nf-core · emmanuel-tan · Jul 21, 2026 · Jul 24, 2026 · Aug 1, 2026 · Aug 1, 2026
diff --git a/main.nf b/main.nf
@@ -103,6 +103,7 @@ workflow NFCORE_RNASEQ {
         params.bowtie2_index,
         params.bbsplit_index,
         params.sortmerna_index,
+        params.bowtie2_rrna_index,
         params.aligner,
         params.pseudo_aligner,
         params.skip_bbsplit,
@@ -126,11 +127,6 @@ workflow NFCORE_RNASEQ {
     // WORKFLOW: Run nf-core/rnaseq workflow
     //
     ch_samplesheet = channel.value(file(params.input, checkIfExists: true))
-
-    // Bowtie2 rRNA index is built on-demand inside the fastq_remove_rrna subworkflow
-    // rather than in PREPARE_GENOME_INDICES, to avoid duplicating the rRNA FASTA preparation logic
-    ch_bowtie2_rrna_index = channel.empty()
-
     def qc_tools = defineQcTools(params)
 
     RNASEQ (
@@ -149,7 +145,7 @@ workflow NFCORE_RNASEQ {
         PREPARE_GENOME_INDICES.out.bbsplit_index,
         PREPARE_GENOME_REFERENCES.out.rrna_fastas,
         PREPARE_GENOME_INDICES.out.sortmerna_index,
-        ch_bowtie2_rrna_index,
+        PREPARE_GENOME_INDICES.out.bowtie2_rrna_index,
         PREPARE_GENOME_INDICES.out.splicesites,
         PREPARE_GENOME_REFERENCES.out.kraken_db,
         qc_tools

diff --git a/nextflow_schema.json b/nextflow_schema.json
@@ -330,6 +330,14 @@
                     "help_text": "Choose between 'sortmerna' (default), 'bowtie2', or 'ribodetector' for rRNA removal.\n\n- **sortmerna**: Reference-based filtering (default). Requires `--ribo_database_manifest`. Note: Default databases include SILVA sequences requiring licensing for commercial use.\n- **bowtie2**: Alignment-based filtering. Requires `--ribo_database_manifest`. Recommended for users with SILVA licensing concerns as it supports custom databases.\n- **ribodetector**: ML-based detection without a reference requirement. Useful for organisms lacking well-characterized rRNA sequences or to avoid database licensing requirements.",
                     "enum": ["sortmerna", "ribodetector", "bowtie2"]
                 },
+                "bowtie2_rrna_index": {
+                    "type": "string",
+                    "format": "path",
+                    "exists": true,
+                    "fa_icon": "fas fa-bezier-curve",
+                    "description": "Path to directory or tar.gz archive for pre-built Bowtie2 index for rRNA removal.",
+                    "help_text": "Optional when using `--remove_ribo_rna` and `--ribo_removal_tool` is set to `bowtie2`. If not provided, the index will be built using the database provided with `--ribo_database_manifest`"
+                },
                 "use_gpu_ribodetector": {
                     "type": "boolean",
                     "description": "Enable GPU acceleration for ribodetector.",

diff --git a/subworkflows/local/prepare_genome_indices/main.nf b/subworkflows/local/prepare_genome_indices/main.nf
@@ -4,6 +4,7 @@
 
 include { UNTAR as UNTAR_BBSPLIT_INDEX      } from '../../../modules/nf-core/untar'
 include { UNTAR as UNTAR_SORTMERNA_INDEX    } from '../../../modules/nf-core/untar'
+include { UNTAR as UNTAR_BOWTIE2_RRNA_INDEX } from '../../../modules/nf-core/untar'
 include { UNTAR as UNTAR_STAR_INDEX         } from '../../../modules/nf-core/untar'
 include { UNTAR as UNTAR_RSEM_INDEX         } from '../../../modules/nf-core/untar'
 include { UNTAR as UNTAR_HISAT2_INDEX       } from '../../../modules/nf-core/untar'
@@ -43,6 +44,7 @@ workflow PREPARE_GENOME_INDICES {
     bowtie2_index            // directory: /path/to/bowtie2/index/
     bbsplit_index            // directory: /path/to/bbsplit/index/
     sortmerna_index          // directory: /path/to/sortmerna/index/
+    bowtie2_rrna_index       // directory: /path/to/bowtie2/index/
     aligner                  // string: Specifies the alignment algorithm to use - available options are 'star_salmon', 'star_rsem', 'hisat2', and 'bowtie2_salmon'
     pseudo_aligner           // string: Specifies the pseudo aligner to use - available options are 'salmon'. Runs in addition to '--aligner'
     skip_bbsplit             // boolean: Skip BBSplit for removal of non-reference genome reads
@@ -62,6 +64,7 @@ workflow PREPARE_GENOME_INDICES {
     def prepare_tool_indices = []
     if (!skip_bbsplit)                                           { prepare_tool_indices << 'bbsplit' }
     if (ribo_removal_tool == 'sortmerna')                        { prepare_tool_indices << 'sortmerna' }
+    if (ribo_removal_tool == 'bowtie2' && bowtie2_rrna_index)    { prepare_tool_indices << 'bowtie2_rrna' } // If no index is provided, this subworkflow does not need to build an index as that is handled by the fastq_remove_rrna subworkflow. 
     if ((!skip_alignment && aligner) || aligner == 'star_rsem')  { prepare_tool_indices << aligner }
     if (!skip_pseudo_alignment && pseudo_aligner)                { prepare_tool_indices << pseudo_aligner }
 
@@ -124,6 +127,19 @@ workflow PREPARE_GENOME_INDICES {
         }
     }
 
+    //-------------------------------------------------------------
+    // 3b) Bowtie2 rRNA index - only handles untar
+    //-------------------------------------------------------------
+    ch_bowtie2_rrna_index = channel.empty()
+    if ('bowtie2_rrna' in prepare_tool_indices) {
+        if (bowtie2_rrna_index.endsWith('.tar.gz')) {
+            ch_bowtie2_rrna_index = UNTAR_BOWTIE2_RRNA_INDEX ([ [:], file(bowtie2_rrna_index, checkIfExists: true) ]).untar.first()
+        } else {
+            ch_bowtie2_rrna_index = channel.value([ [:], file(bowtie2_rrna_index, checkIfExists: true) ])
+        }
+        // No need to build as that is handled in the fastq_remove_rrna subworkflow from nf-core  
+    }
+
     //----------------------------------------------------
     // 4) STAR index (e.g. for 'star_salmon') -> needs FASTA if built
     //----------------------------------------------------
@@ -267,13 +283,14 @@ workflow PREPARE_GENOME_INDICES {
     }
 
     emit:
-    splicesites      = ch_splicesites            // channel: path(genome.splicesites.txt)
-    bbsplit_index    = ch_bbsplit_index          // channel: path(bbsplit/index/)
-    sortmerna_index  = ch_sortmerna_index        // channel: path(sortmerna/index/)
-    star_index       = ch_star_index             // channel: path(star/index/)
-    rsem_index       = ch_rsem_index             // channel: path(rsem/index/)
-    hisat2_index     = ch_hisat2_index           // channel: path(hisat2/index/)
-    bowtie2_index    = ch_bowtie2_index          // channel: path(bowtie2/index/)
-    salmon_index     = ch_salmon_index           // channel: path(salmon/index/)
-    kallisto_index   = ch_kallisto_index         // channel: [ meta, path(kallisto/index/) ]
+    splicesites         = ch_splicesites            // channel: path(genome.splicesites.txt)
+    bbsplit_index       = ch_bbsplit_index          // channel: path(bbsplit/index/)
+    sortmerna_index     = ch_sortmerna_index        // channel: path(sortmerna/index/)
+    bowtie2_rrna_index  = ch_bowtie2_rrna_index     // channel: path(bowtie2/index/)
+    star_index          = ch_star_index             // channel: path(star/index/)
+    rsem_index          = ch_rsem_index             // channel: path(rsem/index/)
+    hisat2_index        = ch_hisat2_index           // channel: path(hisat2/index/)
+    bowtie2_index       = ch_bowtie2_index          // channel: path(bowtie2/index/)
+    salmon_index        = ch_salmon_index           // channel: path(salmon/index/)
+    kallisto_index      = ch_kallisto_index         // channel: [ meta, path(kallisto/index/) ]
 }
diff --git a/tests/remove_ribo_rna.nf.test b/tests/remove_ribo_rna.nf.test
@@ -179,6 +179,91 @@ nextflow_pipeline {
             )
         }
 
+        cleanup {
+            // Only prune/delete on CI - keeping local VM state intact prevents
+            // cross-test launchDir wipes that break nf-test snapshot walks.
+            if (System.getenv("CI") == "true") {
+                if (System.getenv("NFT_PROFILE")?.contains("docker")) {
+                    "docker system prune -a -f --volumes".execute()
+                }
+                new File("${launchDir}").deleteDir()
+            }
+        }
+    }
+    test("Params: --remove_ribo_rna --ribo_removal_tool bowtie2 --bowtie2_rrna_index") {
+
+        when {
+            params {
+                outdir             = "$outputDir"
+                remove_ribo_rna    = true
+                ribo_removal_tool  = 'bowtie2'
+                bowtie2_rrna_index = '' // Pending PR of test dataset on nf-core/test-datasets:rnaseq
+                skip_qc        = true
+                skip_stringtie = true
+                skip_bigwig    = true
+            }
+        }
+
+        then {
+            def stable_name = getAllFilesFromDir(params.outdir, relative: true, includeDir: true, ignore: ['pipeline_info/*.{html,json,txt}', 'seqkit', 'seqkit/**'])
+            def stable_path = getAllFilesFromDir(params.outdir, ignoreFile: 'tests/.nftignore')
+            assertAll(
+                { assert workflow.success},
+                { assert snapshot(
+                    // Number of tasks should be lower than the standard bowtie2 test because the index build step is skipped
+                    workflow.trace.succeeded().size(),
+                    removeFromYamlMap("$outputDir/pipeline_info/nf_core_rnaseq_software_mqc_versions.yml", "Workflow").findAll { it.key != "SEQKIT_REPLACE" && it.key != "SEQKIT_REPLACE_U2T" },
+                    stable_name,
+                    stable_path
+                ).match() }
+            )
+        }
+
+        cleanup {
+            if (System.getenv("CI") == "true") {
+                if (System.getenv("NFT_PROFILE")?.contains("docker")) {
+                    "docker system prune -a -f --volumes".execute()
+                }
+                new File("${launchDir}").deleteDir()
+            }
+        }
+    }
+    test("Params: --remove_ribo_rna --ribo_removal_tool bowtie2 --bowtie2_rrna_index - stub") {
+
+        options "-stub"
+
+        when {
+            params {
+                outdir             = "$outputDir"
+                remove_ribo_rna    = true
+                ribo_removal_tool  = 'bowtie2'
+                bowtie2_rrna_index = '' // Pending PR of test dataset on nf-core/test-datasets:rnaseq
+                skip_qc        = true
+                skip_stringtie = true
+                skip_bigwig    = true
+            }
+        }
+
+        then {
+            // stable_name: All files + folders in ${params.outdir}/ with a stable name
+            def stable_name = getAllFilesFromDir(params.outdir, relative: true, includeDir: true, ignore: ['pipeline_info/*.{html,json,txt}', 'seqkit', 'seqkit/**'])
+            // stable_path: All files in ${params.outdir}/ with stable content
+            def stable_path = getAllFilesFromDir(params.outdir, ignoreFile: 'tests/.nftignore')
+            assertAll(
+                { assert workflow.success},
+                { assert snapshot(
+                    // Number of successful tasks
+                    workflow.trace.succeeded().size(),
+                    // pipeline versions.yml file for multiqc from which Nextflow and pipeline versions are removed (all from the workflow key)
+                    removeFromYamlMap("$outputDir/pipeline_info/nf_core_rnaseq_software_mqc_versions.yml", "Workflow").findAll { it.key != "SEQKIT_REPLACE" && it.key != "SEQKIT_REPLACE_U2T" },
+                    // All stable path name, with a relative path
+                    stable_name,
+                    // All files with stable contents
+                    stable_path
+                ).match() }
+            )
+        }
+
         cleanup {
             // Only prune/delete on CI - keeping local VM state intact prevents
             // cross-test launchDir wipes that break nf-test snapshot walks.

diff --git a/workflows/rnaseq/main.nf b/workflows/rnaseq/main.nf
@@ -203,7 +203,7 @@ workflow RNASEQ {
 
     // Determine if we need to build rRNA removal indexes
     def make_sortmerna_index = !params.sortmerna_index && params.remove_ribo_rna && params.ribo_removal_tool == 'sortmerna'
-    def make_bowtie2_index   = params.remove_ribo_rna && params.ribo_removal_tool == 'bowtie2'
+    def make_bowtie2_index   = !params.bowtie2_rrna_index && params.remove_ribo_rna && params.ribo_removal_tool == 'bowtie2'
 
     FASTQ_QC_TRIM_FILTER_SETSTRANDEDNESS (
         ch_fastq,                                   // ch_reads