diff --git a/CHANGELOG.md b/CHANGELOG.md
index d42fcdde3..d5c45983f 100644
--- a/CHANGELOG.md
+++ b/CHANGELOG.md
@@ -7,9 +7,14 @@ and this project adheres to [Semantic Versioning](https://semver.org/spec/v2.0.0
 
 ### Credits
 
+Special thanks to the following for their contributions to the release:
+
+- [Elad Herzog](https://github.com/EladH1)
+
 ### Enhancements and fixes
 
 - [PR #1608](https://github.com/nf-core/rnaseq/pull/1608) - Bump version after release 3.21.0
+- [PR #1617](https://github.com/nf-core/rnaseq/pull/1617) - Update bbmap/bbsplit module
 
 ## [[3.21.0](https://github.com/nf-core/rnaseq/releases/tag/3.21.0)] - 2025-09-18
 
diff --git a/modules.json b/modules.json
index 8d8669e9a..674b309c8 100644
--- a/modules.json
+++ b/modules.json
@@ -7,7 +7,7 @@
                 "nf-core": {
                     "bbmap/bbsplit": {
                         "branch": "master",
-                        "git_sha": "8864afe586537bf562eac7b83349c26207f3cb4d",
+                        "git_sha": "fa1477128e0ff24675c8f56d6c334fcc323025c4",
                         "installed_by": ["fastq_qc_trim_filter_setstrandedness", "modules"]
                     },
                     "bedtools/genomecov": {
diff --git a/modules/nf-core/bbmap/bbsplit/main.nf b/modules/nf-core/bbmap/bbsplit/main.nf
index da4e0a1b2..2deebbec7 100644
--- a/modules/nf-core/bbmap/bbsplit/main.nf
+++ b/modules/nf-core/bbmap/bbsplit/main.nf
@@ -10,13 +10,13 @@ process BBMAP_BBSPLIT {
 
     input:
     tuple val(meta), path(reads)
-    path  index
+    path  index, name: 'input_index'
     path  primary_ref
     tuple val(other_ref_names), path(other_ref_paths)
     val   only_build_index
 
     output:
-    path "bbsplit"                            , optional:true, emit: index
+    path "bbsplit_index"                      , optional:true, emit: index
     tuple val(meta), path('*primary*fastq.gz'), optional:true, emit: primary_fastq
     tuple val(meta), path('*fastq.gz')        , optional:true, emit: all_fastq
     tuple val(meta), path('*txt')             , optional:true, emit: stats
@@ -46,16 +46,17 @@ process BBMAP_BBSPLIT {
     def fastq_out=''
     def index_files=''
     def refstats_cmd=''
+    def use_index = index ? true : false
 
     if (only_build_index) {
         if (primary_ref && other_ref_names && other_ref_paths) {
-            index_files = 'ref_primary=' +primary_ref + ' ' + other_refs.join(' ') + ' path=bbsplit'
+            index_files = 'ref_primary=' +primary_ref + ' ' + other_refs.join(' ') + ' path=bbsplit_build'
         } else {
             log.error 'ERROR: Please specify as input a primary fasta file along with names and paths to non-primary fasta files.'
         }
     } else {
         if (index) {
-            index_files = "path=$index"
+            index_files = "path=index_writable"
         } else if (primary_ref && other_ref_names && other_ref_paths) {
             index_files = "ref_primary=${primary_ref} ${other_refs.join(' ')}"
         } else {
@@ -67,15 +68,18 @@ process BBMAP_BBSPLIT {
     }
     """
 
-    # When we stage in the index files the time stamps get disturbed, which
-    # bbsplit doesn't like. Fix the time stamps in its summaries. This needs to
-    # be done via Java to match what bbmap does
+    # If using a pre-built index, copy it to avoid modifying input files in place,
+    # then fix timestamps. When we stage in the index files the time stamps get
+    # disturbed, which bbsplit doesn't like. Fix the time stamps in its summaries.
+    # This needs to be done via Java to match what bbmap does.
+    if [ "$use_index" == "true" ]; then
+        cp -rL input_index index_writable
 
-    if [ $index ]; then
-        for summary_file in \$(find $index/ref/genome -name summary.txt); do
-            src=\$(grep '^source' "\$summary_file" | cut -f2- -d\$'\\t' | sed 's|.*/bbsplit|bbsplit|')
+        for summary_file in \$(find index_writable/ref/genome -name summary.txt); do
+            # Extract the path from summary.txt and update it to point to index_writable
+            src=\$(grep '^source' "\$summary_file" | cut -f2- -d\$'\\t' | sed 's|.*/ref/|index_writable/ref/|')
             mod=\$(echo "System.out.println(java.nio.file.Files.getLastModifiedTime(java.nio.file.Paths.get(\\"\$src\\")).toMillis());" | jshell -J-Djdk.lang.Process.launchMechanism=vfork -)
-            sed "s|^last modified.*|last modified\\t\$mod|" "\$summary_file" > \${summary_file}.tmp && mv \${summary_file}.tmp \${summary_file}
+            sed -e 's|bbsplit_index/ref|index_writable/ref|' -e "s|^last modified.*|last modified\\t\$mod|" "\$summary_file" > \${summary_file}.tmp && mv \${summary_file}.tmp \${summary_file}
         done
     fi
 
@@ -92,11 +96,15 @@ process BBMAP_BBSPLIT {
 
     # Summary files will have an absolute path that will make the index
     # impossible to use in other processes- we can fix that
+    if [ -d bbsplit_build/ref/genome ]; then
+        for summary_file in \$(find bbsplit_build/ref/genome -name summary.txt); do
+            src=\$(grep '^source' "\$summary_file" | cut -f2- -d\$'\\t' | sed 's|.*/bbsplit_build|bbsplit_index|')
+            sed "s|^source.*|source\\t\$src|" "\$summary_file" > \${summary_file}.tmp && mv \${summary_file}.tmp \${summary_file}
+        done
 
-    for summary_file in \$(find bbsplit/ref/genome -name summary.txt); do
-        src=\$(grep '^source' "\$summary_file" | cut -f2- -d\$'\\t' | sed 's|.*/bbsplit|bbsplit|')
-        sed "s|^source.*|source\\t\$src|" "\$summary_file" > \${summary_file}.tmp && mv \${summary_file}.tmp \${summary_file}
-    done
+        # Atomically rename the completed index
+        mv bbsplit_build bbsplit_index
+    fi
 
     cat <<-END_VERSIONS > versions.yml
     "${task.process}":
@@ -110,11 +118,14 @@ process BBMAP_BBSPLIT {
     other_ref_names.eachWithIndex { name, index ->
         other_refs += "echo '' | gzip > ${prefix}_${name}.fastq.gz"
     }
+    def will_build_index = only_build_index || (!index && primary_ref && other_ref_names && other_ref_paths)
     """
-    if [ ! -d bbsplit ]; then
-        mkdir bbsplit
+    # Create index directory if building an index (either only_build_index or on-the-fly)
+    if [ "${will_build_index}" == "true" ]; then
+        mkdir -p bbsplit_index
     fi
 
+    # Only create output files if splitting (not just building index)
     if ! (${only_build_index}); then
         echo '' | gzip >  ${prefix}_primary.fastq.gz
         ${other_refs}
diff --git a/modules/nf-core/bbmap/bbsplit/meta.yml b/modules/nf-core/bbmap/bbsplit/meta.yml
index 081aeb2b3..66f87cce0 100644
--- a/modules/nf-core/bbmap/bbsplit/meta.yml
+++ b/modules/nf-core/bbmap/bbsplit/meta.yml
@@ -25,31 +25,33 @@ input:
         description: |
           List of input FastQ files of size 1 and 2 for single-end and paired-end data,
           respectively.
-  - - index:
-        type: directory
-        description: Directory to place generated index
-        pattern: "*"
-  - - primary_ref:
-        type: file
-        description: Path to the primary reference
-        pattern: "*"
+        ontologies: []
+  - index:
+      type: directory
+      description: Directory to place generated index
+      pattern: "*"
+  - primary_ref:
+      type: file
+      description: Path to the primary reference
+      pattern: "*"
+      ontologies: []
   - - other_ref_names:
         type: list
         description: List of other reference ids apart from the primary
     - other_ref_paths:
         type: list
         description: Path to other references paths corresponding to "other_ref_names"
-  - - only_build_index:
-        type: string
-        description: true = only build index; false = mapping
+  - only_build_index:
+      type: string
+      description: true = only build index; false = mapping
 output:
-  - index:
-      - bbsplit:
-          type: directory
-          description: Directory with index files
-          pattern: "bbsplit"
-  - primary_fastq:
-      - meta:
+  index:
+    - bbsplit_index:
+        type: directory
+        description: Directory with index files
+        pattern: "bbsplit_index"
+  primary_fastq:
+    - - meta:
           type: map
           description: |
             Groovy Map containing sample information
@@ -58,8 +60,10 @@ output:
           type: file
           description: Output reads that map to the primary reference
           pattern: "*primary*fastq.gz"
-  - all_fastq:
-      - meta:
+          ontologies:
+            - edam: http://edamontology.org/format_3989 # GZIP format
+  all_fastq:
+    - - meta:
           type: map
           description: |
             Groovy Map containing sample information
@@ -68,8 +72,10 @@ output:
           type: file
           description: All reads mapping to any of the references
           pattern: "*fastq.gz"
-  - stats:
-      - meta:
+          ontologies:
+            - edam: http://edamontology.org/format_3989 # GZIP format
+  stats:
+    - - meta:
           type: map
           description: |
             Groovy Map containing sample information
@@ -78,8 +84,9 @@ output:
           type: file
           description: Tab-delimited text file containing mapping statistics
           pattern: "*.txt"
-  - log:
-      - meta:
+          ontologies: []
+  log:
+    - - meta:
           type: map
           description: |
             Groovy Map containing sample information
@@ -88,14 +95,19 @@ output:
           type: file
           description: Log file
           pattern: "*.log"
-  - versions:
-      - versions.yml:
-          type: file
-          description: File containing software versions
-          pattern: "versions.yml"
+          ontologies: []
+  versions:
+    - versions.yml:
+        type: file
+        description: File containing software versions
+        pattern: "versions.yml"
+        ontologies:
+          - edam: http://edamontology.org/format_3750 # YAML
 authors:
   - "@joseespinosa"
   - "@drpatelh"
+  - "@pinin4fjords"
 maintainers:
   - "@joseespinosa"
   - "@drpatelh"
+  - "@pinin4fjords"
diff --git a/modules/nf-core/bbmap/bbsplit/tests/main.nf.test b/modules/nf-core/bbmap/bbsplit/tests/main.nf.test
index 0674d247f..2f942f39c 100644
--- a/modules/nf-core/bbmap/bbsplit/tests/main.nf.test
+++ b/modules/nf-core/bbmap/bbsplit/tests/main.nf.test
@@ -3,8 +3,12 @@ nextflow_process {
     name "Test Process BBMAP_BBSPLIT"
     script "../main.nf"
     process "BBMAP_BBSPLIT"
+    tag "modules"
+    tag "modules_nfcore"
+    tag "bbmap"
+    tag "bbmap/bbsplit"
 
-    test("sarscov2_se_fastq_fasta_chr22_fasta - index") {
+    test("sarscov2_se_fastq_fasta_chr22_fasta - build index") {
 
         when {
             process {
@@ -31,7 +35,7 @@ nextflow_process {
         }
     }
 
-    test("sarscov2_se_fastq_fasta_chr22_fasta - index - stub") {
+    test("sarscov2_se_fastq_fasta_chr22_fasta - build index - stub") {
 
         options "-stub"
 
@@ -52,13 +56,12 @@ nextflow_process {
 
         then {
             assertAll(
-                { assert process.success },
-                { assert snapshot(process.out).match() }
+                { assert process.success }
             )
         }
     }
 
-    test("sarscov2_se_fastq_fasta_chr22_fasta") {
+    test("sarscov2_se_fastq_fasta_chr22_fasta - split with prebuilt index") {
 
         setup {
 
@@ -87,58 +90,27 @@ nextflow_process {
                         file(params.modules_testdata_base_path + 'genomics/sarscov2/illumina/fastq/test_1.fastq.gz', checkIfExists: true)
                     ])
                     input[1] = BBMAP_BBSPLIT_INDEX.out.index
-                    input[2] = Channel.of(file(params.modules_testdata_base_path + 'genomics/sarscov2/genome/genome.fasta', checkIfExists: true))
-                    input[3] = Channel.of([
-                        [ 'human' ], // meta map
-                        file(params.modules_testdata_base_path + 'genomics/homo_sapiens/genome/chr22/sequence/chr22_23800000-23980000.fa', checkIfExists: true)
-                    ])
+                    input[2] = []
+                    input[3] = [[], []]
                     input[4] = false
                     """
                 }
         }
 
         then {
-            def filesToExclude = [
-                "info.txt",
-                "reflist.txt",
-                "scaffolds.txt.gz",
-                "summary.txt"
-            ]
-
-            def outputFiles = []
-            def outputDirectory = new File(process.out.index[0])
-            outputDirectory.eachFileRecurse { file ->
-                if (file.isFile()){
-                    outputFiles << file
-                    }
-            }
-
-            def filesExist = filesToExclude.every { excludeName ->
-                outputFiles.any { file ->
-                    file.getName().endsWith(excludeName) && file.exists()
-                }
-            }
-
-            def filteredFiles = outputFiles
-                .findAll { file ->
-                    !filesToExclude.any { excludeName ->
-                        file.getName().endsWith(excludeName)
-                    }
-                }.sort{ it.getName()}
-
             assertAll(
                 { assert process.success },
                 { assert path(process.out.log[0][1]).text.contains("If you wish to regenerate the index") },
-                { assert filesExist : "One or more files to exclude do not exist" },
                 { assert snapshot(
-                    filteredFiles,
+                    process.out.primary_fastq,
+                    process.out.stats,
                     process.out.versions
                 ).match()}
             )
         }
     }
 
-    test("sarscov2_se_fastq_fasta_chr22_fasta - stub") {
+    test("sarscov2_se_fastq_fasta_chr22_fasta - split with prebuilt index - stub") {
 
         options "-stub"
 
@@ -169,11 +141,8 @@ nextflow_process {
                         file(params.modules_testdata_base_path + 'genomics/sarscov2/illumina/fastq/test_1.fastq.gz', checkIfExists: true)
                     ])
                     input[1] = BBMAP_BBSPLIT_INDEX.out.index
-                    input[2] = Channel.of(file(params.modules_testdata_base_path + 'genomics/sarscov2/genome/genome.fasta', checkIfExists: true))
-                    input[3] = Channel.of([
-                        [ 'human' ], // meta map
-                        file(params.modules_testdata_base_path + 'genomics/homo_sapiens/genome/chr22/sequence/chr22_23800000-23980000.fa', checkIfExists: true)
-                    ])
+                    input[2] = []
+                    input[3] = [[], []]
                     input[4] = false
                 """
             }
@@ -181,8 +150,7 @@ nextflow_process {
 
         then {
             assertAll(
-                { assert process.success },
-                { assert snapshot(process.out).match() }
+                { assert process.success }
             )
         }
     }
diff --git a/modules/nf-core/bbmap/bbsplit/tests/main.nf.test.snap b/modules/nf-core/bbmap/bbsplit/tests/main.nf.test.snap
index 656d3ed05..7e739a663 100644
--- a/modules/nf-core/bbmap/bbsplit/tests/main.nf.test.snap
+++ b/modules/nf-core/bbmap/bbsplit/tests/main.nf.test.snap
@@ -1,12 +1,23 @@
 {
-    "sarscov2_se_fastq_fasta_chr22_fasta": {
+    "sarscov2_se_fastq_fasta_chr22_fasta - split with prebuilt index": {
         "content": [
             [
-                "chr1.chrom.gz:md5,8fec4c63ec642613ad10adf4cc2e6ade",
-                "chr1_index_k13_c13_b1.block:md5,385913c1e84b77dc7bf36288ee1c8706",
-                "chr1_index_k13_c13_b1.block2.gz:md5,2556b45206835a0ff7078d683b5fd6e2",
-                "merged_ref_9222711925172838098.fa.gz:md5,983cef447fb28394b88a5b49b3579f0c",
-                "namelist.txt:md5,45e7a4cdc7a11a39ada56844ca3a1e30"
+                [
+                    {
+                        "id": "test",
+                        "single_end": true
+                    },
+                    "test_primary.fastq.gz:md5,4161df271f9bfcd25d5845a1e220dbec"
+                ]
+            ],
+            [
+                [
+                    {
+                        "id": "test",
+                        "single_end": true
+                    },
+                    "test.stats.txt:md5,2cbf69b72e5f4f8508306b54e8fe2861"
+                ]
             ],
             [
                 "versions.yml:md5,b75d21d7649b5a512e7b54dd15cd3eba"
@@ -14,11 +25,11 @@
         ],
         "meta": {
             "nf-test": "0.9.2",
-            "nextflow": "24.10.5"
+            "nextflow": "25.10.0"
         },
-        "timestamp": "2025-03-12T18:57:32.958846"
+        "timestamp": "2025-11-07T12:36:23.511852"
     },
-    "sarscov2_se_fastq_fasta_chr22_fasta - index": {
+    "sarscov2_se_fastq_fasta_chr22_fasta - build index": {
         "content": [
             [
                 "versions.yml:md5,b75d21d7649b5a512e7b54dd15cd3eba"
@@ -26,174 +37,8 @@
         ],
         "meta": {
             "nf-test": "0.9.2",
-            "nextflow": "24.10.5"
-        },
-        "timestamp": "2025-03-12T18:57:12.155636"
-    },
-    "sarscov2_se_fastq_fasta_chr22_fasta - index - stub": {
-        "content": [
-            {
-                "0": [
-                    [
-                        
-                    ]
-                ],
-                "1": [
-                    
-                ],
-                "2": [
-                    
-                ],
-                "3": [
-                    
-                ],
-                "4": [
-                    [
-                        {
-                            
-                        },
-                        "null.log:md5,d41d8cd98f00b204e9800998ecf8427e"
-                    ]
-                ],
-                "5": [
-                    "versions.yml:md5,b75d21d7649b5a512e7b54dd15cd3eba"
-                ],
-                "all_fastq": [
-                    
-                ],
-                "index": [
-                    [
-                        
-                    ]
-                ],
-                "log": [
-                    [
-                        {
-                            
-                        },
-                        "null.log:md5,d41d8cd98f00b204e9800998ecf8427e"
-                    ]
-                ],
-                "primary_fastq": [
-                    
-                ],
-                "stats": [
-                    
-                ],
-                "versions": [
-                    "versions.yml:md5,b75d21d7649b5a512e7b54dd15cd3eba"
-                ]
-            }
-        ],
-        "meta": {
-            "nf-test": "0.9.2",
-            "nextflow": "24.10.5"
-        },
-        "timestamp": "2025-03-12T18:57:17.177351"
-    },
-    "sarscov2_se_fastq_fasta_chr22_fasta - stub": {
-        "content": [
-            {
-                "0": [
-                    [
-                        
-                    ]
-                ],
-                "1": [
-                    [
-                        {
-                            "id": "test",
-                            "single_end": true
-                        },
-                        "test_primary.fastq.gz:md5,68b329da9893e34099c7d8ad5cb9c940"
-                    ]
-                ],
-                "2": [
-                    [
-                        {
-                            "id": "test",
-                            "single_end": true
-                        },
-                        [
-                            "test_human.fastq.gz:md5,68b329da9893e34099c7d8ad5cb9c940",
-                            "test_primary.fastq.gz:md5,68b329da9893e34099c7d8ad5cb9c940"
-                        ]
-                    ]
-                ],
-                "3": [
-                    [
-                        {
-                            "id": "test",
-                            "single_end": true
-                        },
-                        "test.stats.txt:md5,d41d8cd98f00b204e9800998ecf8427e"
-                    ]
-                ],
-                "4": [
-                    [
-                        {
-                            "id": "test",
-                            "single_end": true
-                        },
-                        "test.log:md5,d41d8cd98f00b204e9800998ecf8427e"
-                    ]
-                ],
-                "5": [
-                    "versions.yml:md5,b75d21d7649b5a512e7b54dd15cd3eba"
-                ],
-                "all_fastq": [
-                    [
-                        {
-                            "id": "test",
-                            "single_end": true
-                        },
-                        [
-                            "test_human.fastq.gz:md5,68b329da9893e34099c7d8ad5cb9c940",
-                            "test_primary.fastq.gz:md5,68b329da9893e34099c7d8ad5cb9c940"
-                        ]
-                    ]
-                ],
-                "index": [
-                    [
-                        
-                    ]
-                ],
-                "log": [
-                    [
-                        {
-                            "id": "test",
-                            "single_end": true
-                        },
-                        "test.log:md5,d41d8cd98f00b204e9800998ecf8427e"
-                    ]
-                ],
-                "primary_fastq": [
-                    [
-                        {
-                            "id": "test",
-                            "single_end": true
-                        },
-                        "test_primary.fastq.gz:md5,68b329da9893e34099c7d8ad5cb9c940"
-                    ]
-                ],
-                "stats": [
-                    [
-                        {
-                            "id": "test",
-                            "single_end": true
-                        },
-                        "test.stats.txt:md5,d41d8cd98f00b204e9800998ecf8427e"
-                    ]
-                ],
-                "versions": [
-                    "versions.yml:md5,b75d21d7649b5a512e7b54dd15cd3eba"
-                ]
-            }
-        ],
-        "meta": {
-            "nf-test": "0.9.2",
-            "nextflow": "24.10.5"
+            "nextflow": "25.10.0"
         },
-        "timestamp": "2025-03-12T18:57:42.233549"
+        "timestamp": "2025-11-07T11:05:45.517832"
     }
 }
\ No newline at end of file