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nf-core/chipseq Usage

Table of contents

Running the pipeline

The typical command for running the pipeline is as follows:

module load blic-modules
module load nextflow

nextflow_chipseq --reads '*_R{1,2}.fastq.gz' --genome GRCh38

This will launch the pipeline with the legion or myriad configuration profile, depending on where you submit the job from.

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.

Input Data

--reads

Location of the input FastQ files:

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

NB: Must be enclosed in quotes!

Note that the {1,2} parentheses are required to specify paired end data. The file path should be in quotation marks to prevent shell glob expansion.

If left unspecified, the pipeline will assume that the data is in a directory called data in the working directory (data/*{1,2}.fastq.gz).

--singleEnd

By default, the pipeline expects paired-end data. If you have single-end data, specify --singleEnd 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: --singleEnd --reads '*.fastq'

It is not possible to run a mixture of single-end and paired-end files in one run.

--macsconfig

The setup file for peak calling using MACS.

Default: data/macsconfig

Format:

ChIPSampleID1,CtrlSampleID1,AnalysisID1
ChIPSampleID2,CtrlSampleID2,AnalysisID2
ChIPSampleID3,,AnalysisID3
  1. Column 1: ChIP sample name
    • Typically the file base name, shared between both reads (no file type extension)
    • eg. for chip_sample_1_R1.fastq.gz and chip_sample_1_R2.fastq.gz, enter chip_sample_1
  2. Column 2: Control sample name
    • Typically the input file base name, shared between both reads (no file type extension)
    • eg. for chip_input_R1.fastq.gz and chip_input_R2.fastq.gz, enter chip_input
  3. Column 3: Analysis ID
    • The analysis ID. Used for the output directory name.
    • Should be unique for each sample / line in the file.

For single-sample peaking calling without a control sample, leave the second column blank (control sample name).

--broad

Run MACS with the --broad flag. With this flag on, MACS will try to composite broad regions in BED12 ( a gene-model-like format ) by putting nearby highly enriched regions into a broad region with loose cutoff. The broad region is controlled by the default qvalue cutoff 0.1.

--saturation

Run saturation analysis by sub-sampling the sequence reads of ChIP sample from 10% to 100% with 10% interval, then calling ChIP-seq peaks with MACS. For one test there will be 10 sets of ChIP-seq peaks. A summary file (.CSV) will be provided with the numbers of ChIP-seq peaks.

--blacklist_filtering

Specifying this flag instructs the pipeline to use bundled ENCODE blacklist regions to filter out known blacklisted regions in the called ChIP-seq peaks. Please note that this is only supported when --genome is set to GRCh37 or GRCm38.

--blacklist

If you prefer, you can specify the full path to the blacklist regions (should be in .BED format) which will be filtered out from the called ChIP-seq peaks. Please note that --blacklist_filtering is required for using this option.

--blacklist_filtering --blacklist '[path to blacklisted regions]'

--extendReadsLen

Number of base pairs to extend the reads for the deepTools analysis. This number should be based on the length of your reads and the expected fragment length.

Default: 100

Reference Genomes

--genome

The reference genome to use for the analysis, needs to be one of the genome specified in the config file.

  • Human
    • --genome GRCh37
    • --genome GRCh38
  • Mouse
    • --genome GRCm38

--bwa_index

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

--bwa_index '[path to BWA index]'

--fasta

If you don't have a BWA index available, you can pass a FASTA file to the pipeline and a BWA index will be generated for you. Combine with --saveReference to save for future runs.

--fasta '[path to FASTA file]'

--gtf

The full path to GTF file can be specified for annotating peaks. Note that the GTF file should be in the Ensembl format.

--gtf '[path to GTF file]'

--allow_multi_align

Specifying --allow_multi_align will turn off the filtering of secondary alignments and unmapped reads from the BWA output file. Without this option, only primary alignments will be retained.

--saveReference

Supply this parameter to save any generated reference genome files to your results folder. These can then be used for future pipeline runs, reducing processing times.

Adapter Trimming

The pipeline accepts a number of parameters to change how the trimming is done, according to your data type. You can specify custom trimming parameters as follows:

  • --clip_r1 <NUMBER>
    • Instructs Trim Galore to remove bp from the 5' end of read 1 (or single-end reads).
  • --clip_r2 <NUMBER>
    • Instructs Trim Galore to remove bp from the 5' end of read 2 (paired-end reads only).
  • --three_prime_clip_r1 <NUMBER>
    • Instructs Trim Galore to remove bp from the 3' end of read 1 AFTER adapter/quality trimming has been
  • --three_prime_clip_r2 <NUMBER>
    • Instructs Trim Galore to re move bp from the 3' end of read 2 AFTER adapter/quality trimming has been performed.

--notrim

Specifying --notrim will skip the adapter trimming step. Use this if your input FastQ files have already been trimmed outside of the workflow or if you're very confident that there is no adapter contamination in your data.

--saveTrimmed

By default, trimmed FastQ files will not be saved to the results directory. Specify this flag (or set to true in your config file) to copy these files to the results directory when complete.

--saveAlignedIntermediates

By default, intermediate BAM files will not be saved. The final BAM files created after the Picard MarkDuplicates step are always saved. Set to true to also copy out BAM files from BWA and sorting steps.

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 on UPPMAX 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.

Maximum resource requests

All resource requests are checked against the following default limits (standard config profile shown):

--max_memory '128.GB'
--max_cpus '16'
--max_time '240.h'

If a task requests more than this amount, it will be reduced to this threshold.

To adjust these limits, specify them on the command line, eg. --max_memory '64.GB'.

Note that these limits are the maximum to be used per task. Nextflow will automatically attempt to parallelise as many jobs as possible given the available resources.

Other command line parameters

--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 speicfy this on the command line for every run.

--plaintext_email

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

-name

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

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.

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). Useful if using different UPPMAX projects or different sets of reference genomes.

NB: Single hyphen (core Nextflow option)

Note - you can use this to override defaults. For example, we run on UPPMAX but don't want to use the MultiQC environment module as is the default. So we specify a config file using -c that contains the following:

process.$multiqc.module = []

--clusterOptions

Submit arbitrary SLURM options (UPPMAX profile only). For instance, you could use --clusterOptions '-p devcore' to run on the development node (though won't work with default process time requests).

--multiqc_config

If you would like to supply a custom config file to MultiQC, you can specify a path with --multiqc_config. This is used instead of the config file specific to the pipeline.