diff --git a/CHANGELOG.md b/CHANGELOG.md
index bb4155168..05a0bb006 100644
--- a/CHANGELOG.md
+++ b/CHANGELOG.md
@@ -17,6 +17,7 @@ Special thanks to the following for their contributions to the release:
 - [PR #1398](https://github.com/nf-core/rnaseq/pull/1398) - Bump pipeline version to 3.17.0dev
 - [PR #1401](https://github.com/nf-core/rnaseq/pull/1401) - Template update for nf-core/tools v3.0.1
 - [PR #1405](https://github.com/nf-core/rnaseq/pull/1405) - Fix bad variable name in subworkflow
+- [PR #1406](https://github.com/nf-core/rnaseq/pull/1406) - Keep only one samplesheetToList
 
 ### Parameters
 
diff --git a/subworkflows/local/utils_nfcore_rnaseq_pipeline/main.nf b/subworkflows/local/utils_nfcore_rnaseq_pipeline/main.nf
index 16936fe31..aa0dd4ed7 100644
--- a/subworkflows/local/utils_nfcore_rnaseq_pipeline/main.nf
+++ b/subworkflows/local/utils_nfcore_rnaseq_pipeline/main.nf
@@ -12,7 +12,6 @@ import groovy.json.JsonSlurper
 
 include { UTILS_NFSCHEMA_PLUGIN     } from '../../nf-core/utils_nfschema_plugin'
 include { paramsSummaryMap          } from 'plugin/nf-schema'
-include { samplesheetToList         } from 'plugin/nf-schema'
 include { completionEmail           } from '../../nf-core/utils_nfcore_pipeline'
 include { completionSummary         } from '../../nf-core/utils_nfcore_pipeline'
 include { imNotification            } from '../../nf-core/utils_nfcore_pipeline'
@@ -71,28 +70,7 @@ workflow PIPELINE_INITIALISATION {
     //
     validateInputParameters()
 
-    //
-    // Create channel from input file provided through params.input
-    //
-
-    Channel
-        .fromList(samplesheetToList(params.input, "${projectDir}/assets/schema_input.json"))
-        .map {
-            meta, fastq_1, fastq_2 ->
-                if (!fastq_2) {
-                    return [ meta.id, meta + [ single_end:true ], [ fastq_1 ] ]
-                } else {
-                    return [ meta.id, meta + [ single_end:false ], [ fastq_1, fastq_2 ] ]
-                }
-        }
-        .groupTuple()
-        .map {
-            checkSamplesAfterGrouping(it)
-        }
-        .set{ ch_samplesheet }
-
     emit:
-    samplesheet = ch_samplesheet
     versions    = ch_versions
 }
 
diff --git a/workflows/rnaseq/main.nf b/workflows/rnaseq/main.nf
index 2ac6dcf86..84bedaeb6 100755
--- a/workflows/rnaseq/main.nf
+++ b/workflows/rnaseq/main.nf
@@ -121,10 +121,10 @@ workflow RNASEQ {
                 }
         }
         .groupTuple()
-        .map {
-            checkSamplesAfterGrouping(it)
+        .map { samplesheet ->
+            checkSamplesAfterGrouping(samplesheet)
         }
-        .set{ ch_fastq }
+        .set { ch_fastq }
 
     //
     // Run RNA-seq FASTQ preprocessing subworkflow