Introduction Usage Parameters Output Results Releases

Define where the pipeline should find input data and save output data.

Path to comma-separated file containing information about the samples in the experiment.

type: string
pattern: ^\S+\.csv$

You will need to create a design file with information about the samples in your experiment before running the pipeline. Use this parameter to specify its location. It has to be a comma-separated file with 4 columns, and a header row. See usage docs.

The output directory where the results will be saved. You have to use absolute paths to storage on Cloud infrastructure.

type: string

Email address for completion summary.

type: string
pattern: ^([a-zA-Z0-9_\-\.]+)@([a-zA-Z0-9_\-\.]+)\.([a-zA-Z]{2,5})$

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 specify this on the command line for every run.

MultiQC report title. Printed as page header, used for filename if not otherwise specified.

type: string

Reference genome related files and options required for the workflow.

Name of iGenomes reference.

type: string

If using a reference genome configured in the pipeline using iGenomes (not recommended), use this parameter to give the ID for the reference. This is then used to build the full paths for all required reference genome files e.g. --genome GRCh38.

See the nf-core website docs for more details.

Path to FASTA genome file.

type: string
pattern: ^\S+\.fn?a(sta)?(\.gz)?$

This parameter is mandatory if --genome is not specified. If you don't have the appropriate alignment index available this will be generated for you automatically. Combine with --save_reference to save alignment index for future runs.

Path to GTF annotation file.

type: string
pattern: ^\S+\.gtf(\.gz)?$

This parameter is mandatory if --genome is not specified.

Path to GFF3 annotation file.

type: string
pattern: ^\S+\.gff(\.gz)?$

This parameter must be specified if --genome or --gtf are not specified.

Path to BED file containing gene intervals. This will be created from the GTF file if not specified.

type: string
pattern: ^\S+\.bed(\.gz)?$

Path to FASTA transcriptome file.

type: string
pattern: ^\S+\.fn?a(sta)?(\.gz)?$

FASTA file to concatenate to genome FASTA file e.g. containing spike-in sequences.

type: string
pattern: ^\S+\.fn?a(sta)?(\.gz)?$

If provided, the sequences in this file will get concatenated to the existing genome FASTA file, a GTF file will be automatically created using the entire sequence as the gene, transcript, and exon features, and any alignment index will get created from the combined FASTA and GTF. It is recommended to save the reference with --save_reference to re-use the index for future runs so you do not need to create it again.

Splice sites file required for HISAT2.

type: string

Path to directory or tar.gz archive for pre-built STAR index.

type: string

Path to directory or tar.gz archive for pre-built HISAT2 index.

type: string

Path to directory or tar.gz archive for pre-built RSEM index.

type: string

Path to directory or tar.gz archive for pre-built Salmon index.

type: string

Path to directory or tar.gz archive for pre-built Kallisto index.

type: string

Minimum memory required to use splice sites and exons in the HiSAT2 index build process.

type: string
default: 200.GB
pattern: ^\d+(\.\d+)?\.?\s*(K|M|G|T)?B$

HiSAT2 requires a huge amount of RAM to build a genome index for larger genomes, if including splice sites and exons e.g. the human genome might typically require 200GB. If you specify less than this threshold for the HISAT2_BUILD process then the splice sites and exons will be ignored, meaning that the process will require a lot less memory. If you are working with a small genome, set this parameter to a lower value to reduce the threshold for skipping this check. If using a larger genome, consider supplying more memory to the HISAT2_BUILD process.

Specify if your GTF annotation is in GENCODE format.

type: boolean

If your GTF file is in GENCODE format and you would like to run Salmon i.e. --pseudo_aligner salmon, you will need to provide this parameter in order to build the Salmon index appropriately.

By default, the pipeline uses the gene_name field to obtain additional gene identifiers from the input GTF file when running Salmon.

type: string
default: gene_name

This behaviour can be modified by specifying --gtf_extra_attributes when running the pipeline. Note that you can also specify more than one desired value, separated by a comma e.g. --gtf_extra_attributes gene_id,....

Define the attribute type used to group features in the GTF file when running Salmon.

type: string
default: gene_id

The attribute type used to group feature types in the GTF file when generating the biotype plot with featureCounts.

type: string
default: gene_biotype

By default, the pipeline assigns reads based on the 'exon' attribute within the GTF file.

type: string
default: exon

The feature type used from the GTF file when generating the biotype plot with featureCounts.

Do not load the iGenomes reference config.

hidden
type: boolean

Do not load igenomes.config when running the pipeline. You may choose this option if you observe clashes between custom parameters and those supplied in igenomes.config.

The base path to the igenomes reference files

hidden
type: string
default: s3://ngi-igenomes/igenomes/

Options to adjust read trimming criteria.

Specifies the trimming tool to use - available options are 'trimgalore' and 'fastp'.

type: string

Extra arguments to pass to Trim Galore! command in addition to defaults defined by the pipeline.

type: string

Extra arguments to pass to fastp command in addition to defaults defined by the pipeline.

type: string

Minimum number of trimmed reads below which samples are removed from further processing. Some downstream steps in the pipeline will fail if this threshold is too low.

type: integer
default: 10000

Options for filtering reads prior to alignment

Path to comma-separated file containing a list of reference genomes to filter reads against with BBSplit. You have to also explicitly set --skip_bbsplit false if you want to use BBSplit.

type: string

The file should contain 2 columns: short name and full path to reference genome(s) e.g.

mm10,/path/to/mm10.fa
ecoli,/path/to/ecoli.fa

Path to directory or tar.gz archive for pre-built BBSplit index.

type: string

The BBSplit index will have to be built at least once with this pipeline (see --save_reference to save index). It can then be provided via --bbsplit_index for future runs.

Path to directory or tar.gz archive for pre-built sortmerna index.

type: string

The sortmerna index will have to be built at least once with this pipeline (see --save_reference to save index). It can then be provided via --sortmerna_index for future runs.

Enable the removal of reads derived from ribosomal RNA using SortMeRNA.

type: boolean

Any patterns found in the sequences defined by the '--ribo_database_manifest' parameter will be used.

Text file containing paths to fasta files (one per line) that will be used to create the database for SortMeRNA.

type: string
default: ${projectDir}/workflows/rnaseq/assets/rrna-db-defaults.txt

By default, rRNA databases defined in the SortMeRNA GitHub repo are used. You can see an example in the pipeline Github repository in assets/rrna-default-dbs.txt. Please note that commercial/non-academic entities require licensing for SILVA for these default databases.

Options for processing reads with unique molecular identifiers

Enable UMI-based read deduplication.

type: boolean

UMI pattern to use. Can be either 'string' (default) or 'regex'.

type: string
default: string

More details can be found in the UMI-tools documentation.

The UMI barcode pattern to use e.g. 'NNNNNN' indicates that the first 6 nucleotides of the read are from the UMI.

type: string

More details can be found in the UMI-tools documentation.

The UMI barcode pattern to use if the UMI is located in read 2.

type: string

After UMI barcode extraction discard either R1 or R2 by setting this parameter to 1 or 2, respectively.

type: integer

The character that separates the UMI in the read name. Most likely a colon if you skipped the extraction with UMI-tools and used other software.

type: string

Method to use to determine read groups by subsuming those with similar UMIs. All methods start by identifying the reads with the same mapping position, but treat similar yet nonidentical UMIs differently.

type: string

Generate output stats when running "umi_tools dedup".

type: boolean

It can be quite time consuming generating these output stats - see #827.

Options to adjust parameters and filtering criteria for read alignments.

Specifies the alignment algorithm to use - available options are 'star_salmon', 'star_rsem' and 'hisat2'.

type: string

Specifies the pseudo aligner to use - available options are 'salmon'. Runs in addition to '--aligner'.

type: string

Kmer length passed to indexing step of pseudoaligners

type: integer
default: 31

Failure to set a good kmer size could cause issues with quantification with Kallisto or Salmon. This is mostly an issue for short reads (<50bp), where the default kmer size of 31 is an problem.

Create a CSI index for BAM files instead of the traditional BAI index. This will be required for genomes with larger chromosome sizes.

type: boolean

When using pre-built STAR indices do not re-extract and use splice junctions from the GTF file.

type: boolean

Override Salmon library type inferred based on strandedness defined in meta object.

type: string

Minimum percentage of uniquely mapped reads below which samples are removed from further processing.

type: number
default: 5

Some downstream steps in the pipeline will fail if this threshold is too low.

Sequencing center information to be added to read group of BAM files.

type: string

Perform reference-guided de novo assembly of transcripts using StringTie i.e. dont restrict to those in GTF file.

type: boolean

Extra arguments to pass to STAR alignment command in addition to defaults defined by the pipeline. Only available for the STAR-Salmon route.

type: string

Extra arguments to pass to Salmon quant command in addition to defaults defined by the pipeline.

type: string

Extra arguments to pass to Kallisto quant command in addition to defaults defined by the pipeline.

type: string

In single-end mode Kallisto requires an estimated fragment length. Specify a default value for that here. TODO: use existing RSeQC results to do this dynamically.

type: integer
default: 200

In single-end mode, Kallisto requires an estimated standard error for fragment length. Specify a default value for that here. TODO: use existing RSeQC results to do this dynamically.

type: integer
default: 200

The fraction of stranded reads that must be assigned to a strandedness for confident assignment. Must be at least 0.5.

type: number
default: 0.8

The difference in fraction of stranded reads assigned to 'forward' and 'reverse' below which a sample is classified as 'unstranded'. By default the forward and reverse fractions must differ by less than 0.1 for the sample to be called as unstranded.

type: number
default: 0.1

Additional output files produces as intermediates that can be saved

Save FastQ files after merging re-sequenced libraries in the results directory.

type: boolean

If this option is specified, intermediate FastQ and BAM files produced by UMI-tools are also saved in the results directory.

type: boolean

If this option is specified, intermediate FastQ files containing non-rRNA reads will be saved in the results directory.

type: boolean

If this option is specified, FastQ files split by reference will be saved in the results directory.

type: boolean

If generated by the pipeline save the STAR index in the results directory.

type: boolean

If an alignment index is generated by the pipeline use this parameter to save it to your results folder. These can then be used for future pipeline runs, reducing processing times.

Save the trimmed FastQ files in the results directory.

type: boolean

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.

Save the intermediate BAM files from the alignment step.

type: boolean

By default, intermediate BAM files will not be saved. The final BAM files created after the appropriate filtering step are always saved to limit storage usage. Set this parameter to also save other intermediate BAM files.

Where possible, save unaligned reads from either STAR, HISAT2 or Salmon to the results directory.

type: boolean

This may either be in the form of FastQ or BAM files depending on the options available for that particular tool.

Save read-by-read assignments from Kraken2.

type: boolean

--kraken_db parameter must be provided.

Save reads that were not given assignment from Kraken2.

type: boolean

--kraken_db parameter must be provided.

Additional quality control options.

Use vst transformation instead of rlog with DESeq2.

type: boolean
default: true

Specify the RSeQC modules to run.

type: string
default: bam_stat,inner_distance,infer_experiment,junction_annotation,junction_saturation,read_distribution,read_duplication

Tool to use for detecting contaminants in unaligned reads - available options are 'kraken2' and 'kraken2_bracken'

type: string

Database when using Kraken2/Bracken for contaminant screening.

type: string

See the usage tab for more information

Taxonomic level for Bracken abundance estimations.

type: string

First letter of Domain / Phylum / Class / Order / Family / Genus / Species

Options to skip various steps within the workflow.

Skip filtering of GTF for valid scaffolds and/ or transcript IDs.

type: boolean

If you're confident on the validity of the GTF with respect to the genome fasta file, or wish to disregard failures thriggered by the filtering module, activate this option.

Skip the 'transcript_id' checking component of the GTF filtering script used in the pipeline.

type: boolean

Skip BBSplit for removal of non-reference genome reads.

type: boolean
default: true

Skip the UMI extraction from the read in case the UMIs have been moved to the headers in advance of the pipeline run.

type: boolean

Skip the adapter trimming step.

type: boolean

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.

Skip all of the alignment-based processes within the pipeline.

type: boolean

Skip all of the pseudoalignment-based processes within the pipeline.

type: boolean

Skip picard MarkDuplicates step.

type: boolean

Skip bigWig file creation.

type: boolean

Skip StringTie.

type: boolean

Skip FastQC.

type: boolean

Skip Preseq.

type: boolean
default: true

Skip dupRadar.

type: boolean

Skip Qualimap.

type: boolean

Skip RSeQC.

type: boolean

Skip additional featureCounts process for biotype QC.

type: boolean

Skip DESeq2 PCA and heatmap plotting.

type: boolean

Skip MultiQC.

type: boolean

Skip all QC steps except for MultiQC.

type: boolean

Parameters used to describe centralised config profiles. These should not be edited.

Git commit id for Institutional configs.

hidden
type: string
default: master

Base directory for Institutional configs.

hidden
type: string
default: https://raw.githubusercontent.com/nf-core/configs/master

If you're running offline, Nextflow will not be able to fetch the institutional config files from the internet. If you don't need them, then this is not a problem. If you do need them, you should download the files from the repo and tell Nextflow where to find them with this parameter.

Institutional config name.

hidden
type: string

Institutional config description.

hidden
type: string

Institutional config contact information.

hidden
type: string

Institutional config URL link.

hidden
type: string

Less common options for the pipeline, typically set in a config file.

Display version and exit.

hidden
type: boolean

Method used to save pipeline results to output directory.

hidden
type: string

The Nextflow publishDir option specifies which intermediate files should be saved to the output directory. This option tells the pipeline what method should be used to move these files. See Nextflow docs for details.

Email address for completion summary, only when pipeline fails.

hidden
type: string
pattern: ^([a-zA-Z0-9_\-\.]+)@([a-zA-Z0-9_\-\.]+)\.([a-zA-Z]{2,5})$

An email address to send a summary email to when the pipeline is completed - ONLY sent if the pipeline does not exit successfully.

Send plain-text email instead of HTML.

hidden
type: boolean

File size limit when attaching MultiQC reports to summary emails.

hidden
type: string
default: 25.MB

Do not use coloured log outputs.

hidden
type: boolean

Incoming hook URL for messaging service

hidden
type: string

Incoming hook URL for messaging service. Currently, MS Teams and Slack are supported.

Custom config file to supply to MultiQC.

hidden
type: string

Custom logo file to supply to MultiQC. File name must also be set in the MultiQC config file

hidden
type: string

Custom MultiQC yaml file containing HTML including a methods description.

type: string

Boolean whether to validate parameters against the schema at runtime

hidden
type: boolean
default: true

Base URL or local path to location of pipeline test dataset files

hidden
type: string
default: https://raw.githubusercontent.com/nf-core/test-datasets/7f1614baeb0ddf66e60be78c3d9fa55440465ac8/
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