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nfcore/rnaseq Usage

Running the pipeline

The typical command for running the pipeline is as follows:

module load blic-modules
module load nextflow

nextflow_rnaseq --reads '*_R{1,2}.fastq.gz' --genome hg38

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.

Main Arguments

-profile

This parameter is NOT necessary as the shortcut nextflow_rnaseq takes care of selecting the appropiate configuration profile. But just for your information, profiles are used to give configuration presets for different compute environments.

  • legion
    • A generic configuration profile to be used with the UCL cluster legion
  • myriad
    • A generic configuration profile to be used with the UCL cluster myriad

--reads

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

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

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

--singleEnd

By default, the pipeline expects paired-end data. If you have single-end data, you need to 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.

Library strandedness

Three command line flags / config parameters set the library strandedness for a run:

  • --forward_stranded
  • --reverse_stranded
  • --unstranded

If not set, the pipeline will be run as unstranded. Specifying --pico makes the pipeline run in forward_stranded mode.

These flags affect the commands used for several steps in the pipeline - namely HISAT2, featureCounts, RSeQC (RPKM_saturation.py) and StringTie:

  • --forward_stranded
    • HISAT2: --rna-strandness F / --rna-strandness FR
    • featureCounts: -s 1
    • RSeQC: -d ++,-- / -d 1++,1--,2+-,2-+
    • StringTie: --fr
  • --reverse_stranded
    • HISAT2: --rna-strandness R / --rna-strandness RF
    • featureCounts: -s 2
    • RSeQC: -d +-,-+ / -d 1+-,1-+,2++,2--
    • StringTie: --rf

Alignment tool

By default, the pipeline uses STAR to align the raw FastQ reads to the reference genome. STAR is fast and common, but requires a lot of memory to run, typically around 38GB for the Human hg19 reference genome.

If you prefer, you can use HISAT2 as the alignment tool instead. Developed by the same group behind the popular Tophat aligner, HISAT2 has a much smaller memory footprint.

To use HISAT2, use the parameter --aligner hisat2 or set params.aligner = 'hisat2' in your config file.

Additionally, the pipeline runs kallisto for transcript quantification. As it does not need alignment, this step is failry quick, but if you wnat to skip it you cna do it using the --skip_kallisto argument.

Minimum alignment lengths

When using STAR as the aligner then the pipeline sets the minimum alignment length to 15 base pairs. This filters out very short read alignments that can result from soft-clipping.

To customise this threshold, use the pipeline command line parameter --min_aln_length [num], where [num] is the minimum required alignment length in base pairs. Mapping sensitivity can be improved by increasing this value, or made more flexible by decreasing it. The default value in STAR is 0.

Reference Genomes

The pipeline config files come bundled with paths to the following reference genome assemblies: hg19, hg38, mm10. The pipeline has this aprameter set up as false by default, you need to specify it using the --genome flag.

  • Human
    • --genome hg19
    • --genome hg38
  • Mouse
    • --genome mm10

--star_index, --hisat2_index, --fasta, --gtf, --bed12

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

--star_index '[path to STAR index]' \
--hisat2_index '[path to HISAT2 index]' \
--fasta '[path to Fasta reference]' \
--gtf '[path to GTF file]' \
--bed12 '[path to bed12 file]'

Note that only one of --star_index / --hisat2_index are needed depending on which aligner you are using (see below).

The minimum requirements are a Fasta and GTF file. If these are provided and no others, then all other reference files will be automatically generated by the pipeline.

--downloadFasta, --downloadGTF

Instead of a path to a file, a URL can be supplied to download reference Fasta and GTF files at the start of the pipeline. A required STAR index and BED12 files will then be generated from these downloaded files.

--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.

--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 when complete.

--saveAlignedIntermediates

As above, by default intermediate BAM files from the alignment 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 STAR / HISAT2 and sorting steps.

Adapter Trimming

If specific additional trimming is required (for example, from additional tags), you can use any of the following command line parameters. These affect the command used to launch TrimGalore!

--clip_r1 [int]

Instructs Trim Galore to remove bp from the 5' end of read 1 (or single-end reads).

--clip_r2 [int]

Instructs Trim Galore to remove bp from the 5' end of read 2 (paired-end reads only).

--three_prime_clip_r1 [int]

Instructs Trim Galore to remove bp from the 3' end of read 1 AFTER adapter/quality trimming has been performed.

--three_prime_clip_r2 [int]

Instructs Trim Galore to re move bp from the 3' end of read 2 AFTER adapter/quality trimming has been performed.

Library Prep Presets

Some command line options are available to automatically set parameters for common RNA-seq library preparation kits.

Note that these presets override other command line arguments. So if you specify --pico --clip_r1 0, the --clip_r1 bit will be ignored.

If you have a kit that you'd like a preset added for, please let us know!

--pico

Sets trimming and standedness settings for the SMARTer Stranded Total RNA-Seq Kit - Pico Input kit.

Equivalent to: --forward_stranded --clip_r1 3 --three_prime_clip_r2 3

Skipping QC steps

The pipeline contains a large number of quality control steps. Sometimes, it may not be desirable to run all of them if time and compute resources are limited. The following options make this easy:

  • --skip_qc - Skip all QC steps, apart from MultiQC
  • --skip_fastqc - Skip FastQC
  • --skip_rseqc - Skip RSeQC
  • --skip_genebody_coverage - Skip calculating the genebody coverage
  • --skip_preseq - Skip Preseq
  • --skip_dupradar - Skip dupRadar (and Picard MarkDups)
  • --skip_edger - Skip edgeR MDS plot and heatmap
  • --skip_multiqc - Skip MultiQC

Other steps that can be skipped are:

  • --skip_kallisto - Skip kallisto
  • --skip_featurecounts - Skip featureCounts
  • --skip_stringtie - Skip stringtie

The above is useful if for example you are only interested in doing the alignments and nothing else, which cna be done by running the pipeline with the following parameters:

module load blic-modules
module load nextflow

nextflow_rnaseq --reads '*_R{1,2}.fastq.gz' --genome hg38 \
	--skip_qc --skip_kallisto --skip_featurecounts --skip_stringtie --skip_multiqc   \
	--saveAlignedIntermediates

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.

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.

-name

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.

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)

--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.

--sampleLevel

Used to turn of the edgeR MDS and heatmap. Set automatically when running on fewer than 3 samples.

### --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.

--clusterOptions

Submit arbitrary cluster scheduler options (not available for all config profiles). For instance, you could use --clusterOptions '-p devcore' to run on the development node (though won't work with default process time requests).

Stand-alone scripts

The bin directory contains some scripts used by the pipeline which may also be run manually:

  • gtf2bed
    • Script used to generate the BED12 reference files used by RSeQC. Takes a .gtf file as input
  • dupRadar.r
    • dupRadar script used in the dupRadar pipeline process.
  • edgeR_heatmap_MDS.r
    • edgeR script used in the Sample Correlation process
  • RNA-pipeline-from-BAM.sh
    • SLURM script used to mimic pipeline QC steps, taking an aligned BAM file as input.
    • Potentially unmaintained, use at your own risk!