From 0fe30831abbc2ed115e46e92330edf38f56edc3d Mon Sep 17 00:00:00 2001
From: Matthias De Smet <11850640+matthdsm@users.noreply.github.com>
Date: Tue, 19 Mar 2024 13:01:20 +0100
Subject: [PATCH] add cram support to bowtie2 (#5180)

* add cram support to bowtie2

* fix tests

* fastq_align_bowtie2 > nf-test

* fix tests

* more robust test

* fix cram tests
---
 modules/nf-core/bowtie2/align/main.nf         |  26 ++-
 .../bowtie2/align/tests/cram_crai.config      |   5 +
 .../nf-core/bowtie2/align/tests/main.nf.test  | 162 +++++++++----
 .../bowtie2/align/tests/main.nf.test.snap     | 216 +++++++++++-------
 .../nf-core/fastq_align_bowtie2/main.nf       |   6 +-
 .../fastq_align_bowtie2/tests/main.nf.test    |  99 ++++++++
 .../tests/main.nf.test.snap                   | 126 ++++++++++
 .../tests}/nextflow.config                    |   9 -
 .../fastq_align_bowtie2/tests/tags.yml        |   2 +
 subworkflows/nf-core/fastq_align_dna/main.nf  |  12 +-
 tests/config/pytest_modules.yml               |   3 -
 .../nf-core/fastq_align_bowtie2/main.nf       |  44 ----
 .../nf-core/fastq_align_bowtie2/test.yml      |  63 -----
 13 files changed, 507 insertions(+), 266 deletions(-)
 create mode 100644 modules/nf-core/bowtie2/align/tests/cram_crai.config
 create mode 100644 subworkflows/nf-core/fastq_align_bowtie2/tests/main.nf.test
 create mode 100644 subworkflows/nf-core/fastq_align_bowtie2/tests/main.nf.test.snap
 rename {tests/subworkflows/nf-core/fastq_align_bowtie2 => subworkflows/nf-core/fastq_align_bowtie2/tests}/nextflow.config (56%)
 create mode 100644 subworkflows/nf-core/fastq_align_bowtie2/tests/tags.yml
 delete mode 100644 tests/subworkflows/nf-core/fastq_align_bowtie2/main.nf
 delete mode 100644 tests/subworkflows/nf-core/fastq_align_bowtie2/test.yml

diff --git a/modules/nf-core/bowtie2/align/main.nf b/modules/nf-core/bowtie2/align/main.nf
index 8c405ee386c..96a7027ddbe 100644
--- a/modules/nf-core/bowtie2/align/main.nf
+++ b/modules/nf-core/bowtie2/align/main.nf
@@ -10,13 +10,18 @@ process BOWTIE2_ALIGN {
     input:
     tuple val(meta) , path(reads)
     tuple val(meta2), path(index)
+    tuple val(meta3), path(fasta)
     val   save_unaligned
     val   sort_bam
 
     output:
-    tuple val(meta), path("*.{bam,sam}"), emit: aligned
+    tuple val(meta), path("*.sam")      , emit: sam     , optional:true
+    tuple val(meta), path("*.bam")      , emit: bam     , optional:true
+    tuple val(meta), path("*.cram")     , emit: cram    , optional:true
+    tuple val(meta), path("*.csi")      , emit: csi     , optional:true
+    tuple val(meta), path("*.crai")     , emit: crai    , optional:true
     tuple val(meta), path("*.log")      , emit: log
-    tuple val(meta), path("*fastq.gz")  , emit: fastq, optional:true
+    tuple val(meta), path("*fastq.gz")  , emit: fastq   , optional:true
     path  "versions.yml"                , emit: versions
 
     when:
@@ -39,7 +44,10 @@ process BOWTIE2_ALIGN {
 
     def samtools_command = sort_bam ? 'sort' : 'view'
     def extension_pattern = /(--output-fmt|-O)+\s+(\S+)/
-    def extension = (args2 ==~ extension_pattern) ? (args2 =~ extension_pattern)[0][2].toLowerCase() : "bam"
+    def extension_matcher =  (args2 =~ extension_pattern)
+    def extension = extension_matcher.getCount() > 0 ? extension_matcher[0][2].toLowerCase() : "bam"
+    def reference = fasta && extension=="cram"  ? "--reference ${fasta}" : ""
+    if (!fasta && extension=="cram") error "Fasta reference is required for CRAM output"
 
     """
     INDEX=`find -L ./ -name "*.rev.1.bt2" | sed "s/\\.rev.1.bt2\$//"`
@@ -53,7 +61,7 @@ process BOWTIE2_ALIGN {
         $unaligned \\
         $args \\
         2> >(tee ${prefix}.bowtie2.log >&2) \\
-        | samtools $samtools_command $args2 --threads $task.cpus -o ${prefix}.${extension} -
+        | samtools $samtools_command $args2 --threads $task.cpus ${reference} -o ${prefix}.${extension} -
 
     if [ -f ${prefix}.unmapped.fastq.1.gz ]; then
         mv ${prefix}.unmapped.fastq.1.gz ${prefix}.unmapped_1.fastq.gz
@@ -82,9 +90,19 @@ process BOWTIE2_ALIGN {
     } else {
         create_unmapped = save_unaligned ? "touch ${prefix}.unmapped_1.fastq.gz && touch ${prefix}.unmapped_2.fastq.gz" : ""
     }
+    def reference = fasta && extension=="cram"  ? "--reference ${fasta}" : ""
+    if (!fasta && extension=="cram") error "Fasta reference is required for CRAM output"
+
+    def create_index = ""
+    if (extension == "cram") {
+        create_index = "touch ${prefix}.crai"
+    } else if (extension == "bam") {
+        create_index = "touch ${prefix}.csi"
+    }
 
     """
     touch ${prefix}.${extension}
+    ${create_index}
     touch ${prefix}.bowtie2.log
     ${create_unmapped}
 
diff --git a/modules/nf-core/bowtie2/align/tests/cram_crai.config b/modules/nf-core/bowtie2/align/tests/cram_crai.config
new file mode 100644
index 00000000000..03f1d5e511f
--- /dev/null
+++ b/modules/nf-core/bowtie2/align/tests/cram_crai.config
@@ -0,0 +1,5 @@
+process {
+    withName: BOWTIE2_ALIGN {
+        ext.args2 = '--output-fmt cram --write-index'
+    }
+}
diff --git a/modules/nf-core/bowtie2/align/tests/main.nf.test b/modules/nf-core/bowtie2/align/tests/main.nf.test
index a478d17b542..03aeaf9eef3 100644
--- a/modules/nf-core/bowtie2/align/tests/main.nf.test
+++ b/modules/nf-core/bowtie2/align/tests/main.nf.test
@@ -6,9 +6,10 @@ nextflow_process {
     tag "modules"
     tag "modules_nfcore"
     tag "bowtie2"
+    tag "bowtie2/build"
     tag "bowtie2/align"
 
-    test("sarscov2 - fastq, index, false, false - bam") {
+    test("sarscov2 - fastq, index, fasta, false, false - bam") {
 
         setup {
             run("BOWTIE2_BUILD") {
@@ -32,8 +33,9 @@ nextflow_process {
                     file(params.test_data['sarscov2']['illumina']['test_1_fastq_gz'], checkIfExists: true)
                 ]
                 input[1] = BOWTIE2_BUILD.out.index
-                input[2] = false //save_unaligned
-                input[3] = false //sort
+                input[2] = [[ id:'test'], file(params.test_data['sarscov2']['genome']['genome_fasta'], checkIfExists: true)]
+                input[3] = false //save_unaligned
+                input[4] = false //sort
                 """
             }
         }
@@ -42,7 +44,7 @@ nextflow_process {
             assertAll (
                 { assert process.success },
                 { assert snapshot(
-                    file(process.out.aligned[0][1]).name,
+                    file(process.out.bam[0][1]).name,
                     process.out.log,
                     process.out.fastq,
                     process.out.versions
@@ -52,7 +54,7 @@ nextflow_process {
 
     }
 
-    test("sarscov2 - fastq, index, false, false - sam") {
+    test("sarscov2 - fastq, index, fasta, false, false - sam") {
 
         config "./sam.config"
         setup {
@@ -77,8 +79,9 @@ nextflow_process {
                     file(params.test_data['sarscov2']['illumina']['test_1_fastq_gz'], checkIfExists: true)
                 ]
                 input[1] = BOWTIE2_BUILD.out.index
-                input[2] = false //save_unaligned
-                input[3] = false //sort
+                input[2] = [[ id:'test'], file(params.test_data['sarscov2']['genome']['genome_fasta'], checkIfExists: true)]
+                input[3] = false //save_unaligned
+                input[4] = false //sort
                 """
             }
         }
@@ -87,7 +90,7 @@ nextflow_process {
             assertAll (
                 { assert process.success },
                 { assert snapshot(
-                    file(process.out.aligned[0][1]).readLines()[0..4],
+                    file(process.out.sam[0][1]).readLines()[0..4],
                     process.out.log,
                     process.out.fastq,
                     process.out.versions
@@ -97,7 +100,7 @@ nextflow_process {
 
     }
 
-    test("sarscov2 - fastq, index, false, false - sam2") {
+    test("sarscov2 - fastq, index, fasta, false, false - sam2") {
 
         config "./sam2.config"
         setup {
@@ -122,8 +125,9 @@ nextflow_process {
                     file(params.test_data['sarscov2']['illumina']['test_1_fastq_gz'], checkIfExists: true)
                 ]
                 input[1] = BOWTIE2_BUILD.out.index
-                input[2] = false //save_unaligned
-                input[3] = false //sort
+                input[2] = [[ id:'test'], file(params.test_data['sarscov2']['genome']['genome_fasta'], checkIfExists: true)]
+                input[3] = false //save_unaligned
+                input[4] = false //sort
                 """
             }
         }
@@ -132,7 +136,7 @@ nextflow_process {
             assertAll (
                 { assert process.success },
                 { assert snapshot(
-                    file(process.out.aligned[0][1]).readLines()[0..4],
+                    file(process.out.sam[0][1]).readLines()[0..4],
                     process.out.log,
                     process.out.fastq,
                     process.out.versions
@@ -142,7 +146,7 @@ nextflow_process {
 
     }
 
-    test("sarscov2 - fastq, index, false, true - bam") {
+    test("sarscov2 - fastq, index, fasta, false, true - bam") {
 
         setup {
             run("BOWTIE2_BUILD") {
@@ -166,8 +170,9 @@ nextflow_process {
                     file(params.test_data['sarscov2']['illumina']['test_1_fastq_gz'], checkIfExists: true)
                 ]
                 input[1] = BOWTIE2_BUILD.out.index
-                input[2] = false //save_unaligned
-                input[3] = true //sort
+                input[2] = [[ id:'test'], file(params.test_data['sarscov2']['genome']['genome_fasta'], checkIfExists: true)]
+                input[3] = false //save_unaligned
+                input[4] = false //sort
                 """
             }
         }
@@ -176,7 +181,7 @@ nextflow_process {
             assertAll (
                 { assert process.success },
                 { assert snapshot(
-                    file(process.out.aligned[0][1]).name,
+                    file(process.out.bam[0][1]).name,
                     process.out.log,
                     process.out.fastq,
                     process.out.versions
@@ -186,7 +191,7 @@ nextflow_process {
 
     }
 
-    test("sarscov2 - [fastq1, fastq2], index, false, false - bam") {
+    test("sarscov2 - [fastq1, fastq2], index, fasta, false, false - bam") {
 
         setup {
             run("BOWTIE2_BUILD") {
@@ -213,8 +218,9 @@ nextflow_process {
                     ]
                 ]
                 input[1] = BOWTIE2_BUILD.out.index
-                input[2] = false //save_unaligned
-                input[3] = false //sort
+                input[2] = [[ id:'test'], file(params.test_data['sarscov2']['genome']['genome_fasta'], checkIfExists: true)]
+                input[3] = false //save_unaligned
+                input[4] = false //sort
                 """
             }
         }
@@ -223,7 +229,7 @@ nextflow_process {
             assertAll (
                 { assert process.success },
                 { assert snapshot(
-                    file(process.out.aligned[0][1]).name,
+                    file(process.out.bam[0][1]).name,
                     process.out.log,
                     process.out.fastq,
                     process.out.versions
@@ -233,7 +239,7 @@ nextflow_process {
 
     }
 
-    test("sarscov2 - [fastq1, fastq2], index, false, true - bam") {
+    test("sarscov2 - [fastq1, fastq2], index, fasta, false, true - bam") {
 
         setup {
             run("BOWTIE2_BUILD") {
@@ -260,8 +266,9 @@ nextflow_process {
                     ]
                 ]
                 input[1] = BOWTIE2_BUILD.out.index
-                input[2] = false //save_unaligned
-                input[3] = true //sort
+                input[2] = [[ id:'test'], file(params.test_data['sarscov2']['genome']['genome_fasta'], checkIfExists: true)]
+                input[3] = false //save_unaligned
+                input[4] = false //sort
                 """
             }
         }
@@ -270,7 +277,7 @@ nextflow_process {
             assertAll (
                 { assert process.success },
                 { assert snapshot(
-                    file(process.out.aligned[0][1]).name,
+                    file(process.out.bam[0][1]).name,
                     process.out.log,
                     process.out.fastq,
                     process.out.versions
@@ -280,7 +287,7 @@ nextflow_process {
 
     }
 
-    test("sarscov2 - fastq, large_index, false, false - bam") {
+    test("sarscov2 - fastq, large_index, fasta, false, false - bam") {
 
         config "./large_index.config"
         setup {
@@ -305,8 +312,9 @@ nextflow_process {
                     file(params.test_data['sarscov2']['illumina']['test_1_fastq_gz'], checkIfExists: true)
                 ]
                 input[1] = BOWTIE2_BUILD.out.index
-                input[2] = false //save_unaligned
-                input[3] = false //sort
+                input[2] = [[ id:'test'], file(params.test_data['sarscov2']['genome']['genome_fasta'], checkIfExists: true)]
+                input[3] = false //save_unaligned
+                input[4] = false //sort
                 """
             }
         }
@@ -315,7 +323,7 @@ nextflow_process {
             assertAll (
                 { assert process.success },
                 { assert snapshot(
-                    file(process.out.aligned[0][1]).name,
+                    file(process.out.bam[0][1]).name,
                     process.out.log,
                     process.out.fastq,
                     process.out.versions
@@ -325,7 +333,7 @@ nextflow_process {
 
     }
 
-    test("sarscov2 - [fastq1, fastq2], large_index, false, false - bam") {
+    test("sarscov2 - [fastq1, fastq2], large_index, fasta, false, false - bam") {
 
         config "./large_index.config"
         setup {
@@ -353,8 +361,9 @@ nextflow_process {
                     ]
                 ]
                 input[1] = BOWTIE2_BUILD.out.index
-                input[2] = false //save_unaligned
-                input[3] = false //sort
+                input[2] = [[ id:'test'], file(params.test_data['sarscov2']['genome']['genome_fasta'], checkIfExists: true)]
+                input[3] = false //save_unaligned
+                input[4] = false //sort
                 """
             }
         }
@@ -363,7 +372,7 @@ nextflow_process {
             assertAll (
                 { assert process.success },
                 { assert snapshot(
-                    file(process.out.aligned[0][1]).name,
+                    file(process.out.bam[0][1]).name,
                     process.out.log,
                     process.out.fastq,
                     process.out.versions
@@ -373,7 +382,7 @@ nextflow_process {
 
     }
 
-    test("sarscov2 - [fastq1, fastq2], index, true, false - bam") {
+    test("sarscov2 - [fastq1, fastq2], index, fasta, true, false - bam") {
 
         setup {
             run("BOWTIE2_BUILD") {
@@ -400,8 +409,9 @@ nextflow_process {
                     ]
                 ]
                 input[1] = BOWTIE2_BUILD.out.index
-                input[2] = true //save_unaligned
-                input[3] = false //sort
+                input[2] = [[ id:'test'], file(params.test_data['sarscov2']['genome']['genome_fasta'], checkIfExists: true)]
+                input[3] = false //save_unaligned
+                input[4] = false //sort
                 """
             }
         }
@@ -410,7 +420,7 @@ nextflow_process {
             assertAll (
                 { assert process.success },
                 { assert snapshot(
-                    file(process.out.aligned[0][1]).name,
+                    file(process.out.bam[0][1]).name,
                     process.out.log,
                     process.out.fastq,
                     process.out.versions
@@ -420,7 +430,7 @@ nextflow_process {
 
     }
 
-    test("sarscov2 - fastq, index, true, false - bam") {
+    test("sarscov2 - fastq, index, fasta, true, false - bam") {
 
         setup {
             run("BOWTIE2_BUILD") {
@@ -444,8 +454,9 @@ nextflow_process {
                     file(params.test_data['sarscov2']['illumina']['test_1_fastq_gz'], checkIfExists: true)
                 ]
                 input[1] = BOWTIE2_BUILD.out.index
-                input[2] = true //save_unaligned
-                input[3] = false //sort
+                input[2] = [[ id:'test'], file(params.test_data['sarscov2']['genome']['genome_fasta'], checkIfExists: true)]
+                input[3] = false //save_unaligned
+                input[4] = false //sort
                 """
             }
         }
@@ -454,7 +465,7 @@ nextflow_process {
             assertAll (
                 { assert process.success },
                 { assert snapshot(
-                    file(process.out.aligned[0][1]).name,
+                    file(process.out.bam[0][1]).name,
                     process.out.log,
                     process.out.fastq,
                     process.out.versions
@@ -465,7 +476,54 @@ nextflow_process {
 
     }
 
-    test("sarscov2 - [fastq1, fastq2], index, false, false - stub") {
+    test("sarscov2 - [fastq1, fastq2], index, fasta, true, true - cram") {
+
+        config "./cram_crai.config"
+        setup {
+            run("BOWTIE2_BUILD") {
+                script "../../build/main.nf"
+                process {
+                    """
+                    input[0] = [
+                        [ id:'test'],
+                        file(params.test_data['sarscov2']['genome']['genome_fasta'], checkIfExists: true)
+                    ]
+                    """
+                }
+            }
+        }
+
+        when {
+            process {
+                """
+                input[0] = [
+                    [ id:'test', single_end:false ], // meta map
+                    [
+                        file(params.test_data['sarscov2']['illumina']['test_1_fastq_gz'], checkIfExists: true),
+                        file(params.test_data['sarscov2']['illumina']['test_2_fastq_gz'], checkIfExists: true)
+                    ]
+                ]
+                input[1] = BOWTIE2_BUILD.out.index
+                input[2] = [[ id:'test'], file(params.test_data['sarscov2']['genome']['genome_fasta'], checkIfExists: true)]
+                input[3] = false //save_unaligned
+                input[4] = true //sort
+                """
+            }
+        }
+
+        then {
+            assertAll (
+                { assert process.success },
+                { assert snapshot(
+                    file(process.out.cram[0][1]).name,
+                    file(process.out.crai[0][1]).name
+                ).match() }
+            )
+        }
+
+    }
+
+    test("sarscov2 - [fastq1, fastq2], index, fasta, false, false - stub") {
 
         options "-stub"
         setup {
@@ -493,8 +551,9 @@ nextflow_process {
                     ]
                 ]
                 input[1] = BOWTIE2_BUILD.out.index
-                input[2] = false //save_unaligned
-                input[3] = false //sort
+                input[2] = [[ id:'test'], file(params.test_data['sarscov2']['genome']['genome_fasta'], checkIfExists: true)]
+                input[3] = false //save_unaligned
+                input[4] = false //sort
                 """
             }
         }
@@ -503,7 +562,8 @@ nextflow_process {
             assertAll (
                 { assert process.success },
                 { assert snapshot(
-                    file(process.out.aligned[0][1]).name,
+                    file(process.out.bam[0][1]).name,
+                    file(process.out.csi[0][1]).name,
                     file(process.out.log[0][1]).name,
                     process.out.fastq,
                     process.out.versions
@@ -513,7 +573,7 @@ nextflow_process {
 
     }
 
-    test("sarscov2 - fastq, index, true, false - stub") {
+    test("sarscov2 - fastq, index, fasta, true, false - stub") {
 
         options "-stub"
         setup {
@@ -538,8 +598,9 @@ nextflow_process {
                     file(params.test_data['sarscov2']['illumina']['test_1_fastq_gz'], checkIfExists: true)
                 ]
                 input[1] = BOWTIE2_BUILD.out.index
-                input[2] = true //save_unaligned
-                input[3] = false //sort
+                input[2] = [[ id:'test'], file(params.test_data['sarscov2']['genome']['genome_fasta'], checkIfExists: true)]
+                input[3] = false //save_unaligned
+                input[4] = false //sort
                 """
             }
         }
@@ -548,14 +609,15 @@ nextflow_process {
             assertAll (
                 { assert process.success },
                 { assert snapshot(
-                    file(process.out.aligned[0][1]).name,
+                    file(process.out.bam[0][1]).name,
+                    file(process.out.csi[0][1]).name,
                     file(process.out.log[0][1]).name,
-                    file(process.out.fastq[0][1]).name,
+                    process.out.fastq,
                     process.out.versions
                 ).match() }
             )
         }
 
     }
-    
+
 }
diff --git a/modules/nf-core/bowtie2/align/tests/main.nf.test.snap b/modules/nf-core/bowtie2/align/tests/main.nf.test.snap
index 883dc7ecba1..028e7da6898 100644
--- a/modules/nf-core/bowtie2/align/tests/main.nf.test.snap
+++ b/modules/nf-core/bowtie2/align/tests/main.nf.test.snap
@@ -1,34 +1,38 @@
 {
-    "sarscov2 - fastq, index, false, false - sam2": {
+    "sarscov2 - [fastq1, fastq2], large_index, fasta, false, false - bam": {
         "content": [
-            [
-                "ERR5069949.2151832\t16\tMT192765.1\t17453\t42\t150M\t*\t0\t0\tACGCACATTGCTAACTAAGGGCACACTAGAACCAGAATATTTCAATTCAGTGTGTAGACTTATGAAAACTATAGGTCCAGACATGTTCCTCGGAACTTGTCGGCGTTGTCCTGCTGAAATTGTTGACACTGTGAGTGCTTTGGTTTATGA\tAAAA<EEEEEEAEEEAEAAAAEEEEEEEEEAAAEE<EEEEEAAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAEEEEAAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAAAAAA\tAS:i:0\tXN:i:0\tXM:i:0\tXO:i:0\tXG:i:0\tNM:i:0\tMD:Z:150\tYT:Z:UU",
-                "ERR5069949.576388\t16\tMT192765.1\t5798\t42\t77M\t*\t0\t0\tGCATAGACGGTGCTTTACTTACAAAGTCCTCAGAATACAAAGGTCCTATTACGGATGTTTTCTACAAAGAAAACAGT\tEA/AEEE/<EEEEEEEEEEEAA<EEEEEEEEEEEEEEEEEEEEEAEEEEEAEEEAEE6/EEEAEEEEEEEEEA6AAA\tAS:i:0\tXN:i:0\tXM:i:0\tXO:i:0\tXG:i:0\tNM:i:0\tMD:Z:77\tYT:Z:UU",
-                "ERR5069949.501486\t16\tMT192765.1\t5423\t42\t146M\t*\t0\t0\tTAGGTGAGTTAGGTGATGTTAGAGAAACAATGAGTTACTTGTTTCAACATGCCAATTTAGATTCTTGCAAAAGAGTCTTGAACGTGGTGTGTAAAACTTGTGGACAACAGCAGACAACCCTTAAGGGTGTAGAAGCTGTTATGTAC\tEAAAAEAEEEE6E<AEEEEEEEE<EEEEEEAAEE/EEEEEE/<EEEEEEAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAEEEEEEEEEEAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAAAAA\tAS:i:0\tXN:i:0\tXM:i:0\tXO:i:0\tXG:i:0\tNM:i:0\tMD:Z:146\tYT:Z:UU",
-                "ERR5069949.1331889\t0\tMT192765.1\t13010\t42\t132M\t*\t0\t0\tGTCTACAAGCTGGTAATGCAACAGAAGTGCCTGCCAATTCAACTGTATTATCTTTCTGTGCTTTTGCTGTAGATGCTGCTAAAGCTTACAAAGATTATCTAGCTAGTGGGGGACAACCAATCACTAATTGTG\tA/AAAEEEEEEEEEEEEEEEEEEEAEEEEAEEEEEEEEEEEAEEEEEEEEEEEEEEE/EEEEE<AEAEEEEE/EAEAEEE/AEEEEEEEEEEEEEEEEEEEEAE/EEEEEEEEEEEEEEEEEEEEEEEA<EE\tAS:i:0\tXN:i:0\tXM:i:0\tXO:i:0\tXG:i:0\tNM:i:0\tMD:Z:132\tYT:Z:UU",
-                "ERR5069949.2161340\t0\tMT192765.1\t17482\t42\t80M\t*\t0\t0\tAACCAGAATATTTCAATTCAGTGTGTAGACTTATGAAAACTATAGGTCCAGACATGTTCCTCGGAACTTGTCGGCGTTGT\tA/AA//EEAEA/E/AEEEE6EE/EEEA/6AEEEEEEEEE6EEEAEAEE//A/EEEEEE//E/E/A//E/E/<<EE</E/E\tAS:i:0\tXN:i:0\tXM:i:0\tXO:i:0\tXG:i:0\tNM:i:0\tMD:Z:80\tYT:Z:UU"
-            ],
+            "test.bam",
             [
                 [
                     {
                         "id": "test",
-                        "single_end": true
+                        "single_end": false
                     },
-                    "test.bowtie2.log:md5,7b8a9e61b7646da1089b041333c41a87"
+                    "test.bowtie2.log:md5,bd89ce1b28c93bf822bae391ffcedd19"
                 ]
             ],
             [
-
+                
             ],
             [
                 "versions.yml:md5,01d18ab035146ea790e9a0f70adb758f"
             ]
         ],
-        "timestamp": "2023-11-23T11:31:43.71534618"
+        "meta": {
+            "nf-test": "0.8.4",
+            "nextflow": "23.10.1"
+        },
+        "timestamp": "2024-03-18T13:19:25.337323"
     },
-    "sarscov2 - fastq, index, false, false - bam": {
+    "sarscov2 - fastq, index, fasta, false, false - sam2": {
         "content": [
-            "test.bam",
+            [
+                "ERR5069949.2151832\t16\tMT192765.1\t17453\t42\t150M\t*\t0\t0\tACGCACATTGCTAACTAAGGGCACACTAGAACCAGAATATTTCAATTCAGTGTGTAGACTTATGAAAACTATAGGTCCAGACATGTTCCTCGGAACTTGTCGGCGTTGTCCTGCTGAAATTGTTGACACTGTGAGTGCTTTGGTTTATGA\tAAAA<EEEEEEAEEEAEAAAAEEEEEEEEEAAAEE<EEEEEAAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAEEEEAAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAAAAAA\tAS:i:0\tXN:i:0\tXM:i:0\tXO:i:0\tXG:i:0\tNM:i:0\tMD:Z:150\tYT:Z:UU",
+                "ERR5069949.576388\t16\tMT192765.1\t5798\t42\t77M\t*\t0\t0\tGCATAGACGGTGCTTTACTTACAAAGTCCTCAGAATACAAAGGTCCTATTACGGATGTTTTCTACAAAGAAAACAGT\tEA/AEEE/<EEEEEEEEEEEAA<EEEEEEEEEEEEEEEEEEEEEAEEEEEAEEEAEE6/EEEAEEEEEEEEEA6AAA\tAS:i:0\tXN:i:0\tXM:i:0\tXO:i:0\tXG:i:0\tNM:i:0\tMD:Z:77\tYT:Z:UU",
+                "ERR5069949.501486\t16\tMT192765.1\t5423\t42\t146M\t*\t0\t0\tTAGGTGAGTTAGGTGATGTTAGAGAAACAATGAGTTACTTGTTTCAACATGCCAATTTAGATTCTTGCAAAAGAGTCTTGAACGTGGTGTGTAAAACTTGTGGACAACAGCAGACAACCCTTAAGGGTGTAGAAGCTGTTATGTAC\tEAAAAEAEEEE6E<AEEEEEEEE<EEEEEEAAEE/EEEEEE/<EEEEEEAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAEEEEEEEEEEAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAAAAA\tAS:i:0\tXN:i:0\tXM:i:0\tXO:i:0\tXG:i:0\tNM:i:0\tMD:Z:146\tYT:Z:UU",
+                "ERR5069949.1331889\t0\tMT192765.1\t13010\t42\t132M\t*\t0\t0\tGTCTACAAGCTGGTAATGCAACAGAAGTGCCTGCCAATTCAACTGTATTATCTTTCTGTGCTTTTGCTGTAGATGCTGCTAAAGCTTACAAAGATTATCTAGCTAGTGGGGGACAACCAATCACTAATTGTG\tA/AAAEEEEEEEEEEEEEEEEEEEAEEEEAEEEEEEEEEEEAEEEEEEEEEEEEEEE/EEEEE<AEAEEEEE/EAEAEEE/AEEEEEEEEEEEEEEEEEEEEAE/EEEEEEEEEEEEEEEEEEEEEEEA<EE\tAS:i:0\tXN:i:0\tXM:i:0\tXO:i:0\tXG:i:0\tNM:i:0\tMD:Z:132\tYT:Z:UU",
+                "ERR5069949.2161340\t0\tMT192765.1\t17482\t42\t80M\t*\t0\t0\tAACCAGAATATTTCAATTCAGTGTGTAGACTTATGAAAACTATAGGTCCAGACATGTTCCTCGGAACTTGTCGGCGTTGT\tA/AA//EEAEA/E/AEEEE6EE/EEEA/6AEEEEEEEEE6EEEAEAEE//A/EEEEEE//E/E/A//E/E/<<EE</E/E\tAS:i:0\tXN:i:0\tXM:i:0\tXO:i:0\tXG:i:0\tNM:i:0\tMD:Z:80\tYT:Z:UU"
+            ],
             [
                 [
                     {
@@ -39,66 +43,57 @@
                 ]
             ],
             [
-
+                
             ],
             [
                 "versions.yml:md5,01d18ab035146ea790e9a0f70adb758f"
             ]
         ],
-        "timestamp": "2023-11-23T10:51:15.152469637"
+        "meta": {
+            "nf-test": "0.8.4",
+            "nextflow": "23.10.1"
+        },
+        "timestamp": "2024-03-18T12:31:40.337129939"
     },
-    "sarscov2 - [fastq1, fastq2], index, true, false - bam": {
+    "sarscov2 - [fastq1, fastq2], index, fasta, true, true - cram": {
+        "content": [
+            "test.cram",
+            "test.cram.crai"
+        ],
+        "meta": {
+            "nf-test": "0.8.4",
+            "nextflow": "23.10.1"
+        },
+        "timestamp": "2024-03-19T08:46:08.033126014"
+    },
+    "sarscov2 - fastq, index, fasta, false, false - bam": {
         "content": [
             "test.bam",
             [
                 [
                     {
                         "id": "test",
-                        "single_end": false
+                        "single_end": true
                     },
-                    "test.bowtie2.log:md5,bd89ce1b28c93bf822bae391ffcedd19"
+                    "test.bowtie2.log:md5,7b8a9e61b7646da1089b041333c41a87"
                 ]
             ],
             [
-                [
-                    {
-                        "id": "test",
-                        "single_end": false
-                    },
-                    [
-                        "test.unmapped_1.fastq.gz:md5,84b4ab15e6ffc9f29d0a3f1b79003744",
-                        "test.unmapped_2.fastq.gz:md5,40bbe14edd48a98cca6f53574b2198fd"
-                    ]
-                ]
+                
             ],
             [
                 "versions.yml:md5,01d18ab035146ea790e9a0f70adb758f"
             ]
         ],
-        "timestamp": "2023-11-23T11:32:26.773226801"
+        "meta": {
+            "nf-test": "0.8.4",
+            "nextflow": "23.10.1"
+        },
+        "timestamp": "2024-03-18T12:58:55.482166476"
     },
-    "sarscov2 - [fastq1, fastq2], index, false, false - stub": {
+    "sarscov2 - fastq, index, fasta, true, false - bam": {
         "content": [
             "test.bam",
-            "test.bowtie2.log",
-            [
-
-            ],
-            [
-                "versions.yml:md5,01d18ab035146ea790e9a0f70adb758f"
-            ]
-        ],
-        "timestamp": "2023-11-23T11:38:10.240204508"
-    },
-    "sarscov2 - fastq, index, false, false - sam": {
-        "content": [
-            [
-                "ERR5069949.2151832\t16\tMT192765.1\t17453\t42\t150M\t*\t0\t0\tACGCACATTGCTAACTAAGGGCACACTAGAACCAGAATATTTCAATTCAGTGTGTAGACTTATGAAAACTATAGGTCCAGACATGTTCCTCGGAACTTGTCGGCGTTGTCCTGCTGAAATTGTTGACACTGTGAGTGCTTTGGTTTATGA\tAAAA<EEEEEEAEEEAEAAAAEEEEEEEEEAAAEE<EEEEEAAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAEEEEAAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAAAAAA\tAS:i:0\tXN:i:0\tXM:i:0\tXO:i:0\tXG:i:0\tNM:i:0\tMD:Z:150\tYT:Z:UU",
-                "ERR5069949.576388\t16\tMT192765.1\t5798\t42\t77M\t*\t0\t0\tGCATAGACGGTGCTTTACTTACAAAGTCCTCAGAATACAAAGGTCCTATTACGGATGTTTTCTACAAAGAAAACAGT\tEA/AEEE/<EEEEEEEEEEEAA<EEEEEEEEEEEEEEEEEEEEEAEEEEEAEEEAEE6/EEEAEEEEEEEEEA6AAA\tAS:i:0\tXN:i:0\tXM:i:0\tXO:i:0\tXG:i:0\tNM:i:0\tMD:Z:77\tYT:Z:UU",
-                "ERR5069949.501486\t16\tMT192765.1\t5423\t42\t146M\t*\t0\t0\tTAGGTGAGTTAGGTGATGTTAGAGAAACAATGAGTTACTTGTTTCAACATGCCAATTTAGATTCTTGCAAAAGAGTCTTGAACGTGGTGTGTAAAACTTGTGGACAACAGCAGACAACCCTTAAGGGTGTAGAAGCTGTTATGTAC\tEAAAAEAEEEE6E<AEEEEEEEE<EEEEEEAAEE/EEEEEE/<EEEEEEAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAEEEEEEEEEEAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAAAAA\tAS:i:0\tXN:i:0\tXM:i:0\tXO:i:0\tXG:i:0\tNM:i:0\tMD:Z:146\tYT:Z:UU",
-                "ERR5069949.1331889\t0\tMT192765.1\t13010\t42\t132M\t*\t0\t0\tGTCTACAAGCTGGTAATGCAACAGAAGTGCCTGCCAATTCAACTGTATTATCTTTCTGTGCTTTTGCTGTAGATGCTGCTAAAGCTTACAAAGATTATCTAGCTAGTGGGGGACAACCAATCACTAATTGTG\tA/AAAEEEEEEEEEEEEEEEEEEEAEEEEAEEEEEEEEEEEAEEEEEEEEEEEEEEE/EEEEE<AEAEEEEE/EAEAEEE/AEEEEEEEEEEEEEEEEEEEEAE/EEEEEEEEEEEEEEEEEEEEEEEA<EE\tAS:i:0\tXN:i:0\tXM:i:0\tXO:i:0\tXG:i:0\tNM:i:0\tMD:Z:132\tYT:Z:UU",
-                "ERR5069949.2161340\t0\tMT192765.1\t17482\t42\t80M\t*\t0\t0\tAACCAGAATATTTCAATTCAGTGTGTAGACTTATGAAAACTATAGGTCCAGACATGTTCCTCGGAACTTGTCGGCGTTGT\tA/AA//EEAEA/E/AEEEE6EE/EEEA/6AEEEEEEEEE6EEEAEAEE//A/EEEEEE//E/E/A//E/E/<<EE</E/E\tAS:i:0\tXN:i:0\tXM:i:0\tXO:i:0\tXG:i:0\tNM:i:0\tMD:Z:80\tYT:Z:UU"
-            ],
             [
                 [
                     {
@@ -109,15 +104,19 @@
                 ]
             ],
             [
-
+                
             ],
             [
                 "versions.yml:md5,01d18ab035146ea790e9a0f70adb758f"
             ]
         ],
-        "timestamp": "2023-11-23T10:54:41.68497071"
+        "meta": {
+            "nf-test": "0.8.4",
+            "nextflow": "23.10.1"
+        },
+        "timestamp": "2024-03-18T13:19:55.741244"
     },
-    "sarscov2 - fastq, index, false, true - bam": {
+    "sarscov2 - fastq, large_index, fasta, false, false - bam": {
         "content": [
             "test.bam",
             [
@@ -130,15 +129,19 @@
                 ]
             ],
             [
-
+                
             ],
             [
                 "versions.yml:md5,01d18ab035146ea790e9a0f70adb758f"
             ]
         ],
-        "timestamp": "2023-11-23T11:31:51.235960009"
+        "meta": {
+            "nf-test": "0.8.4",
+            "nextflow": "23.10.1"
+        },
+        "timestamp": "2024-03-18T13:19:09.604979"
     },
-    "sarscov2 - fastq, index, true, false - bam": {
+    "sarscov2 - fastq, index, fasta, false, true - bam": {
         "content": [
             "test.bam",
             [
@@ -151,74 +154,111 @@
                 ]
             ],
             [
-                [
-                    {
-                        "id": "test",
-                        "single_end": true
-                    },
-                    "test.unmapped.fastq.gz:md5,d17d9a4ff728b6a6afa6203a746b7a01"
-                ]
+                
             ],
             [
                 "versions.yml:md5,01d18ab035146ea790e9a0f70adb758f"
             ]
         ],
-        "timestamp": "2023-11-23T11:32:34.218783018"
+        "meta": {
+            "nf-test": "0.8.4",
+            "nextflow": "23.10.1"
+        },
+        "timestamp": "2024-03-18T13:18:22.547322"
     },
-    "sarscov2 - fastq, index, true, false - stub": {
+    "sarscov2 - fastq, index, fasta, true, false - stub": {
         "content": [
             "test.bam",
+            "test.csi",
             "test.bowtie2.log",
-            "test.unmapped.fastq.gz",
+            [
+                
+            ],
             [
                 "versions.yml:md5,01d18ab035146ea790e9a0f70adb758f"
             ]
         ],
-        "timestamp": "2023-11-23T11:47:34.072444428"
+        "meta": {
+            "nf-test": "0.8.4",
+            "nextflow": "23.10.1"
+        },
+        "timestamp": "2024-03-18T13:02:09.557518106"
     },
-    "sarscov2 - fastq, large_index, false, false - bam": {
+    "sarscov2 - [fastq1, fastq2], index, fasta, false, false - bam": {
         "content": [
             "test.bam",
             [
                 [
                     {
                         "id": "test",
-                        "single_end": true
+                        "single_end": false
                     },
-                    "test.bowtie2.log:md5,7b8a9e61b7646da1089b041333c41a87"
+                    "test.bowtie2.log:md5,bd89ce1b28c93bf822bae391ffcedd19"
                 ]
             ],
             [
-
+                
             ],
             [
                 "versions.yml:md5,01d18ab035146ea790e9a0f70adb758f"
             ]
         ],
-        "timestamp": "2023-11-23T11:32:11.560260131"
+        "meta": {
+            "nf-test": "0.8.4",
+            "nextflow": "23.10.1"
+        },
+        "timestamp": "2024-03-18T13:18:38.78064"
     },
-    "sarscov2 - [fastq1, fastq2], large_index, false, false - bam": {
+    "sarscov2 - [fastq1, fastq2], index, fasta, false, false - stub": {
         "content": [
             "test.bam",
+            "test.csi",
+            "test.bowtie2.log",
+            [
+                
+            ],
+            [
+                "versions.yml:md5,01d18ab035146ea790e9a0f70adb758f"
+            ]
+        ],
+        "meta": {
+            "nf-test": "0.8.4",
+            "nextflow": "23.10.1"
+        },
+        "timestamp": "2024-03-18T13:20:26.047111"
+    },
+    "sarscov2 - fastq, index, fasta, false, false - sam": {
+        "content": [
+            [
+                "ERR5069949.2151832\t16\tMT192765.1\t17453\t42\t150M\t*\t0\t0\tACGCACATTGCTAACTAAGGGCACACTAGAACCAGAATATTTCAATTCAGTGTGTAGACTTATGAAAACTATAGGTCCAGACATGTTCCTCGGAACTTGTCGGCGTTGTCCTGCTGAAATTGTTGACACTGTGAGTGCTTTGGTTTATGA\tAAAA<EEEEEEAEEEAEAAAAEEEEEEEEEAAAEE<EEEEEAAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAEEEEAAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAAAAAA\tAS:i:0\tXN:i:0\tXM:i:0\tXO:i:0\tXG:i:0\tNM:i:0\tMD:Z:150\tYT:Z:UU",
+                "ERR5069949.576388\t16\tMT192765.1\t5798\t42\t77M\t*\t0\t0\tGCATAGACGGTGCTTTACTTACAAAGTCCTCAGAATACAAAGGTCCTATTACGGATGTTTTCTACAAAGAAAACAGT\tEA/AEEE/<EEEEEEEEEEEAA<EEEEEEEEEEEEEEEEEEEEEAEEEEEAEEEAEE6/EEEAEEEEEEEEEA6AAA\tAS:i:0\tXN:i:0\tXM:i:0\tXO:i:0\tXG:i:0\tNM:i:0\tMD:Z:77\tYT:Z:UU",
+                "ERR5069949.501486\t16\tMT192765.1\t5423\t42\t146M\t*\t0\t0\tTAGGTGAGTTAGGTGATGTTAGAGAAACAATGAGTTACTTGTTTCAACATGCCAATTTAGATTCTTGCAAAAGAGTCTTGAACGTGGTGTGTAAAACTTGTGGACAACAGCAGACAACCCTTAAGGGTGTAGAAGCTGTTATGTAC\tEAAAAEAEEEE6E<AEEEEEEEE<EEEEEEAAEE/EEEEEE/<EEEEEEAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAEEEEEEEEEEAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAAAAA\tAS:i:0\tXN:i:0\tXM:i:0\tXO:i:0\tXG:i:0\tNM:i:0\tMD:Z:146\tYT:Z:UU",
+                "ERR5069949.1331889\t0\tMT192765.1\t13010\t42\t132M\t*\t0\t0\tGTCTACAAGCTGGTAATGCAACAGAAGTGCCTGCCAATTCAACTGTATTATCTTTCTGTGCTTTTGCTGTAGATGCTGCTAAAGCTTACAAAGATTATCTAGCTAGTGGGGGACAACCAATCACTAATTGTG\tA/AAAEEEEEEEEEEEEEEEEEEEAEEEEAEEEEEEEEEEEAEEEEEEEEEEEEEEE/EEEEE<AEAEEEEE/EAEAEEE/AEEEEEEEEEEEEEEEEEEEEAE/EEEEEEEEEEEEEEEEEEEEEEEA<EE\tAS:i:0\tXN:i:0\tXM:i:0\tXO:i:0\tXG:i:0\tNM:i:0\tMD:Z:132\tYT:Z:UU",
+                "ERR5069949.2161340\t0\tMT192765.1\t17482\t42\t80M\t*\t0\t0\tAACCAGAATATTTCAATTCAGTGTGTAGACTTATGAAAACTATAGGTCCAGACATGTTCCTCGGAACTTGTCGGCGTTGT\tA/AA//EEAEA/E/AEEEE6EE/EEEA/6AEEEEEEEEE6EEEAEAEE//A/EEEEEE//E/E/A//E/E/<<EE</E/E\tAS:i:0\tXN:i:0\tXM:i:0\tXO:i:0\tXG:i:0\tNM:i:0\tMD:Z:80\tYT:Z:UU"
+            ],
             [
                 [
                     {
                         "id": "test",
-                        "single_end": false
+                        "single_end": true
                     },
-                    "test.bowtie2.log:md5,bd89ce1b28c93bf822bae391ffcedd19"
+                    "test.bowtie2.log:md5,7b8a9e61b7646da1089b041333c41a87"
                 ]
             ],
             [
-
+                
             ],
             [
                 "versions.yml:md5,01d18ab035146ea790e9a0f70adb758f"
             ]
         ],
-        "timestamp": "2023-11-23T11:32:19.342811204"
+        "meta": {
+            "nf-test": "0.8.4",
+            "nextflow": "23.10.1"
+        },
+        "timestamp": "2024-03-18T12:31:26.668053751"
     },
-    "sarscov2 - [fastq1, fastq2], index, false, false - bam": {
+    "sarscov2 - [fastq1, fastq2], index, fasta, true, false - bam": {
         "content": [
             "test.bam",
             [
@@ -231,15 +271,19 @@
                 ]
             ],
             [
-
+                
             ],
             [
                 "versions.yml:md5,01d18ab035146ea790e9a0f70adb758f"
             ]
         ],
-        "timestamp": "2023-11-23T11:40:46.647809118"
+        "meta": {
+            "nf-test": "0.8.4",
+            "nextflow": "23.10.1"
+        },
+        "timestamp": "2024-03-18T13:19:40.606135"
     },
-    "sarscov2 - [fastq1, fastq2], index, false, true - bam": {
+    "sarscov2 - [fastq1, fastq2], index, fasta, false, true - bam": {
         "content": [
             "test.bam",
             [
@@ -252,12 +296,16 @@
                 ]
             ],
             [
-
+                
             ],
             [
                 "versions.yml:md5,01d18ab035146ea790e9a0f70adb758f"
             ]
         ],
-        "timestamp": "2023-11-23T11:40:54.988626035"
+        "meta": {
+            "nf-test": "0.8.4",
+            "nextflow": "23.10.1"
+        },
+        "timestamp": "2024-03-18T13:18:54.194648"
     }
-}
+}
\ No newline at end of file
diff --git a/subworkflows/nf-core/fastq_align_bowtie2/main.nf b/subworkflows/nf-core/fastq_align_bowtie2/main.nf
index ba4420f7d3e..cafaa9bf931 100644
--- a/subworkflows/nf-core/fastq_align_bowtie2/main.nf
+++ b/subworkflows/nf-core/fastq_align_bowtie2/main.nf
@@ -20,17 +20,17 @@ workflow FASTQ_ALIGN_BOWTIE2 {
     //
     // Map reads with Bowtie2
     //
-    BOWTIE2_ALIGN ( ch_reads, ch_index, save_unaligned, sort_bam )
+    BOWTIE2_ALIGN ( ch_reads, ch_index, ch_fasta, save_unaligned, sort_bam )
     ch_versions = ch_versions.mix(BOWTIE2_ALIGN.out.versions)
 
     //
     // Sort, index BAM file and run samtools stats, flagstat and idxstats
     //
-    BAM_SORT_STATS_SAMTOOLS ( BOWTIE2_ALIGN.out.aligned, ch_fasta )
+    BAM_SORT_STATS_SAMTOOLS ( BOWTIE2_ALIGN.out.bam, ch_fasta )
     ch_versions = ch_versions.mix(BAM_SORT_STATS_SAMTOOLS.out.versions)
 
     emit:
-    bam_orig         = BOWTIE2_ALIGN.out.aligned      // channel: [ val(meta), aligned ]
+    bam_orig         = BOWTIE2_ALIGN.out.bam          // channel: [ val(meta), aligned ]
     log_out          = BOWTIE2_ALIGN.out.log          // channel: [ val(meta), log     ]
     fastq            = BOWTIE2_ALIGN.out.fastq        // channel: [ val(meta), fastq   ]
 
diff --git a/subworkflows/nf-core/fastq_align_bowtie2/tests/main.nf.test b/subworkflows/nf-core/fastq_align_bowtie2/tests/main.nf.test
new file mode 100644
index 00000000000..b5e84f512a3
--- /dev/null
+++ b/subworkflows/nf-core/fastq_align_bowtie2/tests/main.nf.test
@@ -0,0 +1,99 @@
+nextflow_workflow {
+
+    name "Test Subworkflow FASTQ_ALIGN_BOWTIE2"
+    script "../main.nf"
+    config "./nextflow.config"
+    workflow "FASTQ_ALIGN_BOWTIE2"
+
+    tag "subworkflows"
+    tag "subworkflows_nfcore"
+    tag "subworkflows/fastq_align_bowtie2"
+    tag "subworkflows/bam_sort_stats_samtools"
+    tag "bowtie2"
+    tag "bowtie2/build"
+    tag "bowtie2/align"
+
+    test("test_align_bowtie2_single_end") {
+        setup {
+            run("BOWTIE2_BUILD") {
+                script "../../../../modules/nf-core/bowtie2/build/main.nf"
+                process {
+                    """
+                    input[0] = Channel.value([ [ id:'genome' ],file(params.test_data['sarscov2']['genome']['genome_fasta'], checkIfExists: true)])
+                    """
+                }
+            }
+        }
+        when {
+            workflow {
+                """
+                input[0] = Channel.of([[ id:'test', single_end:true ], [ file(params.test_data['sarscov2']['illumina']['test_1_fastq_gz'], checkIfExists: true) ]])
+                input[1] = BOWTIE2_BUILD.out.index
+                input[2] = false
+                input[3] = false
+                input[4] = Channel.value([ [ id:'genome' ],file(params.test_data['sarscov2']['genome']['genome_fasta'], checkIfExists: true)])
+                """
+            }
+        }
+
+        then {
+            assertAll(
+                { assert workflow.success},
+                { assert snapshot(
+                    file(workflow.out.bam_orig[0][1]).name,
+                    workflow.out.fastq,
+                    workflow.out.log_out,
+                    file(workflow.out.bam[0][1]).name,
+                    file(workflow.out.bai[0][1]).name,
+                    workflow.out.csi,
+                    workflow.out.stats,
+                    workflow.out.flagstat,
+                    workflow.out.idxstats,
+                    workflow.out.versions,
+                ).match()}
+            )
+        }
+    }
+
+    test("test_align_bowtie2_paired_end") {
+        setup {
+            run("BOWTIE2_BUILD") {
+                script "../../../../modules/nf-core/bowtie2/build/main.nf"
+                process {
+                    """
+                    input[0] = Channel.value([ [ id:'genome' ],file(params.test_data['sarscov2']['genome']['genome_fasta'], checkIfExists: true)])
+                    """
+                }
+            }
+        }
+        when {
+            workflow {
+                """
+                input[0] = Channel.of([[ id:'test', single_end:false ], [file(params.test_data['sarscov2']['illumina']['test_1_fastq_gz'], checkIfExists: true), file(params.test_data['sarscov2']['illumina']['test_2_fastq_gz'], checkIfExists: true)]])
+                input[1] = BOWTIE2_BUILD.out.index
+                input[2] = false
+                input[3] = false
+                input[4] = Channel.value([ [ id:'genome' ],file(params.test_data['sarscov2']['genome']['genome_fasta'], checkIfExists: true)])
+                """
+            }
+        }
+
+        then {
+            assertAll(
+                { assert workflow.success},
+                { assert snapshot(
+                    file(workflow.out.bam_orig[0][1]).name,
+                    workflow.out.fastq,
+                    workflow.out.log_out,
+                    file(workflow.out.bam[0][1]).name,
+                    file(workflow.out.bai[0][1]).name,
+                    workflow.out.csi,
+                    workflow.out.stats,
+                    workflow.out.flagstat,
+                    workflow.out.idxstats,
+                    workflow.out.versions,
+                ).match()}
+            )
+        }
+    }
+}
diff --git a/subworkflows/nf-core/fastq_align_bowtie2/tests/main.nf.test.snap b/subworkflows/nf-core/fastq_align_bowtie2/tests/main.nf.test.snap
new file mode 100644
index 00000000000..c0f3f8bfb75
--- /dev/null
+++ b/subworkflows/nf-core/fastq_align_bowtie2/tests/main.nf.test.snap
@@ -0,0 +1,126 @@
+{
+    "test_align_bowtie2_single_end": {
+        "content": [
+            "test.bam",
+            [
+                
+            ],
+            [
+                [
+                    {
+                        "id": "test",
+                        "single_end": true
+                    },
+                    "test.bowtie2.log:md5,7b8a9e61b7646da1089b041333c41a87"
+                ]
+            ],
+            "test.sorted.bam",
+            "test.sorted.bam.bai",
+            [
+                
+            ],
+            [
+                [
+                    {
+                        "id": "test",
+                        "single_end": true
+                    },
+                    "test.sorted.bam.stats:md5,9a65272e49581873b1ea211f738e992f"
+                ]
+            ],
+            [
+                [
+                    {
+                        "id": "test",
+                        "single_end": true
+                    },
+                    "test.sorted.bam.flagstat:md5,e9ce9093133116bc54fd335cfe698372"
+                ]
+            ],
+            [
+                [
+                    {
+                        "id": "test",
+                        "single_end": true
+                    },
+                    "test.sorted.bam.idxstats:md5,e16eb632f7f462514b0873c7ac8ac905"
+                ]
+            ],
+            [
+                "versions.yml:md5,5d5ab1d650a93d8bb5ed142943798a6a",
+                "versions.yml:md5,666dbae2343fc479e483656c35d3d8a1",
+                "versions.yml:md5,aab337e63eac9055aadb9a35cec16053",
+                "versions.yml:md5,c27f74d9c37fbb3365c437a9f7e81c27",
+                "versions.yml:md5,eb9364a9f1745d6a345b8b4b03aebe25",
+                "versions.yml:md5,f982efa9031f340ace29f76dd47a8ce1"
+            ]
+        ],
+        "meta": {
+            "nf-test": "0.8.4",
+            "nextflow": "23.10.1"
+        },
+        "timestamp": "2024-03-18T14:40:54.318808117"
+    },
+    "test_align_bowtie2_paired_end": {
+        "content": [
+            "test.bam",
+            [
+                
+            ],
+            [
+                [
+                    {
+                        "id": "test",
+                        "single_end": false
+                    },
+                    "test.bowtie2.log:md5,bd89ce1b28c93bf822bae391ffcedd19"
+                ]
+            ],
+            "test.sorted.bam",
+            "test.sorted.bam.bai",
+            [
+                
+            ],
+            [
+                [
+                    {
+                        "id": "test",
+                        "single_end": false
+                    },
+                    "test.sorted.bam.stats:md5,1086d408391af2a5c80c6dee0efa7e59"
+                ]
+            ],
+            [
+                [
+                    {
+                        "id": "test",
+                        "single_end": false
+                    },
+                    "test.sorted.bam.flagstat:md5,49f3d51a8804ce58fe9cecd2549d279b"
+                ]
+            ],
+            [
+                [
+                    {
+                        "id": "test",
+                        "single_end": false
+                    },
+                    "test.sorted.bam.idxstats:md5,29ff2fa56d35b2a47625b8f517f1a947"
+                ]
+            ],
+            [
+                "versions.yml:md5,5d5ab1d650a93d8bb5ed142943798a6a",
+                "versions.yml:md5,666dbae2343fc479e483656c35d3d8a1",
+                "versions.yml:md5,aab337e63eac9055aadb9a35cec16053",
+                "versions.yml:md5,c27f74d9c37fbb3365c437a9f7e81c27",
+                "versions.yml:md5,eb9364a9f1745d6a345b8b4b03aebe25",
+                "versions.yml:md5,f982efa9031f340ace29f76dd47a8ce1"
+            ]
+        ],
+        "meta": {
+            "nf-test": "0.8.4",
+            "nextflow": "23.10.1"
+        },
+        "timestamp": "2024-03-18T14:41:11.243874685"
+    }
+}
\ No newline at end of file
diff --git a/tests/subworkflows/nf-core/fastq_align_bowtie2/nextflow.config b/subworkflows/nf-core/fastq_align_bowtie2/tests/nextflow.config
similarity index 56%
rename from tests/subworkflows/nf-core/fastq_align_bowtie2/nextflow.config
rename to subworkflows/nf-core/fastq_align_bowtie2/tests/nextflow.config
index 6ec89fcff3f..2f85e8075fc 100644
--- a/tests/subworkflows/nf-core/fastq_align_bowtie2/nextflow.config
+++ b/subworkflows/nf-core/fastq_align_bowtie2/tests/nextflow.config
@@ -1,17 +1,8 @@
 process {
-
-    publishDir = { "${params.outdir}/${task.process.tokenize(':')[-1].tokenize('_')[0].toLowerCase()}" }
-
-    withName: BOWTIE2_BUILD {
-        publishDir = [ enabled: false ]
-    }
-
     withName: '.*:BAM_SORT_STATS_SAMTOOLS:SAMTOOLS_.*' {
         ext.prefix = { "${meta.id}.sorted" }
     }
-
     withName: '.*:BAM_SORT_STATS_SAMTOOLS:BAM_STATS_SAMTOOLS:.*' {
         ext.prefix = { "${meta.id}.sorted.bam" }
     }
-
 }
diff --git a/subworkflows/nf-core/fastq_align_bowtie2/tests/tags.yml b/subworkflows/nf-core/fastq_align_bowtie2/tests/tags.yml
new file mode 100644
index 00000000000..267bcc772c0
--- /dev/null
+++ b/subworkflows/nf-core/fastq_align_bowtie2/tests/tags.yml
@@ -0,0 +1,2 @@
+subworkflows/fastq_align_bowtie2:
+  - subworkflows/nf-core/fastq_align_bowtie2/**
diff --git a/subworkflows/nf-core/fastq_align_dna/main.nf b/subworkflows/nf-core/fastq_align_dna/main.nf
index aa86d46aa0f..f907b16e47a 100644
--- a/subworkflows/nf-core/fastq_align_dna/main.nf
+++ b/subworkflows/nf-core/fastq_align_dna/main.nf
@@ -31,29 +31,29 @@ workflow FASTQ_ALIGN_DNA {
         // Align fastq files to reference genome and (optionally) sort
         switch (aligner) {
             case 'bowtie2':
-                BOWTIE2_ALIGN(ch_reads, ch_aligner_index, false, sort) // if aligner is bowtie2
-                ch_bam = ch_bam.mix(BOWTIE2_ALIGN.out.aligned)
+                BOWTIE2_ALIGN(ch_reads, ch_aligner_index, ch_fasta, false, sort) // if aligner is bowtie2
+                ch_bam = ch_bam.mix(BOWTIE2_ALIGN.out.bam)
                 ch_versions = ch_versions.mix(BOWTIE2_ALIGN.out.versions)
                 break
             case 'bwamem':
-                BWAMEM1_MEM  (ch_reads, ch_aligner_index, ch_fasta, sort) // If aligner is bwa-mem
+                BWAMEM1_MEM  (ch_reads, ch_aligner_index, ch_fasta, sort)        // If aligner is bwa-mem
                 ch_bam = ch_bam.mix(BWAMEM1_MEM.out.bam)
                 ch_bam_index = ch_bam_index.mix(BWAMEM1_MEM.out.csi)
                 ch_versions = ch_versions.mix(BWAMEM1_MEM.out.versions)
                 break
             case 'bwamem2':
-                BWAMEM2_MEM  (ch_reads, ch_aligner_index, sort)        // If aligner is bwa-mem2
+                BWAMEM2_MEM  (ch_reads, ch_aligner_index, sort)                  // If aligner is bwa-mem2
                 ch_bam = ch_bam.mix(BWAMEM2_MEM.out.bam)
                 ch_versions = ch_versions.mix(BWAMEM2_MEM.out.versions)
                 break
             case 'dragmap':
-                DRAGMAP_ALIGN(ch_reads, ch_aligner_index, sort)        // If aligner is dragmap
+                DRAGMAP_ALIGN(ch_reads, ch_aligner_index, sort)                 // If aligner is dragmap
                 ch_bam = ch_bam.mix(DRAGMAP_ALIGN.out.bam)
                 ch_reports = ch_reports.mix(DRAGMAP_ALIGN.out.log)
                 ch_versions = ch_versions.mix(DRAGMAP_ALIGN.out.versions)
                 break
             case 'snap':
-                SNAP_ALIGN   (ch_reads, ch_aligner_index)              // If aligner is snap
+                SNAP_ALIGN   (ch_reads, ch_aligner_index)                       // If aligner is snap
                 ch_bam = ch_bam.mix(SNAP_ALIGN.out.bam)
                 ch_bam_index.mix(SNAP_ALIGN.out.bai)
                 ch_versions = ch_versions.mix(SNAP_ALIGN.out.versions)
diff --git a/tests/config/pytest_modules.yml b/tests/config/pytest_modules.yml
index fa95fc6f07e..4011a5e99d2 100644
--- a/tests/config/pytest_modules.yml
+++ b/tests/config/pytest_modules.yml
@@ -2154,9 +2154,6 @@ subworkflows/fasta_newick_epang_gappa:
 subworkflows/fastq_align_bamcmp_bwa:
   - subworkflows/nf-core/fastq_align_bamcmp_bwa/**
   - tests/subworkflows/nf-core/fastq_align_bamcmp_bwa/**
-subworkflows/fastq_align_bowtie2:
-  - subworkflows/nf-core/fastq_align_bowtie2/**
-  - tests/subworkflows/nf-core/fastq_align_bowtie2/**
 subworkflows/fastq_align_bwaaln:
   - subworkflows/nf-core/fastq_align_bwaaln/**
   - tests/subworkflows/nf-core/fastq_align_bwaaln/**
diff --git a/tests/subworkflows/nf-core/fastq_align_bowtie2/main.nf b/tests/subworkflows/nf-core/fastq_align_bowtie2/main.nf
deleted file mode 100644
index 7c4508f742a..00000000000
--- a/tests/subworkflows/nf-core/fastq_align_bowtie2/main.nf
+++ /dev/null
@@ -1,44 +0,0 @@
-#!/usr/bin/env nextflow
-
-nextflow.enable.dsl = 2
-
-include { BOWTIE2_BUILD } from '../../../../modules/nf-core/bowtie2/build/main.nf'
-include { FASTQ_ALIGN_BOWTIE2 } from '../../../../subworkflows/nf-core/fastq_align_bowtie2/main.nf'
-
-
-workflow test_align_bowtie2_single_end {
-    input = [
-        [ id:'test', single_end:true ], // meta map
-        [ file(params.test_data['sarscov2']['illumina']['test_1_fastq_gz'], checkIfExists: true) ]
-    ]
-    fasta = [
-        [id: 'test'],
-        file(params.test_data['sarscov2']['genome']['genome_fasta'], checkIfExists: true)
-    ]
-    save_unaligned = false
-    sort = false
-
-    BOWTIE2_BUILD ( fasta )
-    FASTQ_ALIGN_BOWTIE2 ( input, BOWTIE2_BUILD.out.index, save_unaligned, sort, [[],[]] )
-}
-
-workflow test_align_bowtie2_paired_end {
-    input = [
-        [ id:'test', single_end:false ], // meta map
-        [
-            file(params.test_data['sarscov2']['illumina']['test_1_fastq_gz'], checkIfExists: true),
-            file(params.test_data['sarscov2']['illumina']['test_2_fastq_gz'], checkIfExists: true)
-        ]
-    ]
-
-    fasta = [
-        [id: 'test'],
-        file(params.test_data['sarscov2']['genome']['genome_fasta'], checkIfExists: true)
-    ]
-    save_unaligned = false
-    sort = false
-
-    BOWTIE2_BUILD ( fasta )
-    FASTQ_ALIGN_BOWTIE2 ( input, BOWTIE2_BUILD.out.index, save_unaligned, sort, [[],[]] )
-}
-
diff --git a/tests/subworkflows/nf-core/fastq_align_bowtie2/test.yml b/tests/subworkflows/nf-core/fastq_align_bowtie2/test.yml
deleted file mode 100644
index cc5626f9ec6..00000000000
--- a/tests/subworkflows/nf-core/fastq_align_bowtie2/test.yml
+++ /dev/null
@@ -1,63 +0,0 @@
-- name: align bowtie2 single-end
-  command: nextflow run ./tests/subworkflows/nf-core/fastq_align_bowtie2 -entry test_align_bowtie2_single_end -c tests/config/nextflow.config -c ./tests/subworkflows/nf-core/fastq_align_bowtie2/nextflow.config
-  tags:
-    - subworkflows
-    - subworkflows/fastq_align_bowtie2
-    - subworkflows/bam_sort_stats_samtools
-    - subworkflows/bam_stats_samtools
-    # Modules
-    - bowtie2
-    - bowtie2/align
-    - samtools
-    - samtools/index
-    - samtools/sort
-    - samtools/stats
-    - samtools/idxstats
-    - samtools/flagstat
-  files:
-    - path: ./output/bowtie2/test.bam
-    - path: ./output/bowtie2/test.bowtie2.log
-    - path: ./output/bowtie2/versions.yml
-      md5sum: 3f547f37f48a6aeb573ba5255d30f210
-      # samtools sort
-    - path: ./output/samtools/test.sorted.bam
-    - path: ./output/samtools/test.sorted.bam.bai
-      # samtools stats
-    - path: ./output/samtools/test.sorted.bam.flagstat
-      md5sum: e9ce9093133116bc54fd335cfe698372
-    - path: ./output/samtools/test.sorted.bam.idxstats
-      md5sum: e16eb632f7f462514b0873c7ac8ac905
-    - path: ./output/samtools/test.sorted.bam.stats
-    - path: ./output/samtools/versions.yml
-
-- name: align bowtie2 paired-end
-  command: nextflow run ./tests/subworkflows/nf-core/fastq_align_bowtie2 -entry test_align_bowtie2_paired_end -c tests/config/nextflow.config -c ./tests/subworkflows/nf-core/fastq_align_bowtie2/nextflow.config
-  tags:
-    - subworkflows
-    - subworkflows/bam_sort_stats_samtools
-    - subworkflows/fastq_align_bowtie2
-    - subworkflows/bam_stats_samtools
-    # Modules
-    - bowtie2
-    - bowtie2/align
-    - samtools
-    - samtools/index
-    - samtools/sort
-    - samtools/stats
-    - samtools/idxstats
-    - samtools/flagstat
-  files:
-    - path: ./output/bowtie2/test.bam
-    - path: ./output/bowtie2/test.bowtie2.log
-    - path: ./output/bowtie2/versions.yml
-      md5sum: 2b6500ccfa1eeb1e8a9a158ca997a41d
-      # samtools sort
-    - path: ./output/samtools/test.sorted.bam
-    - path: ./output/samtools/test.sorted.bam.bai
-      # samtools stats
-    - path: ./output/samtools/test.sorted.bam.flagstat
-      md5sum: 49f3d51a8804ce58fe9cecd2549d279b
-    - path: ./output/samtools/test.sorted.bam.idxstats
-      md5sum: 29ff2fa56d35b2a47625b8f517f1a947
-    - path: ./output/samtools/test.sorted.bam.stats
-    - path: ./output/bowtie2/versions.yml