CLIP sequencing analysis pipeline for QC, pre-mapping, genome mapping, UMI deduplication, and multiple peak-calling options.
You will need to create a design file with information about the samples in your experiment before running the pipeline. Only single end reads are currently supported. Use this parameter to specify its location.
It has to be a comma-separated file with 2 columns, and a header row as shown in the examples below. The column headers must be
fastq. By naming the
sample rows uniquely, one can identify and simultaneously run multiple replicates and samples:
|Unique identifier for read, which may include information about sample and replicate.
|Full path to FastQ file for read. File has to be zipped and have the extension “.fastq.gz” or “.fq.gz”.
Full path to fasta file containing reference genome (mandatory if —genome is not specified). If you don’t have a STAR index available this will be generated for you automatically. Alternatively, it can be set using
--genome (using iGenomes)
There are 31 different species supported in the iGenomes references. To run the pipeline, you must specify which to use with the
--genome flag. If you have the iGenomes references locally available you can set
--igenome_base, otherwise they will be automatically obtained from AWS-iGenomes. You can find the keys to specify the genomes in the iGenomes config file. Common genomes that are supported are:
- H. sapiens
- M. musculus
- D. melanogaster
- S. cerevisiae
There are numerous others - check the config file for more.
Note that you can use the same configuration setup to save sets of reference files for your own use, even if they are not part of the iGenomes resource. See the Nextflow documentation for instructions on where to save such a file.
The syntax for this reference configuration is as follows:
Premapping to rRNA and tRNA will be automatically triggered if there is a reference available for the iGenomes reference chosen. See smallRNA config file for availability. Alternatively, this can be set by
--smrna_fasta as shown below.
The pipeline comes equipped with some ‘smallRNA’ FASTA references for premapping. This includes rRNA and tRNA sequences, the sources of which can be viewed here. The purpose of this premapping is to capture abundant ncRNA that are present in multiple similar copies in the genome, making them hard to assign reads to. tRNA can occur within genes and without proper handling can result in misassignment of reads to mRNA in certain situations. The user may also be interested in the tRNA and rRNA binding of their protein, and this premapping enables simple assessment of this binding. These are available for the following organisms:
Alternatively, the RNA premapping reference can be supplied by the user by giving the path to the reference FASTA:
By default, peak calling on identified crosslinks is not performed unless specified using the
--peakcaller argument. Currently the following peak callers are implemented:
Multiple peak callers can specified separated using a comma, e.g.
--peakcaller icount,paraclu. As a short-hand, all peak callers can be specified using
Running the pipeline
The typical command for running the pipeline is as follows:
This will launch the pipeline with the
docker configuration profile. See below for more information about profiles.
Note that the pipeline will create the following files in your working directory:
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:
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/clipseq 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.
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.
Core Nextflow arguments
NB: These options are part of Nextflow and use a single hyphen (pipeline parameters use a double-hyphen).
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, Podman, Shifter, Charliecloud, 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.
-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.
- A profile with a complete configuration for automated testing
- Includes links to test data so needs no other parameters
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.
Specify the path to a specific config file (this is a core Nextflow command). See the nf-core website documentation for more information.
Custom resource requests
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.
Whilst these default requirements will hopefully work for most people with most data, you may find that you want to customise the compute resources that the pipeline requests. You can do this by creating a custom config file. For example, to give the workflow process
star 32GB of memory, you could use the following config:
To find the exact name of a process you wish to modify the compute resources, check the live-status of a nextflow run displayed on your terminal or check the nextflow error for a line like so:
Error executing process > 'bwa'. In this case the name to specify in the custom config file is
See the main Nextflow documentation for more information.
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 above). 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.
Running in the background
Nextflow handles job submissions and supervises the running jobs. The Nextflow process must run until the pipeline is finished.
-bg flag launches Nextflow in the background, detached from your terminal so that the workflow does not stop if you log out of your session. The logs are saved to a file.
Alternatively, you can use
tmux or similar tool to create a detached session which you can log back into at a later time.
Some HPC setups also allow you to run nextflow within a cluster job submitted your job scheduler (from where it submits more jobs).
Nextflow memory requirements
In some cases, the Nextflow Java virtual machines can start to request a large amount of memory.
We recommend adding the following line to your environment to limit this (typically in