Introduction Usage Output Results Releases

Introduction

Nextflow handles job submissions on SLURM or other environments, and supervises running the jobs. Thus the Nextflow process must run until the pipeline is finished. We recommend that you put the process running in the background through screen / tmux or similar tool. Alternatively you can run nextflow within a cluster job submitted your job scheduler.

It is recommended to limit the Nextflow Java virtual machines memory. We recommend adding the following line to your environment (typically in ~/.bashrc or ~./bash_profile):

NXF_OPTS='-Xms1g -Xmx4g'

Running the pipeline

The typical command for running the pipeline is as follows.

Running the pipeline using Docker

nextflow run nf-core/rnafusion \
  -profile docker \
  --reads '*_R{1,2}.fastq.gz' \
  --arriba \
  --star_fusion \
  --fusioncatcher \
  --ericscript \
  --pizzly \
  --squid \
  --arriba_vis \
  --fusion_inspector

Running the pipeline using Singularity

nextflow run nf-core/rnafusion/download-singularity-img.nf --download_all --outdir /path

If the nextflow download script crashes (network issue), please use the bash script instead.

cd utils && sh download-singularity-img.sh /path/to/images

The command bellow will launch the pipeline using singularity.

nextflow run nf-core/rnafusion \
  -profile singularity \
  --reads '*_R{1,2}.fastq.gz' \
  --arriba \
  --star_fusion \
  --fusioncatcher \
  --ericscript \
  --pizzly \
  --squid \
  --arriba_vis \
  --fusion_inspector

Running specific tools

nextflow run nf-core/rnafusion \
  -profile singularity -c 'example/custom-singularity.config' \
  --reads '*_R{1,2}.fastq.gz' \
  --arriba \
  --squid

Note that the pipeline will create the following files in your working directory:

work            # Directory containing the nextflow working files
results         # Finished results (configurable, see below)
.nextflow_log   # Log file from Nextflow
# Other nextflow hidden files, eg. history of pipeline runs and old logs.

Updating the pipeline

When you run the above command, Nextflow automatically pulls the pipeline code from GitHub and stores it as a cached version. When running the pipeline after this, it will always use the cached version if available - even if the pipeline has been updated since. To make sure that you’re running the latest version of the pipeline, make sure that you regularly update the cached version of the pipeline:

nextflow pull nf-core/rnafusion

Reproducibility

It’s a good idea to specify a pipeline version when running the pipeline on your data. This ensures that a specific version of the pipeline code and software are used when you run your pipeline. If you keep using the same tag, you’ll be running the same version of the pipeline, even if there have been changes to the code since.

First, go to the nf-core/rnafusion releases page and find the latest version number - numeric only (eg. 1.3.1). Then specify this when running the pipeline with -r (one hyphen) - eg. -r 1.3.1.

This version number will be logged in reports when you run the pipeline, so that you’ll know what you used when you look back in the future.

Main arguments

-profile

Use this parameter to choose a configuration profile. Profiles can give configuration presets for different compute environments.

Several generic profiles are bundled with the pipeline which instruct the pipeline to use software packaged using different methods (Docker, Singularity, Conda) - see below.

We highly recommend the use of Docker or Singularity containers for full pipeline reproducibility, however when this is not possible, Conda is also supported.

The pipeline also dynamically loads configurations from https://github.com/nf-core/configs when it runs, making multiple config profiles for various institutional clusters available at run time. For more information and to see if your system is available in these configs please see the nf-core/configs documentation.

Note that multiple profiles can be loaded, for example: -profile test,docker - the order of arguments is important! They are loaded in sequence, so later profiles can overwrite earlier profiles.

If -profile is not specified, the pipeline will run locally and expect all software to be installed and available on the PATH. This is not recommended.

  • docker
  • singularity
  • test
    • A profile with a complete configuration for automated testing
    • Includes links to test data so needs no other parameters

--reads

Use this to specify the location of your input FastQ files. For example:

--reads 'path/to/data/sample_*_{1,2}.fastq.gz'

Please note the following requirements:

  1. The path must be enclosed in quotes
  2. The path must have at least one * wildcard character
  3. When using the pipeline with paired end data, the path must use {1,2} notation to specify read pairs.

If left unspecified, a default pattern is used: data/*{1,2}.fastq.gz

--single_end

By default, the pipeline expects paired-end data. If you have single-end data, you need to specify --single_end on the command line when you launch the pipeline. A normal glob pattern, enclosed in quotation marks, can then be used for --reads. For example:

--single_end --reads '*.fastq'

Tool flags

--arriba

If enabled, executes Arriba tool.

  • --arriba_opt
    • Specify additional parameters. For more info, please refer to the documentation of the tool.

--ericscript

If enabled, executes Ericscript tool.

  • --ericscript_opt
    • Specify additional parameters. For more info, please refer to the documentation of the tool.

--fusioncatcher

If enabled, executes Fusioncatcher tool.

  • --fusioncatcher_opt
    • Specify additional parameters. For more info, please refer to the documentation of the tool.

--fusion_report

If enabled, download databases for fusion-report.

  • fusion_report_opt
    • Specify additional parameters. For more info, please refer to the documentation of the tool.

--pizzly

If enabled, executes Pizzly tool.

  • --pizzly_k
    • Number of k-mers. Deafult 31.

--squid

If enabled, executes Squid tool.

--star_fusion

If enabled, executes STAR-Fusion tool.

  • --star_fusion_opt
    • Parameter for specifying additional parameters. For more info, please refer to the documentation of the tool.

Visualization flags

--arriba_vis

If enabled, executes build in Arriba visualization tool.

--fusion_inspector

If enabled, executes Fusion-Inspector tool.

Reference genomes

--arriba_ref

--arriba_ref '[path to Arriba reference]'

--databases

Required databases in order to run fusion-report.

--databases '[path to fusion-report databases]'

--ericscript_ref

Required reference in order to run EricScript.

--ericscript_ref '[path to EricScript reference]'

--fasta

If you prefer, you can specify the full path to your reference genome when you run the pipeline:

--fasta '[path to Fasta reference]'

--fusioncatcher_ref

Required reference in order to run Fusioncatcher.

--fusioncatcher_ref '[path to Fusioncatcher reference]'

--genome

This pipeline uses only Homo Sapiens version GRCh38. Also make sure to specify --genomes_base.

--genome 'GRCh38' --genome_base '/path/to/references'

--gtf

Required annotation file.

--gtf '[path to GTF annotation]'

--reference_release

Ensembl version.

# ftp://ftp.ensembl.org/pub/release-97/fasta/homo_sapiens/
--reference_release '97'

--star_index

If you prefer, you can specify the full path for STAR index when you run the pipeline. If not specified, the pipeline will build the index using for reads with length 100bp (can be adjusted with parameter --read_length).

--star_index '[path to STAR index]'

--star_fusion_ref

Required reference in order to run STAR-Fusion.

--star_fusion_ref '[path to STAR-Fusion reference]'

--transcript

Required transcript file.

--transcript '[path to transcript reference]'

Job resources

Automatic resubmission

Each step in the pipeline has a default set of requirements for number of CPUs, memory and time. For most of the steps in the pipeline, if the job exits with an error code of 143 (exceeded requested resources) it will automatically resubmit with higher requests (2 x original, then 3 x original). If it still fails after three times then the pipeline is stopped.

Custom resource requests

Wherever process-specific requirements are set in the pipeline, the default value can be changed by creating a custom config file. See the files hosted at nf-core/configs for examples.

If you are likely to be running nf-core pipelines regularly it may be a good idea to request that your custom config file is uploaded to the nf-core/configs git repository. Before you do this please can you test that the config file works with your pipeline of choice using the -c parameter (see definition below). You can then create a pull request to the nf-core/configs repository with the addition of your config file, associated documentation file (see examples in nf-core/configs/docs), and amending nfcore_custom.config to include your custom profile.

If you have any questions or issues please send us a message on Slack.

AWS Batch specific parameters

Running the pipeline on AWS Batch requires a couple of specific parameters to be set according to your AWS Batch configuration. Please use -profile awsbatch and then specify all of the following parameters.

--awsqueue

The JobQueue that you intend to use on AWS Batch.

--awsregion

The AWS region in which to run your job. Default is set to eu-west-1 but can be adjusted to your needs.

--awscli

The AWS CLI path in your custom AMI. Default: /home/ec2-user/miniconda/bin/aws.

The AWS region to run your job in. Default is set to eu-west-1 but can be adjusted to your needs. Please make sure to also set the -w/--work-dir and --outdir parameters to a S3 storage bucket of your choice - you’ll get an error message notifying you if you didn’t.

Other command line parameters

--debug

To run only a specific tool (testing freshly implemented tool) just add --debug parameter. This parameter only works on fusion tools only!

--read_length

Length is used to build a STAR index. Default is 100bp (Illumina).

--outdir

The output directory where the results will be saved.

--email

Set this parameter to your e-mail address to get a summary e-mail with details of the run sent to you when the workflow exits. If set in your user config file (~/.nextflow/config) then you don’t need to specify this on the command line for every run.

--email_on_fail

This works exactly as with --email, except emails are only sent if the workflow is not successful.

--max_multiqc_email_size

Threshold size for MultiQC report to be attached in notification email. If file generated by pipeline exceeds the threshold, it will not be attached (Default: 25MB).

-name

Name for the pipeline run. If not specified, Nextflow will automatically generate a random mnemonic.

Name for the pipeline run. If not specified, Nextflow will automatically generate a random mnemonic. This is used in the MultiQC report (if not default) and in the summary HTML / e-mail (always).

NB: Single hyphen (core Nextflow option)

-resume

Specify this when restarting a pipeline. Nextflow will used cached results from any pipeline steps where the inputs are the same, continuing from where it got to previously.

Specify this when restarting a pipeline. Nextflow will used cached results from any pipeline steps where the inputs are the same, continuing from where it got to previously. You can also supply a run name to resume a specific run: -resume [run-name]. Use the nextflow log command to show previous run names.

NB: Single hyphen (core Nextflow option)

-c

Specify the path to a specific config file (this is a core NextFlow command).

NB: Single hyphen (core Nextflow option)

Note - you can use this to override pipeline defaults.

--custom_config_version

Provide git commit id for custom Institutional configs hosted at nf-core/configs. This was implemented for reproducibility purposes. Default: master.

## Download and use config file with following git commid id
--custom_config_version d52db660777c4bf36546ddb188ec530c3ada1b96

--custom_config_base

If you’re running offline, nextflow will not be able to fetch the institutional config files from the internet. If you don’t need them, then this is not a problem. If you do need them, you should download the files from the repo and tell nextflow where to find them with the custom_config_base option. For example:

## Download and unzip the config files
cd /path/to/my/configs
wget https://github.com/nf-core/configs/archive/master.zip
unzip master.zip
 
## Run the pipeline
cd /path/to/my/data
nextflow run /path/to/pipeline/ --custom_config_base /path/to/my/configs/configs-master/

Note that the nf-core/tools helper package has a download command to download all required pipeline files + singularity containers + institutional configs in one go for you, to make this process easier.

--max_memory

Use to set a top-limit for the default memory requirement for each process. Should be a string in the format integer-unit. eg. --max_memory '8.GB'

--max_time

Use to set a top-limit for the default time requirement for each process. Should be a string in the format integer-unit. eg. --max_time '2.h'

--max_cpus

Use to set a top-limit for the default CPU requirement for each process. Should be a string in the format integer-unit. eg. --max_cpus 1

--plaintext_email

Set to receive plain-text e-mails instead of HTML formatted.

--monochrome_logs

Set to disable colourful command line output and live life in monochrome.

--multiqc_config

Specify a path to a custom MultiQC configuration file.
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