rnasplice
nf-core/rnasplice
Alternative splicing analysis using RNA-seq.
Key tools
Run it
nfclaw run rnasplice --input samplesheet.csv --outdir results --contrasts <contrasts> -profile dockernextflow run pipelines/rnasplice/upstream -profile docker --input samplesheet.csv --outdir results --contrasts <contrasts>nfclaw run rnasplice --demo --outdir results # adds the upstream test profile (-profile test,docker)Inputs
| column | type | required | allowed values | constraints |
|---|---|---|---|---|
sample | string | yes | matches ^(?!\.\.\d+)(?!\.$)[a-zA-Z.]([a-zA-Z0-9._]*)?$ | |
fastq_1 | string (file path) | yes | matches ^([\S\s]*\/)?[^\s\/]+\.f(ast)?q\.gz$ | |
fastq_2 | string (file path) | no | matches ^([\S\s]*\/)?[^\s\/]+\.f(ast)?q\.gz$ | |
strandedness | string | yes | forward, reverse, unstranded | |
condition | string | yes | matches ^\S+$ |
--input must match ^\S+\.csv$.
The samplesheet is a CSV with this header (the columns the schema requires); fill each value per the table above and reference.md (no example value is invented here):
sample,fastq_1,strandedness,conditionAny of the optional columns above may be appended to the header when your data needs them: fastq_2.
From data files. --input may also be the data files — a directory (searched recursively), a quoted glob, or files (--input once per file) — from which nfclaw writes this samplesheet: sample from file names; fastq_1/fastq_2 from FastQ files (mates paired by _R1/_R2, or by _1/_2 when their read names show they are mates). Set the required columns no file determines with --sheet-set COLUMN=VALUE (a constant or a {sample}/{lane} template): strandedness (one of forward, reverse, unstranded); condition (matching ^\S+$). Preview it with --check; a run records it in <outdir>/provenance/ for replay.
Reference genome
No reference genome is set by default: supply your own reference. The schema declares AWS iGenomes at s3://ngi-igenomes/igenomes/; selecting --genome <id> uses the paths in the pipeline's reference configuration. Access to that remote source is required. To supply your own reference, use e.g. --fasta; the reference_genome_options group in reference.md lists every reference option. Set --igenomes-ignore true to disable the lookup entirely. Confirm the selected reference's paths and source in the pinned upstream/nextflow.config and upstream/conf/ before running.
Resources
Resource requests follow the pipeline's configs and selected profiles. nf-core's resource labels often assume a server — a single step can request far more memory than a workstation has, and retry rules can increase requests. If a run fails with Process requirement exceeds available memory (or CPUs), cap every request, and every retry, at what this machine actually has:
nfclaw run rnasplice --input samplesheet.csv --outdir results --contrasts <contrasts> -profile docker \ --limit-cpus 4 --limit-memory 15.GBnfclaw turns those into Nextflow's process.resourceLimits and passes them as a -c config — the mechanism nf-core prescribes for exactly this (docs). Set them to the machine's real capacity. --limit-time DURATION also caps how long any one task may run: a task that needs longer is stopped, and so is every retry, so set it only as a deliberate ceiling, never as a guess at how long the analysis takes. The generated config is kept in <outdir>/provenance/, so commands.sh replays the run under the same ceiling.
Nextflow engine
This release declares nextflowVersion = '!>=26.04.0'.
To pin this release's declared minimum engine — useful when a newer Nextflow has incompatible config parsing:
nfclaw run rnasplice ... --nxf-ver 26.04.0--nxf-ver is recorded in <outdir>/provenance/, so the replay uses the same engine. See known-issues.
Demo
nfclaw run rnasplice --demo --outdir results # adds the upstream test profile (-profile test,docker)Samplesheet
A CSV with this header (the columns the schema requires). Fill each value per the constraints below; optional columns from the table may be appended.
sample,fastq_1,strandedness,condition| Column | Type | Required | Allowed / constraints |
|---|---|---|---|
sample | string | required | matches ^(?!\.\.\d+)(?!\.$)[a-zA-Z.]([a-zA-Z0-9._]*)?$ |
fastq_1 | string (file path) | required | matches ^([\S\s]*\/)?[^\s\/]+\.f(ast)?q\.gz$ |
fastq_2 | string (file path) | optional | matches ^([\S\s]*\/)?[^\s\/]+\.f(ast)?q\.gz$ |
strandedness | string | required | forwardreverseunstranded |
condition | string | required | matches ^\S+$ |
Required parameters
--inputstring (file path)Path to comma-separated file containing information about the samples in the experiment.
Constraint · matches ^\S+\.csv$
--contrastsstringPath to comma-separated file containing information about the contrasts in the experiment.
--sourcestringSource of input files.
fastqgenome_bamtranscriptome_bamsalmon_results--outdirstring (directory path)The output directory where the results will be saved. You have to use absolute paths to storage on Cloud infrastructure.
Parameter explorer
127 parameters · 15 groupsEvery parameter from the pinned nextflow_schema.json. nfclaw checks explicit values before launch; complex rules use the pinned pipeline's runtime validation. Search, filter by group, and expand any parameter for its default, allowed values and constraints.
108 parameters · 19 hidden
Alignment options9
Specifies the alignment algorithm to use.
Create a CSI index for BAM files instead of the traditional BAI index. This will be required for genomes with larger chromosome sizes.
Specifies the pseudo aligner to use. Runs in addition to --aligner.
Override Salmon library type inferred based on strandedness defined in meta object.
Save the intermediate BAM files from the alignment step.
Where possible, save unaligned reads from either STAR or Salmon to the results directory.
Sequencing center information to be added to read group of BAM files.
Skip all of the alignment-based processes within the pipeline.
When using pre-built STAR indices do not re-extract and use splice junctions from the GTF file.
Dexseq Deu options7
Combine overlapping genes into a single aggregate gene.
Minimum alignment quality required for reads to be counted.
Run DEXSeq differential exon usage workflow.
Path to GFF3 annotation file.
Plot the N most significant genes from the DEXSeq results.
Save pre-processed GFF annotation file.
Save plots of the per gene DEXSeq results.
Dexseq Dtu options9
Run DEXSeq differential transcript usage workflow.
Abundance estimates used to generate the estimated counts.
Ignore transcript version numbers when matching Salmon quantification output to tx2gene file.
Minimal feature expression.
Minimal proportion for feature expression. This value should be between 0 and 1.
Minimal gene expression.
Minimal number of samples where features should be expressed.
Minimal number of samples where features should have at least the proportion --min_feature_prop.
Minimal number of samples where genes should be expressed.
Edger Deu options3
Run edgeR workflow.
Plot the N most significant genes from the edgeR results.
Save plots of the per gene edgeR results.
Generic options4
Display the help message.
Display the full detailed help message.
Custom MultiQC yaml file containing HTML including a methods description.
Display hidden parameters in the help message (only works when --help or --help_full are provided).
Input Output options7
Path to comma-separated file containing information about the contrasts in the experiment.
Email address for completion summary.
Path to comma-separated file containing information about the samples in the experiment.
MultiQC report title. Printed as page header, used for filename if not otherwise specified.
The output directory where the results will be saved. You have to use absolute paths to storage on Cloud infrastructure.
Save FastQ files after merging re-sequenced libraries in the results directory.
Source of input files.
Isoformswitchanalyzer3
Run the IsoformSwitchAnalyzeR workflow.
Alpha value for differential isoform expression.
dIF cutoff value for differential isoform expression.
Leafcutter options1
Run the LeafCutter workflow.
Miso6
Sashimi figure height (inches).
Sashimi figure width (inches).
List containing identifiers of genes to plot.
New-line separate file containing identifiers of genes to plot.
Read length used to calculate coverage.
Create sashimi plots using MISO.
Qc options2
Skip bigWig file creation.
Skip FastQC.
Read Trimming options9
Instructs Trim Galore to remove bp from the 5' end of read 1 (or single-end reads).
Instructs Trim Galore to remove bp from the 5' end of read 2 (paired-end reads only).
Minimum number of trimmed reads below which samples are flagged in multiqc output.
Save the trimmed FastQ files in the results directory.
Skip TrimGalore! FastQC.
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.
Instructs Trim Galore to remove bp from the 3' end of read 1 AFTER adapter/quality trimming has been performed.
Instructs Trim Galore to remove bp from the 3' end of read 2 AFTER adapter/quality trimming has been performed.
Instructs Trim Galore to apply the --nextseq=X option, to trim based on quality after removing poly-G tails.
Reference Genome options11
Path to FASTA genome file.
Specify if your transcript FASTA file is in GENCODE format.
Name of iGenomes reference.
Path to GFF3 annotation file.
Path to GTF annotation file.
By default, the pipeline uses the gene_name field to obtain additional gene identifiers from the input GTF file when running Salmon.
Define the attribute type used to group features in the GTF file when running Salmon.
Path to directory or tar.gz archive for pre-built Salmon index.
If generated by the pipeline save the STAR index in the results directory.
Path to directory or tar.gz archive for pre-built STAR index.
Path to FASTA transcriptome file.
Rmats options7
Run rMATS workflow.
Maximum exon length.
Minimum Intron Length.
Detect splicing events that involve an unannotated splice site.
Use paired statistical model. It requires that the same individuals are measured in both conditions.
The length of each read.
The cutoff used in the null hypothesis test for differential splicing.
Suppa options30
Lower-bound for the absolute delta PSI value to cluster.
Maximum distance (between 0 and 1) to consider two events as members of the same cluster.
Cluster transcripts according to PSI values across conditions
Cluster events according to PSI values across conditions
Clustering method to use.
Distance metric.
Minimum number of events required per cluster.
Maximum distance in PSI space of an event to a cluster.
P-value threshold to consider an event significant from the dpsi file.
Family-wise error rate to use for the multiple test correction.
Number of points in the local area of the delta PSI - average TPM distribution.
Correct the p-values by gene.
Calculate differential splicing for differential transcript usage across multiple conditions with replicates
Calculate differential splicing for AS events across multiple conditions with replicates.
Lower-bound for the absolute delta PSI value to test for significance.
Use the median to calculate the Delta PSI, instead of the mean.
The method to use to calculate the significance.
Proportion of samples with nan values allowed per condition to calculate a delta PSI.
Indicates if replicates across conditions are paired.
Minimum expression (calculated as average TPM value within-replicates and between-conditions) to be included in the analysis.
Boundary type (only used for local AS events).
Space separated list of events to generate.
Defines the number of nucleotides to display in the output GTF.
Redefine genes by clustering together transcripts by genomic stranded overlap and sharing at least one exon.
Variability threshold.
Minimum total expression of the transcripts involved in the event.
Run SUPPA workflow.
Quantify isoform inclusion levels (PSIs) from multiple samples.
Quantify event inclusion levels (PSIs) from multiple samples.
Expression file containing the abundances of all transcripts (ideally in TPM units).
Outputs
Results land in --outdir; the files and directory layout depend on the selected workflow, parameters and publication settings. Run metadata is normally placed in <outdir>/pipeline_info/ (execution report, software versions), when those outputs are enabled. The pinned tree includes MultiQC support; a report is produced only when its workflow step runs. nfclaw run also writes <outdir>/provenance/ with the exact params file and the run log, <outdir>/provenance/logs/run.log — the whole launch, whose last line states the outcome (Nextflow's own log is <outdir>/.nextflow.log); unless --no-provenance it adds a run manifest (pinned version, commit and exact command), input/output SHA-256 checksums, and a replayable commands.sh.
The exact output files and directory layout for this release are documented upstream: github.com/nf-core/rnasplice/blob/2.0.0/docs/output.md
Check a run — in the foreground or the background — with nfclaw status <outdir>: success, still running, how it ended (with the error), or stopped without an outcome; exit 0 success, 3 running, 1 otherwise.