bashrc && \
    echo 'export PATH=/usr/local/go/bin:${PATH}:${GOPATH}/bin' >> ~/.bashrc && \
    source ~/.bashrc

Install Singularity

# download singularity
export VERSION=3.9.6 && # adjust this as necessary \
    wget https://github.com/sylabs/singularity/releases/download/v${VERSION}/singularity-ce-${VERSION}.tar.gz && \
    tar -xzf singularity-ce-${VERSION}.tar.gz && \
    cd singularity-ce-${VERSION}

#compile Singularity
./mconfig && \
    make -C ./builddir && \
    sudo make -C ./builddir install

# test installation
singularity  --version

install Conda

cd ~/bin && mkdir -p miniconda3
wget https://repo.anaconda.com/miniconda/Miniconda3-latest-Linux-x86_64.sh -O miniconda3/miniconda.sh
bash miniconda3/miniconda.sh -b -u -p miniconda3
rm miniconda3/miniconda.sh

#### add PATH to ~/.bashrc
echo 'export PATH=${PATH}:~/bin/miniconda3/bin' >> ~/.bashrc && \
    source ~/.bashrc

add bioconda to conda channel

conda config --add channels bioconda
conda config --add channels conda-forge

install nf-core tools

# chosing to install nf-core to a new conda environment called 'nf-core', with nexflow as well
conda create --name nf-core python=3.12 nf-core

# initiate conda 
conda init

# if prompted close terminal and reopen
exit
# reopen with ssh command

# see whether the environment is there
conda env list

#activate this environment
conda activate nf-core

# install  version 2.14.1 (nf-core version 3 just came out)
conda install nf-core=2.14.1

# test installation
nextflow -v
nf-core -v

Samtools

cd ~/bin
wget https://github.com/samtools/samtools/releases/download/1.21/samtools-1.21.tar.bz2
tar -xvjf samtools-1.21.tar.bz2
rm samtools-1.21.tar.bz2
cd samtools-1.21/
make
make install
export PATH=$PATH:/home/workshop/bin/bin

link to RNAseq Background slides

Setup

Introduction to RNAseq Slides

Instructions to set up your personal computer