nf-core/callingcards

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Introduction

Calling Cards experiments may be performed in both yeast and mammalian cells. The appropriate workflow is selected with the datatype parameter. Suggested default parameters for yeast and mammalian processing runs are provided through the profiles yeast and mammal. These may be used by simply including them with the -profile flag. See Running the pipeline for examples of submission commands. See -profile for more details on available profiles.

Samplesheet input

You will need to create a samplesheet with information about the samples you would like to analyse before running the pipeline. Use the input parameter to specify its location. It has to be a comma-separated file with 4 columns, and a header row as shown in the examples below.

Note: Currently, the mammals workflow supports only fastq_1. fastq_2 should simply be left blank. The yeast workflow requires both fastq_1 and fastq_2.

Full samplesheet

The pipeline will auto-detect whether a sample is single- or paired-end using the information provided in the samplesheet. The samplesheet can have as many columns as you desire, however, there is a strict requirement for the first 4 columns to match those defined in the table below.

A final samplesheet file consisting of single end mammalian reads would look like so:

sample,fastq_1,fastq_2,barcode_details
mouse_AY60-6_50,mouse/test_data/AY60-6_50k_downsampled_mouse.fastq.gz,,mouse/barcode_details.json
mouse_AY60-6_100,mouse/test_data/AY60-6_100k_downsampled_mouse.fastq.gz,,mouse/barcode_details.json
mouse_AY09-1_50_lowQuality,mouse/test_data/AY09-1_50k_downsampled_mouse_lowQuality.fastq.gz,,mouse/barcode_details.json
mouse_AY09-1_100_lowQuality,mouse/test_data/AY09-1_100k_downsampled_mouse_lowQuality.fastq.gz,,mouse/barcode_details.json
 

sample,fastq_1,fastq_2,barcode_details
mouse_AY60-6_50,mouse/test_data/AY60-6_50k_downsampled_mouse.fastq.gz,,mouse/barcode_details.json
mouse_AY60-6_100,mouse/test_data/AY60-6_100k_downsampled_mouse.fastq.gz,,mouse/barcode_details.json
mouse_AY09-1_50_lowQuality,mouse/test_data/AY09-1_50k_downsampled_mouse_lowQuality.fastq.gz,,mouse/barcode_details.json
mouse_AY09-1_100_lowQuality,mouse/test_data/AY09-1_100k_downsampled_mouse_lowQuality.fastq.gz,,mouse/barcode_details.json
 

An example samplesheet has been provided with the pipeline.

Barcode Details

The barcode details json stores data which allows the pipeline to relate sequence barcodes in the calling cards reads to a given transcription factor.

The file specificiations for both the yeast and mammals barcode details file may be found here

Running the pipeline

The typical command for running the mammals workflow is as follows:

nextflow run nf-core/callingcards \
    -profile default_mammals,singularity \
    --input /path/to/your_samplesheet.csv \
    --fasta /path/to/genome.fa \
    --gtf /path/to/genes.gtf \
    --outdir results

nextflow run nf-core/callingcards \
    -profile default_mammals,singularity \
    --input /path/to/your_samplesheet.csv \
    --fasta /path/to/genome.fa \
    --gtf /path/to/genes.gtf \
    --outdir results

A typical command for running the yeast workflow is as follows:

nextflow run nf-core/callingcards \
    -profile default_yeast,singularity \
    --input /path/to/your_samplesheet.csv \
    --outdir results

nextflow run nf-core/callingcards \
    -profile default_yeast,singularity \
    --input /path/to/your_samplesheet.csv \
    --outdir results

This will launch the pipeline with the specified profile(s). Note that the pipeline will create the following files in your working directory:

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

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

General parameters

The following describes a selected set of parameters that are common to both the yeast and mammalian workflows. For a full list of parameters, please see the parameters section of the nf-core/callingcards site

• The datatype parameter accepts either yeast or mammals and determines which workflow to run.

• The aligner parameter accepts either bwa, bwamem2, bowtie, or bowtie2

• split_fastq_by_size or split_fastq_by_part controls how the fastq files are split for parallel processing. Set one or the other, not both.

• min_mapq sets the minimal mapping quality for reads to be considered ‘passing’ in the hops counting stage. By default, this is 10.

• r1_crop determines how much of R1 will be passed onto alignment. The read is cropped after extracting the non-genomic sequence.

• save_genome_intermediate, save_sequence_intermediate, and save_alignment_intermediate may be set to save intermediate files from each of the corresponding steps of the workflows.

Mammals specific parameters

The mammals workflow requires that the following parameters be set. Note that these parameters are set in the default_mammals profile. But, you should confirm that these are correct for your data.

• r1_bc_pattern describes the barcode pattern that will be extracted by UMITools on R1.

Yeast specific parameters

There are no yeast specific parameters — rather the yeast specific steps are entirely contained within the data provided in the barcode_details.json and handled by callingCardsTools. You should examine the default_yeast configuration settings prior to using this profile.

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/callingcards

nextflow pull nf-core/callingcards

Reproducibility

It is 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/callingcards releases page and find the latest pipeline version - numeric only (eg. 1.3.1). Then specify this when running the pipeline with -r (one hyphen) - eg. -r 1.3.1. Of course, you can switch to another version by changing the number after the -r flag.

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. For example, at the bottom of the MultiQC reports.

To further assist in reproducbility, you can use share and re-use parameter files to repeat pipeline runs with the same settings without having to write out a command with every single parameter.

Core Nextflow 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, Podman, Shifter, Charliecloud, Apptainer, Conda) - see below.

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, since it can lead to different results on different machines dependent on the computer enviroment.

• test
  • A profile with a complete configuration for automated testing
  • Includes links to test data so needs no other parameters
• docker
  • A generic configuration profile to be used with Docker
• singularity
  • A generic configuration profile to be used with Singularity
• podman
  • A generic configuration profile to be used with Podman
• shifter
  • A generic configuration profile to be used with Shifter
• charliecloud
  • A generic configuration profile to be used with Charliecloud
• apptainer
  • A generic configuration profile to be used with Apptainer
• conda
  • A generic configuration profile to be used with Conda. Please only use Conda as a last resort i.e. when it’s not possible to run the pipeline with Docker, Singularity, Podman, Shifter, Charliecloud, or Apptainer.

Calling cards specific profiles

• default_mammals
  • A profile with suggested configuration for human and mouse data
• default_yeast
  • A profile with suggested configuration for human and mouse data
• test
  • A minimal test profile for the yeast workflow
• test_mammals
  • A minimal test profile for the mammalian workflow
• test_full
  • A minimal test profile for the full workflow — mammals data

-resume

Specify this when restarting a pipeline. Nextflow will use cached results from any pipeline steps where the inputs are the same, continuing from where it got to previously. For input to be considered the same, not only the names must be identical but the files’ contents as well. For more info about this parameter, see this blog post.

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.

-c

Specify the path to a specific config file (this is a core Nextflow command). See the nf-core website documentation for more information.

Custom configuration

Resource requests

Whilst the default requirements set within the pipeline will hopefully work for most people and with most input data, you may find that you want to customise the compute resources that the pipeline 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 any of the error codes specified here it will automatically be resubmitted with higher requests (2 x original, then 3 x original). If it still fails after the third attempt then the pipeline execution is stopped.

To change the resource requests, please see the max resources and tuning workflow resources section of the nf-core website.

Custom Containers

In some cases you may wish to change which container or conda environment a step of the pipeline uses for a particular tool. By default nf-core pipelines use containers and software from the biocontainers or bioconda projects. However in some cases the pipeline specified version maybe out of date.

To use a different container from the default container or conda environment specified in a pipeline, please see the updating tool versions section of the nf-core website.

Custom Tool Arguments

A pipeline might not always support every possible argument or option of a particular tool used in pipeline. Fortunately, nf-core pipelines provide some freedom to users to insert additional parameters that the pipeline does not include by default.

To learn how to provide additional arguments to a particular tool of the pipeline, please see the customising tool arguments section of the nf-core website.

nf-core/configs

In most cases, you will only need to create a custom config as a one-off but if you and others within your organisation are likely to be running nf-core pipelines regularly and need to use the same settings 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. 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.

See the main Nextflow documentation for more information about creating your own configuration files.

If you have any questions or issues please send us a message on Slack on the #configs channel.

Azure Resource Requests

To be used with the azurebatch profile by specifying the -profile azurebatch. We recommend providing a compute params.vm_type of Standard_D16_v3 VMs by default but these options can be changed if required.

Note that the choice of VM size depends on your quota and the overall workload during the analysis. For a thorough list, please refer the Azure Sizes for virtual machines in Azure.

Running in the background

Nextflow handles job submissions and supervises the running jobs. The Nextflow process must run until the pipeline is finished.

The Nextflow -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 screen / 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 ~/.bashrc or ~./bash_profile):

NXF_OPTS='-Xms1g -Xmx4g'

NXF_OPTS='-Xms1g -Xmx4g'