Merge pull request #1607 from nf-core/dev

Dev -> Master for 3.21.0

Author: pinin4fjords

.nf-core.yml

@@ -14,11 +14,11 @@ nf_core_version: 3.3.2
 repository_type: pipeline
 template:
   author: "Harshil Patel, Phil Ewels, Rickard Hammarén"
-  description: RNA sequencing analysis pipeline for gene/isoform quantification
-    and extensive quality control.
+  description: RNA sequencing analysis pipeline for gene/isoform quantification and
+    extensive quality control.
   force: false
   is_nfcore: true
   name: rnaseq
   org: nf-core
   outdir: .
-  version: 3.20.0
+  version: 3.21.0

CHANGELOG.md

@@ -3,7 +3,32 @@
 The format is based on [Keep a Changelog](https://keepachangelog.com/en/1.0.0/)
 and this project adheres to [Semantic Versioning](https://semver.org/spec/v2.0.0.html).
 
-## 3.20.0
+## [[3.21.0](https://github.com/nf-core/rnaseq/releases/tag/3.21.0)] - 2025-09-18
+
+### Credits
+
+Special thanks to the following for their contributions to the release:
+
+- [Edmund Miller](https://github.com/edmundmiller)
+- [Friederike Hanssen](https://github.com/friederikehanssen)
+- [Maxime Garcia](https://github.com/maxulysse)
+- [Jonathan Manning](https://github.com/pinin4fjords)
+
+### Enhancements & fixes
+
+- [PR #1597](https://github.com/nf-core/rnaseq/pull/1597) - Bump version after release 3.20.0
+- [PR #1603](https://github.com/nf-core/rnaseq/pull/1603) - Add bam input pathway
+- [PR #1604](https://github.com/nf-core/rnaseq/pull/1604) - Enable BAM input for RSEM
+- [PR #1605](https://github.com/nf-core/rnaseq/pull/1605) - Fix default for umi_discard_read to prevent validation errors in Platform
+- [PR #1606](https://github.com/nf-core/rnaseq/pull/1606) - Bump version to 3.21.0 ahead of release
+
+### Software dependencies
+
+| Dependency | Old version | New version |
+| ---------- | ----------- | ----------- |
+| `MultiQC`  | 1.30        | 1.31        |
+
+## [[3.20.0](https://github.com/nf-core/rnaseq/releases/tag/3.20.0)] - 2025-08-18
 
 ### Credits
 

README.md

@@ -20,7 +20,7 @@
 
 ## Introduction
 
-**nf-core/rnaseq** is a bioinformatics pipeline that can be used to analyse RNA sequencing data obtained from organisms with a reference genome and annotation. It takes a samplesheet and FASTQ files as input, performs quality control (QC), trimming and (pseudo-)alignment, and produces a gene expression matrix and extensive QC report.
+**nf-core/rnaseq** is a bioinformatics pipeline that can be used to analyse RNA sequencing data obtained from organisms with a reference genome and annotation. It takes a samplesheet with FASTQ files or pre-aligned BAM files as input, performs quality control (QC), trimming and (pseudo-)alignment, and produces a gene expression matrix and extensive QC report.
 
 ![nf-core/rnaseq metro map](docs/images/nf-core-rnaseq_metro_map_grey_animated.svg)
 
@@ -76,6 +76,8 @@ CONTROL_REP1,AEG588A1_S1_L004_R1_001.fastq.gz,AEG588A1_S1_L004_R2_001.fastq.gz,a
 
 Each row represents a fastq file (single-end) or a pair of fastq files (paired end). Rows with the same sample identifier are considered technical replicates and merged automatically. The strandedness refers to the library preparation and will be automatically inferred if set to `auto`.
 
+The pipeline supports a two-step reprocessing workflow using BAM files from previous runs. Run initially with `--save_align_intermeds` to generate a samplesheet with BAM paths, then reprocess using `--skip_alignment` for efficient downstream analysis without repeating expensive alignment steps. This feature is designed specifically for pipeline-generated BAMs.
+
 > [!WARNING]
 > Please provide pipeline parameters via the CLI or Nextflow `-params-file` option. Custom config files including those provided by the `-c` Nextflow option can be used to provide any configuration _**except for parameters**_; see [docs](https://nf-co.re/docs/usage/getting_started/configuration#custom-configuration-files).
 

assets/schema_input.json

@@ -32,6 +32,27 @@
                 "errorMessage": "Strandedness must be provided and be one of 'auto', 'forward', 'reverse' or 'unstranded'",
                 "enum": ["forward", "reverse", "unstranded", "auto"],
                 "meta": ["strandedness"]
+            },
+            "genome_bam": {
+                "type": "string",
+                "format": "file-path",
+                "exists": true,
+                "pattern": "^([\\S\\s]*\\/)?[^\\s\\/]+\\.(bam|BAM)$",
+                "errorMessage": "Genome BAM file cannot contain spaces and must have extension '.bam'"
+            },
+            "transcriptome_bam": {
+                "type": "string",
+                "format": "file-path",
+                "exists": true,
+                "pattern": "^([\\S\\s]*\\/)?[^\\s\\/]+\\.(bam|BAM)$",
+                "errorMessage": "Transcriptome BAM file cannot contain spaces and must have extension '.bam'"
+            },
+            "percent_mapped": {
+                "type": "number",
+                "minimum": 0,
+                "maximum": 100,
+                "errorMessage": "Percent mapped must be a number between 0 and 100",
+                "meta": "percent_mapped"
             }
         },
         "required": ["sample", "fastq_1", "strandedness"]

conf/test.config

@@ -35,7 +35,6 @@ params {
     bbsplit_fasta_list = 'https://raw.githubusercontent.com/nf-core/test-datasets/626c8fab639062eade4b10747e919341cbf9b41a/reference/bbsplit_fasta_list.txt'
     hisat2_index       = 'https://raw.githubusercontent.com/nf-core/test-datasets/626c8fab639062eade4b10747e919341cbf9b41a/reference/hisat2.tar.gz'
     salmon_index       = 'https://raw.githubusercontent.com/nf-core/test-datasets/626c8fab639062eade4b10747e919341cbf9b41a/reference/salmon.tar.gz'
-    rsem_index         = 'https://raw.githubusercontent.com/nf-core/test-datasets/626c8fab639062eade4b10747e919341cbf9b41a/reference/rsem.tar.gz'
 
     // Other parameters
     skip_bbsplit        = false

docs/output.md

@@ -10,6 +10,10 @@ nextflow run nf-core/rnaseq -profile test_full,<docker/singularity/institute>
 
 The directories listed below will be created in the results directory after the pipeline has finished. All paths are relative to the top-level results directory.
 
+:::tip
+Many of the BAM files produced by this pipeline can be reused as input for future runs with `--skip_alignment`. This is particularly useful for reprocessing data or running downstream analysis steps without repeating computationally expensive alignment. See the [usage documentation](https://nf-co.re/rnaseq/usage#bam-input-for-reprocessing-workflow) for details on using BAM files as input.
+:::
+
 ## Pipeline overview
 
 The pipeline is built using [Nextflow](https://www.nextflow.io/) and processes data using the following steps:
@@ -213,8 +217,8 @@ When `--remove_ribo_rna` is specified, the pipeline uses [SortMeRNA](https://git
 <summary>Output files</summary>
 
 - `star_salmon/`
-  - `*.Aligned.out.bam`: If `--save_align_intermeds` is specified the original BAM file containing read alignments to the reference genome will be placed in this directory.
-  - `*.Aligned.toTranscriptome.out.bam`: If `--save_align_intermeds` is specified the original BAM file containing read alignments to the transcriptome will be placed in this directory.
+  - `*.Aligned.out.bam`: If `--save_align_intermeds` is specified the original BAM file containing read alignments to the reference genome will be placed in this directory. These files can be reused as `genome_bam` input in future pipeline runs.
+  - `*.Aligned.toTranscriptome.out.bam`: If `--save_align_intermeds` is specified the original BAM file containing read alignments to the transcriptome will be placed in this directory. These files can be reused as `transcriptome_bam` input in future pipeline runs.
   - `salmon.merged.gene_counts.tsv`: Matrix of gene-level raw counts across all samples.
   - `salmon.merged.gene_tpm.tsv`: Matrix of gene-level TPM values across all samples.
   - `salmon.merged.gene.SummarizedExperiment.rds`: RDS object that can be loaded in R that contains a [SummarizedExperiment](https://bioconductor.org/packages/release/bioc/html/SummarizedExperiment.html) container with the abundance TPM (`tpm`), estimated counts (`counts`) and gene length (`length`), estimated library size-scaled counts (`counts_scaled`), estimated length-scaled counts (`counts_length_scaled`) in the assays slot for genes.
@@ -276,16 +280,16 @@ The STAR section of the MultiQC report shows a bar plot with alignment rates: go
   - `rsem.merged.transcript_tpm.tsv`: Matrix of isoform-level TPM values across all samples.
   - `*.genes.results`: RSEM gene-level quantification results for each sample.
   - `*.isoforms.results`: RSEM isoform-level quantification results for each sample.
-  - `*.STAR.genome.bam`: If `--save_align_intermeds` is specified the original BAM file containing read alignments to the reference genome will be placed in this directory.
-  - `*.transcript.bam`: If `--save_align_intermeds` is specified the original BAM file containing read alignments to the transcriptome will be placed in this directory.
+  - `*.STAR.genome.bam`: If `--save_align_intermeds` is specified the BAM file from STAR alignment containing read alignments to the reference genome will be placed in this directory. These files can be reused as `genome_bam` input in future pipeline runs.
+  - `*.transcript.bam`: If `--save_align_intermeds` is specified the BAM file from STAR alignment containing read alignments to the transcriptome will be placed in this directory. These files can be reused as `transcriptome_bam` input in future pipeline runs.
 - `star_rsem/<SAMPLE>.stat/`
   - `*.cnt`, `*.model`, `*.theta`: RSEM counts and statistics for each sample.
   - `star_rsem/log/`
   - `*.log`: STAR alignment report containing the mapping results summary.
 
 </details>
 
-[RSEM](https://github.com/deweylab/RSEM) is a software package for estimating gene and isoform expression levels from RNA-seq data. It has been widely touted as one of the most accurate quantification tools for RNA-seq analysis. RSEM wraps other popular tools to map the reads to the genome (i.e. STAR, Bowtie2, HISAT2; STAR is used in this pipeline) which are then subsequently filtered relative to a transcriptome before quantifying at the gene- and isoform-level. Other advantages of using RSEM are that it performs both the alignment and quantification in a single package and its ability to effectively use ambiguously-mapping reads.
+[RSEM](https://github.com/deweylab/RSEM) is a software package for estimating gene and isoform expression levels from RNA-seq data. It has been widely touted as one of the most accurate quantification tools for RNA-seq analysis. When using `--aligner star_rsem`, the pipeline first runs STAR alignment with RSEM-compatible parameters to generate genome and transcriptome BAM files, then RSEM quantifies expression using these pre-aligned BAMs via the `--alignments` mode. This approach ensures optimal compatibility while maintaining RSEM's ability to effectively use ambiguously-mapping reads.
 
 You can choose to align and quantify your data with RSEM by providing the `--aligner star_rsem` parameter.
 
@@ -299,7 +303,7 @@ You can choose to align and quantify your data with RSEM by providing the `--ali
 <summary>Output files</summary>
 
 - `hisat2/`
-  - `<SAMPLE>.bam`: If `--save_align_intermeds` is specified the original BAM file containing read alignments to the reference genome will be placed in this directory.
+  - `<SAMPLE>.bam`: If `--save_align_intermeds` is specified the original BAM file containing read alignments to the reference genome will be placed in this directory. These files can be reused as `genome_bam` input in future pipeline runs.
 - `hisat2/log/`
   - `*.log`: HISAT2 alignment report containing the mapping results summary.
 - `hisat2/unmapped/`
@@ -323,7 +327,7 @@ The pipeline has been written in a way where all the files generated downstream
 <summary>Output files</summary>
 
 - `<ALIGNER>/`
-  - `<SAMPLE>.sorted.bam`: If `--save_align_intermeds` is specified the original coordinate sorted BAM file containing read alignments will be placed in this directory.
+  - `<SAMPLE>.sorted.bam`: If `--save_align_intermeds` is specified the original coordinate sorted BAM file containing read alignments will be placed in this directory. These files can be reused as `genome_bam` input in future pipeline runs.
   - `<SAMPLE>.sorted.bam.bai`: If `--save_align_intermeds` is specified the BAI index file for the original coordinate sorted BAM file will be placed in this directory.
   - `<SAMPLE>.sorted.bam.csi`: If `--save_align_intermeds --bam_csi_index` is specified the CSI index file for the original coordinate sorted BAM file will be placed in this directory.
 - `<ALIGNER>/samtools_stats/`
@@ -864,6 +868,8 @@ A number of genome-specific files are generated by the pipeline because they are
   - Reports generated by the pipeline: `pipeline_report.html`, `pipeline_report.txt` and `software_versions.yml`. The `pipeline_report*` files will only be present if the `--email` / `--email_on_fail` parameter's are used when running the pipeline.
   - Reformatted samplesheet files used as input to the pipeline: `samplesheet.valid.csv`.
   - Parameters used by the pipeline run: `params.json`.
+- `samplesheets/`
+  - `samplesheet_with_bams.csv`: **Auto-generated samplesheet for BAM reprocessing** (only created when using `--save_align_intermeds`) containing all samples with BAM file paths. For samples processed from FASTQ, includes paths to newly generated BAMs; for samples that were BAM input, preserves the original input paths. This samplesheet can be used directly for future pipeline runs with `--skip_alignment`, enabling efficient reprocessing without re-alignment.
 
 </details>