nf-core/demultiplex

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Introduction

• The pipeline samplesheet is a file provided as input to the nf-core pipeline itself. It contains the overall configuration for your run, specifying the paths to individual flowcell samplesheets, flowcell directories, and other metadata required to manage multiple sequencing runs. This is the primary configuration file that directs the pipeline on how to process your data.
• The flowcell samplesheet is specific to a particular sequencing run. It is typically created by the sequencing facility and contains the sample information, including barcodes, lane numbers, and indexes. The typical name is SampleSheet.csv. Each demultiplexer may require a different format for this file, which must be adhered to for proper data processing.

Pipeline samplesheet input

You will need to create a pipeline samplesheet with information about the samples you would like to analyse before running the pipeline. Use this parameter to specify its location. It has to be a comma-separated file with at least 4 columns, and a header row as shown in the examples below. The input pipeline samplesheet is a comma-separated file that contains four columns: id, samplesheet, lane, flowcell.

When using the demultiplexer fqtk, the pipeline samplesheet must contain an additional column per_flowcell_manifest. The column per_flowcell_manifest must contain two headers fastq and read_structure. As shown in the example provided each row must contain one fastq file name and the correlating read structure.

--input '[path to pipeline samplesheet file]'

Example: Pipeline samplesheet

id,samplesheet,lane,flowcell
DDMMYY_SERIAL_NUMBER_FC,/path/to/SampleSheet.csv,1,/path/to/sequencer/output
DDMMYY_SERIAL_NUMBER_FC,/path/to/SampleSheet.csv,2,/path/to/sequencer/output
DDMMYY_SERIAL_NUMBER_FC2,/path/to/SampleSheet2.csv,1,/path/to/sequencer/output2
DDMMYY_SERIAL_NUMBER_FC3,/path/to/SampleSheet3.csv,3,/path/to/sequencer/output3

An example pipeline samplesheet has been provided with the pipeline.

Note that the run directory in the flowcell column must lead to a tar.gz for compatibility with the demultiplexers sgdemux and fqtk.

Example: Pipeline samplesheet for fqtk

id,samplesheet,lane,flowcell,per_flowcell_manifest
DDMMYY_SERIAL_NUMBER_FC,/path/to/SampleSheet.csv,1,/path/to/sequencer/output,/path/to/flowcell/manifest.csv
DDMMYY_SERIAL_NUMBER_FC,/path/to/SampleSheet1.csv,2,/path/to/sequencer/output,/path/to/flowcell/manifest1.csv
DDMMYY_SERIAL_NUMBER_FC2,/path/to/SampleSheet2.csv,1,/path/to/sequencer/output2,/path/to/flowcell/manifest2.csv
DDMMYY_SERIAL_NUMBER_FC3,/path/to/SampleSheet3.csv,3,/path/to/sequencer/output3,/path/to/flowcell/manifest3.csv

Flowcell samplesheet

Each demultiplexing software uses a distinct flowcell samplesheet format. Below are examples for demultiplexer-specific flowcell samplesheets. Please see the following examples to format the flowcell SampleSheet.csv:

Running the pipeline

The typical command for running the pipeline is as follows:

nextflow run nf-core/demultiplex \
    --input pipeline_samplesheet.csv \
    --outdir results \
    -profile docker

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:

work                # Directory containing the nextflow working files
<OUTDIR>            # Finished results in specified location (defined with --outdir)
.nextflow_log       # Log file from Nextflow
# Other nextflow hidden files, eg. history of pipeline runs and old logs.

If you wish to repeatedly use the same parameters for multiple runs, rather than specifying each flag in the command, you can specify these in a params file.

Pipeline settings can be provided in a yaml or json file via -params-file <file>.

The above pipeline run specified with a params file in yaml format:

nextflow run nf-core/demultiplex -profile docker -params-file params.yaml

with:

input: './samplesheet.csv'
outdir: './results/'
genome: 'GRCh37'
<...>

You can also generate such YAML/JSON files via nf-core/launch.

Optional parameters

checkQC

If you are running this pipeline with the bcl2fastq demultiplexer, the checkqc module is run. In this case, the default run will include the default config file for checkqc, but you can additionally provide your own checkqc config file using the parameter --checkqc_config and a path to a yml. See an example of a config file in the checkqc repository.

Trimming

The trimming process in our demultiplexing pipeline has been updated to ensure compatibility with 10x Genomics recommendations. By default, trimming in the pipeline is performed using fastp, which reliably auto-detects and removes adapter sequences without the need for storing adapter sequences. As users can also supply adapter sequences in a samplesheet and thereby triggering trimming in any bcl2fastq or bclconvert subworkflows, we have added a new parameter, remove_adapter, which is set to true by default. When remove_adapter is true, the pipeline automatically removes any adapter sequences listed in the [Settings] section of the Illumina sample sheet, replacing them with an empty string in order to not provoke this behaviour. This approach aligns with 10x Genomics’ guidelines, as they advise against pre-processing FASTQ reads before inputting them into their software pipelines. If the remove_adapter setting is true but no adapter is removed, a warning will be displayed; however, this does not necessarily indicate an error, as some sample sheets may already lack these adapter sequences. Users can disable this behavior by setting --remove_adapter false in the command line, though this is not recommended.

samshee (Samplesheet validator)

samshee ensures the integrity of Illumina v2 Sample Sheets by allowing users to apply custom validation rules. The module can be used together with the parameter --json_schema_validator, which accepts a JSON schema validation string; the --name_schema_validator, which accepts a schema name string; and the --file_schema_validator which accepts a JSON schema validation file. Users can specify additional validation rules beyond the default ones provided by the tool using all or any of these parameters, this enables tailored validation of Sample Sheets to meet specific requirements or standards relevant to your sequencing workflow. For more information refer to Samshee on GitHub.

When indicating --json_schema_validator or --name_schema_validator, please note that it expects a JSON reference value in string format. For example:

--json_schema_validator '{"required": ["Data"]}'
--name_schema_validator '{"$ref": "urn:samshee:illuminav2/v1"}'

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/demultiplex

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/demultiplex 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
• wave
  • A generic configuration profile to enable Wave containers. Use together with one of the above (requires Nextflow 24.03.0-edge or later).
• 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.

-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.

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'