This page:

Summary

This page covers information on how to use the 'Pawsey Bio - Ubuntu 22.04 - 2023-XX' image for Nimbus. Instructions on how to choose this image when creating your instance can be found here. This Bio-image is created to cater to bioinformatics users who prefer to have their instances pre-installed with software, tools and datasets commonly used in the bioinformatics domain, including over 8000 Biocontainer tools.

Commonly used software

Software	Information	Usage/Notes
Ansible	An automation platform that Pawsey uses to automate a number of software deployment
CernVM-FS	A read-only file system for accessing files on shared repositories	See 1. Biocontainers and Reference Genome data
Docker	A popular container engine
Google Chrome	A web browser	Make sure to SSH in to your instance with X11 forwarding, i.e. `ssh -X (or -Y) ubuntu@146.XX.XXX,` and have XQuart installed if you are using MacOS
Jupyter Notebook	For using an interactive Jupyter Notebook	See Run Jupyter Notebook Interactively
Lmod	A modules environment that we use at Pawsey for loading sotware
Nextflow	A popular workflow manager - "Nextflow enables scalable and reproducible scientific workflows using software containers. It allows the adaptation of pipelines written in the most common scripting languages."	Nextflow can leverage the containers from /cvmfs/singularity.galaxyproject.org or container modules - See 4. Using Nextflow
Pip	A Python package installer
Python3	A popular programming language used in many Bioinformatics software
RStudio	For using R interactively	See Run RStudio Interactively
Singularity	A popular container engine that can be used on HPC
Singularity-HPC	A container modules installer	See 2. Using Biocontainers
Spack	A package management tool
X2go	A virtual desktop application	X2go server has been pre-installed on the image. To use it, you will need to install X2go client on your local machine - see Installing X2go Client

Instructions

On this page, we will only cover instructions for how to use access and use Biocontainers and reference genome data sets. For instructions for the software listed above, please see the software's original documentation page.

1. Biocontainers and Reference Genome data

CernVM-FS is a read-only file system that was developed by another supercomputing centre (Cern). It allows files such as container tools, reference datasets and other shared resources that are commonly used by many researchers to be accessed, added to, and updated in the one location. At Pawsey, we currently cache the Biocontainer tools and reference genome datasets that are on Galaxy Project's repositories. The list of Biocontainer tools available can be searched on https://biocontainers.pro/registry.

To use the Biocontainer tools, you can skip this step and proceed to the next section, 2. Using the Biocontainer tools.

To view these repositories, you can do the following:

List the Biocontainer tools repository:

Note: It may take a minute or two to load the folders. When you have done it once, it will not take as long to show again.

The /cvmfs/singularity.galaxyproject.org/all subdirectory is where the entire list of 8000+ Biocontainers can be found, with the alphabetical subdirectories being symlinks to them.

$ ls -la /cvmfs/singularity.galaxyproject.org

total 140
drwxr-xr-x 33 cvmfs cvmfs 4096 Mar 25  2020 .
drwxr-xr-x  3 cvmfs cvmfs 4096 May 12  2022 1
drwxr-xr-x  3 cvmfs cvmfs 4096 Jun 29  2020 2
drwxr-xr-x  4 cvmfs cvmfs 4096 Feb 10 07:09 3
drwxr-xr-x 24 cvmfs cvmfs 4096 Feb 23  2021 a
drwxr-xr-x  4 cvmfs cvmfs 4096 Mar  7 19:06 all
drwxr-xr-x 23 cvmfs cvmfs 4096 Sep  2  2022 b
drwxr-xr-x 26 cvmfs cvmfs 4096 Feb 10 15:05 c
drwxr-xr-x 22 cvmfs cvmfs 4096 Feb 10 15:05 d
drwxr-xr-x 23 cvmfs cvmfs 4096 May  1  2020 e
drwxr-xr-x 21 cvmfs cvmfs 4096 Feb 10 19:06 f
drwxr-xr-x 25 cvmfs cvmfs 4096 Feb  5  2022 g
drwxr-xr-x 19 cvmfs cvmfs 4096 Feb 10 21:21 h
drwxr-xr-x 18 cvmfs cvmfs 4096 Feb 10 21:21 i
drwxr-xr-x 14 cvmfs cvmfs 4096 Jul 10  2020 j
drwxr-xr-x 21 cvmfs cvmfs 4096 Feb 25  2021 k
drwxr-xr-x 16 cvmfs cvmfs 4096 Feb 10 22:54 l
drwxr-xr-x 26 cvmfs cvmfs 4096 Feb 10 22:54 m
drwxr-xr-x 20 cvmfs cvmfs 4096 Feb 11 05:13 n
drwxr-xr-x 14 cvmfs cvmfs 4096 May  7  2020 o
drwxr-xr-x 24 cvmfs cvmfs 4096 Aug 24  2021 p
drwxr-xr-x 12 cvmfs cvmfs 4096 Feb 27  2021 q
drwxr-xr-x 27 cvmfs cvmfs 4096 Jun 28  2022 r
drwxr-xr-x 26 cvmfs cvmfs 4096 Apr  7  2020 s
drwxr-xr-x 24 cvmfs cvmfs 4096 Feb 11 23:17 t
drwxr-xr-x 13 cvmfs cvmfs 4096 Feb 11 23:17 u
drwxr-xr-x 18 cvmfs cvmfs 4096 Feb 11 23:17 v
drwxr-xr-x 17 cvmfs cvmfs 4096 Jul 28  2021 w
drwxr-xr-x 16 cvmfs cvmfs 4096 Apr  7  2020 x
drwxr-xr-x  4 cvmfs cvmfs 4096 Feb 28  2021 y
drwxr-xr-x 10 cvmfs cvmfs 4096 Feb 11 23:17 z

List the reference genome sets and other data files:

$ ls -la /cvmfs/data.galaxyproject.org/

total 14
drwxr-xr-x   4 cvmfs cvmfs 4096 Mar 31  2016 .
-rw-r--r--   1 cvmfs cvmfs   21 Oct 24  2018 .cvmfsdirtab
drwxr-xr-x 210 cvmfs cvmfs 4096 Apr 21  2022 byhand
drwxr-xr-x  18 cvmfs cvmfs 4096 Nov 24  2020 managed

Please note that the data sets may not be comprehensive, and this service is not meant to replace your current methods for accessing public datasets.

To use these data files for your analyses, copy the absolute file path in your workflow/pipeline. For example, with the reference genome Hg38, the file can be found in the following location, and specifically under the seq sub directory:

$ ls -la /cvmfs/data.galaxyproject.org/byhand/hg38

total 46
drwxrwxr-x  10 cvmfs cvmfs 4096 Apr 22  2016 .
drwxr-xr-x 210 cvmfs cvmfs 4096 Apr 21  2022 ..
-rw-r--r--   1 cvmfs cvmfs    0 Apr 22  2016 .cvmfscatalog
drwxrwxr-x   3 cvmfs cvmfs 4096 Jan 21  2015 download
drwxrwxr-x   6 cvmfs cvmfs 4096 Jan 20  2015 hg38canon
drwxrwxr-x   6 cvmfs cvmfs 4096 Jan 20  2015 hg38female
drwxrwxr-x   6 cvmfs cvmfs 4096 Jan 20  2015 hg38full
drwxrwxr-x   2 cvmfs cvmfs 4096 Mar 18  2014 liftOver
drwxrwxr-x   2 cvmfs cvmfs 4096 Mar 18  2014 picard_index
drwxrwxr-x   2 cvmfs cvmfs 4096 Mar 18  2014 sam_index
drwxrwxr-x   2 cvmfs cvmfs 4096 Apr  1  2016 seq


$ ls -la /cvmfs/data.galaxyproject.org/byhand/hg38/seq

total 10108046
drwxrwxr-x  2 cvmfs cvmfs       4096 Apr  1  2016 .
drwxrwxr-x 10 cvmfs cvmfs       4096 Apr 22  2016 ..
-rw-rw-r--  1 cvmfs cvmfs        136 Mar 18  2014 README
-rw-rw-r--  1 cvmfs cvmfs  835393456 Mar 18  2014 hg38.2bit
lrwxrwxrwx  1 cvmfs cvmfs         11 May 17  2014 hg38.fa -> hg38full.fa
-rw-r--r--  1 cvmfs cvmfs      19327 Aug 24  2015 hg38.fa.fai
-rw-rw-r--  1 cvmfs cvmfs 3150052305 Mar 17  2014 hg38canon.fa
-rw-rw-r--  1 cvmfs cvmfs 3091680335 Mar 17  2014 hg38female.fa
-rw-r--r--  1 cvmfs cvmfs        757 Apr  1  2016 hg38female.fa.fai
-rw-rw-r--  1 cvmfs cvmfs 3273481150 Mar 18  2014 hg38full.fa

So the full absolute path for the Hg38 sequence file would be:
```
/cvmfs/data.galaxyproject.org/byhand/hg38/seq/hg38full.fa
```

If you run into any errors with accessing the file system, run the following to re-install it:

sudo apt-get autoremove cvmfs
sudo apt-get purge cvmfs
sudo rm -rf /etc/cvmfs/ 
cd /home/ubuntu
git clone https://github.com/PawseySC/Pawsey-CernVM-FS.git
cd Pawsey-CernVM-FS
sudo ./install-cvmfs.sh install

If it is still causing errors, you may need to reboot your instance.

2. Using the Biocontainer tools

Singularity-HPC (SHPC) is a software for container modules. In this Pawsey Bio - Ubuntu 22.04 - 2023-XX image, we have integrated the use of SHPC seamlessly with CernVM-FS. This means that you can easily access and use over 8000 Biocontainers (and up to the latest versions) without needing to understand container syntax.

If you are using the now deprecated 'Pawsey Bio - Ubuntu 20.04 - 2021-11' image, you will not be able to seamlessly use Biocontainer tools without first installing them using SHPC in the next section, 3. Adding a local SHPC registry. To avoid that, we recommend that you recreate a new instance with the 'Pawsey Bio - Ubuntu 22.04 - 2023-XX' image.

Data directories

When using Biocontainer tools, you will be required to export the paths for your data directory(ies) to Singularity, so that they can be readable by the container. For example, if your data directory is /data, then you would run the following to add it to the Singularity bind path:

export SINGULARITY_BINDPATH=/data
echo 'export SINGULARITY_BINDPATH=/data' >> ~/.bashrc

To search for versions and information on a particular tool, e.g. for cuttlefish, use shpc show:

$ shpc show quay.io/biocontainers/cuttlefish

url: https://biocontainers.pro/tools/cuttlefish
maintainer: '@vsoch'
description: shpc-registry automated BioContainers addition for cuttlefish
latest:
  2.2.0--hf1761c0_0: sha256:63cdd7778b144a37684ae53b8e760ed00852f3010aa79292b3f1a6a6470f0992
tags:
  2.1.0--hf1761c0_0: sha256:aa009abd48c372125e060d39f49f1690be74b6dac276d451bf1cc4c847a914d6
  2.1.1--hf1761c0_0: sha256:8bccced83dd6bbf87843cf08851563c812ef7c36afda2efbcf0d54f9102b913f
  2.2.0--hf1761c0_0: sha256:63cdd7778b144a37684ae53b8e760ed00852f3010aa79292b3f1a6a6470f0992
docker: quay.io/biocontainers/cuttlefish
aliases:
  cuttlefish: /usr/local/bin/cuttlefish

To check for availability and to load the tool, use the module avail and module load commands:

Note that on first use, the tool might take a 30 seconds or so to run the command as the container is being accessed from the filesystem for the first time

$ module avail cuttlefish

------------------------------- /home/ubuntu/singularity-hpc/modules -------------------------------
   quay.io/biocontainers/cuttlefish/2.2.0--hf1761c0_0/module

$ module load quay.io/biocontainers/cuttlefish/2.2.0--hf1761c0_0/module

$ cuttlefish --version
cuttlefish 2.2.0
Supported commands: `build`, `help`, `version`.
Usage:
	cuttlefish build [options]

To check for the list of modules loaded:

This list will be cleared whenever you log out of your instance. After logging back in, you will need to reload the module for it to be on the list for use.
```
$ module list 

Currently Loaded Modules:
  1) quay.io/biocontainers/cuttlefish/2.2.0--hf1761c0_0/module 
```

To install another version not available as a module:

Expand...

To use another version of the tool available from the above shpc show list, use shpc to install the module, ensuring to use and keep the cvmfs path of the container:

$ sudo shpc install quay.io/biocontainers/cuttlefish:2.1.1--hf1761c0_0 /cvmfs/singularity.galaxyproject.org/all/cuttlefish:2.1.1--hf1761c0_0 --keep-path 
Module quay.io/biocontainers/cuttlefish:2.1.1--hf1761c0_0 was created.

Update the modules cache, then, run the same module commands to load the tool in this version:

$ /usr/local/lmod/lmod/libexec/update_lmod_system_cache_files -d /opt/mData/cacheDir -t /opt/mData/cacheTS.txt /home/ubuntu/singularity-hpc/modules

 $ module avail cuttlefish

------------------------------- /home/ubuntu/singularity-hpc/modules -------------------------------
   quay.io/biocontainers/cuttlefish/2.1.1--hf1761c0_0/module
   quay.io/biocontainers/cuttlefish/2.2.0--hf1761c0_0/module (L,D)

  Where:
   L:  Module is loaded
   D:  Default Module

 $ module load quay.io/biocontainers/cuttlefish/2.1.1--hf1761c0_0/module

The following have been reloaded with a version change:
  1) quay.io/biocontainers/cuttlefish/2.2.0--hf1761c0_0/module => quay.io/biocontainers/cuttlefish/2.1.1--hf1761c0_0/module

You will notice that the previous version of the tool is now swapped out for the version you just loaded

$ module list   

Currently Loaded Modules:
  1) quay.io/biocontainers/cuttlefish/2.1.1--hf1761c0_0/module

If you prefer to use the biocontainers without SHPC, you can do so by using the absolute path for each of the biocontainers. Note that you would require knowledge on how to use Singularity to do so. The version of Singularity installed on the Nimbus Bio image is 3.8.7 and instructions can be found here: Singularity exec.

For example, to use cuttlefish version 2.2.0–hf1761c0_0:

$ ls /cvmfs/singularity.galaxyproject.org/all/cuttlefish:2.2.0*
/cvmfs/singularity.galaxyproject.org/all/cuttlefish:2.2.0--h6a68c12_1
/cvmfs/singularity.galaxyproject.org/all/cuttlefish:2.2.0--h6a68c12_2
/cvmfs/singularity.galaxyproject.org/all/cuttlefish:2.2.0--hf1761c0_0
/cvmfs/singularity.galaxyproject.org/all/cuttlefish:2.2.0--hf1761c0_1

$ singularity exec /cvmfs/singularity.galaxyproject.org/all/cuttlefish:2.2.0--hf1761c0_0 cuttlefish

cuttlefish 2.2.0
Supported commands: `build`, `help`, `version`.
Usage:
	cuttlefish build [options]

When using Singularity, if you run into an issue with no loop devices found, please use the solution provided here: Using Containers#Commonissues

3. Adding a local SHPC registry

If there are versions (usually older ones) of a Biocontainer tool that is present in the cvmfs repository but not on the shpc show list (i.e. the default recipe), you can create a local SHPC registry and add/update a recipe file for the Biocontainer tool.

Clone the remote SHPC-registry and add it as a local registry:

$ cd /home/ubuntu

$ git clone https://github.com/singularityhub/shpc-registry.git

$ sudo shpc config add registry /home/ubuntu/shpc-registry/
Warning: Check with shpc config edit - ordering of list can change.
Added registry to /home/ubuntu/shpc-registry/

Look up for available versions of the tool, e.g. cuttlefish:

$ ls /cvmfs/singularity.galaxyproject.org/all/cuttlefish*
/cvmfs/singularity.galaxyproject.org/all/cuttlefish:1.0.0--h2e03b76_0
/cvmfs/singularity.galaxyproject.org/all/cuttlefish:1.0.0--h2e03b76_1
/cvmfs/singularity.galaxyproject.org/all/cuttlefish:2.0.0--h95f258a_0
/cvmfs/singularity.galaxyproject.org/all/cuttlefish:2.0.0--hf1761c0_1
/cvmfs/singularity.galaxyproject.org/all/cuttlefish:2.1.0--hf1761c0_0
/cvmfs/singularity.galaxyproject.org/all/cuttlefish:2.1.1--hf1761c0_0
/cvmfs/singularity.galaxyproject.org/all/cuttlefish:2.2.0--hf1761c0_0

To add a different version to the recipe file for the tool, e.g. 2.0.0--hf1761c0_1:

$ shpc add /cvmfs/singularity.galaxyproject.org/all/cuttlefish:2.0.0--hf1761c0_1 quay.io/biocontainers/cuttlefish:2.0.0--hf1761c0_1 --registry /home/ubuntu/shpc-registry/

quay.io/biocontainers/cuttlefish:2.0.0--hf1761c0_1 already exists and will be updated!

Registry entry quay.io/biocontainers/cuttlefish was added! Before shpc install, edit:
/home/ubuntu/shpc-registry/quay.io/biocontainers/cuttlefish/container.yaml

To install the tool:

If you are using the now deprecated 'Pawsey Bio - Ubuntu 20.04 - 2021-11' image, run the following steps first:

cd /home/ubuntu

git clone https://github.com/singularityhub/singularity-hpc.git

mkdir /home/ubuntu/singularity-hpc/modules

sudo shpc config set module_base /home/ubuntu/singularity-hpc/modules

cat >> ~/.bashrc <<'EOF' 
module use /home/ubuntu/singularity-hpc/modules
EOF

source ~/.bashrc

By setting your module_base to this new location, all new container modules will be installed to this path.

$ sudo shpc install quay.io/biocontainers/cuttlefish:2.0.0--hf1761c0_1 /cvmfs/singularity.galaxyproject.org/all/cuttlefish:2.0.0--hf1761c0_1 --keep-path
Module biocontainers/cuttlefish:2.0.0--hf1761c0_1 was created.

Now when you do a module avail, the newly installed 2.0.0--hf1761c0_1 version will be available:

$ module avail cuttlefish  

------------------------------- /home/ubuntu/singularity-hpc/modules -------------------------------
   quay.io/biocontainers/cuttlefish/2.0.0--hf1761c0_1/module

Since you have created your own local registry, shpc will default to your local registry whenever you do a look up with shpc show. To look up the full list of Biocontainer tools with the latest versions, you will need to add a flag to point to the remote (Github) shpc-registry in your search:

The shpc-registry is kept up-to-date with the latest versions of all Biocontainers on a nightly update.
```
$ shpc show quay.io/biocontainers/cuttlefish --registry https://github.com/singularityhub/shpc-registry
```

4. Using Nextflow

Nextflow makes use of containers to run your workflows sequentially. Each step of your workflow is called a process. For each process, Nextflow pulls the appropriate container required for that step to run it. You can prevent Nextflow from pulling the container and using what is present on your instance to save time and space.

To do so, you would create an additional config file to point Nextflow to either 1) the paths of the containers on /cvmfs/singularity.galaxyproject.org, or 2) the module paths on your instance. Nextflow prioritises this custom config file above the default nextflow.config file(s), if present, in other directories for your workflow.

Nextflow pipelines for Bioinformatics

Nextflow has a repository of pipelines that are available through https://nf-co.re. These are becoming increasingly popular, as more peer-reviewed pipelines are added by the community. A couple of popular ones include:

Configuring Nextflow to use Biocontainers

Please note this is only an example of how you can configure your Nextflow workflow to use the containers available from your instance.

Suppose you are using the nfcore/rnaseq pipeline. Note that in the main.nf for the FASTQC process, there are a few parameters for the tool. These will be over-written by the config file that you will create in the next step.

$ cat rnaseq/modules/nf-core/fastqc/main.nf

process FASTQC {
    tag "$meta.id"
    label 'process_medium'

    conda "bioconda::fastqc=0.11.9"
    container "${ workflow.containerEngine == 'singularity' && !task.ext.singularity_pull_docker_container ?
        'https://depot.galaxyproject.org/singularity/fastqc:0.11.9--0' :
        'quay.io/biocontainers/fastqc:0.11.9--0' }"

    input:
    tuple val(meta), path(reads)

    output:
    tuple val(meta), path("*.html"), emit: html
    tuple val(meta), path("*.zip") , emit: zip
    path  "versions.yml"           , emit: versions

    when:
    task.ext.when == null || task.ext.when

    script:
    def args = task.ext.args ?: ''
    def prefix = task.ext.prefix ?: "${meta.id}"
    // Make list of old name and new name pairs to use for renaming in the bash while loop
    def old_new_pairs = reads instanceof Path || reads.size() == 1 ? [[ reads, "${prefix}.${reads.extension}" ]] : reads.withIndex().collect { entry, index -> [ entry, "${prefix}_${index + 1}.${entry.extension}" ] }
    def rename_to = old_new_pairs*.join(' ').join(' ')
    def renamed_files = old_new_pairs.collect{ old_name, new_name -> new_name }.join(' ')
    """
    printf "%s %s\\n" $rename_to | while read old_name new_name; do
        [ -f "\${new_name}" ] || ln -s \$old_name \$new_name
    done
    fastqc $args --threads $task.cpus $renamed_files

    cat <<-END_VERSIONS > versions.yml
    "${task.process}":
        fastqc: \$( fastqc --version | sed -e "s/FastQC v//g" )
    END_VERSIONS
    """

    stub:
    def prefix = task.ext.prefix ?: "${meta.id}"
    """
    touch ${prefix}.html
    touch ${prefix}.zip

    cat <<-END_VERSIONS > versions.yml
    "${task.process}":
        fastqc: \$( fastqc --version | sed -e "s/FastQC v//g" )
    END_VERSIONS
    """
}

Nextflow prioritises a custom config file over any other config files or values defined in the workflow files. To ensure that Nextflow uses the existing container for fastqc, you would create and use a custom config file, choosing either of the two ways:

For the other processes in your workflow, you will need to also add to the corresponding *_path.config file to ensure that every process has the path for the existing container to run that part of the workflow from. More info on config files for processes can be found here: https://www.nextflow.io/docs/latest/config.html#scope-process