From b308ac05c09212f993129c19d64923a7632026bc Mon Sep 17 00:00:00 2001
From: Jonathan Manning <jonathan.manning@healx.io>
Date: Mon, 21 Nov 2022 14:56:04 +0000
Subject: [PATCH 01/12] Add control feature handling for deseq2

---
 modules/nf-core/deseq2/differential/main.nf   |  1 +
 .../deseq2/differential/templates/deseq_de.R  |  7 +++
 .../nf-core/deseq2/differential/main.nf       | 62 ++++++++++++++++++-
 .../deseq2/differential/nextflow.config       |  3 +
 .../nf-core/deseq2/differential/test.yml      | 31 +++++++++-
 5 files changed, 98 insertions(+), 6 deletions(-)

diff --git a/modules/nf-core/deseq2/differential/main.nf b/modules/nf-core/deseq2/differential/main.nf
index 3ebf9fbfd9c..19a16359590 100644
--- a/modules/nf-core/deseq2/differential/main.nf
+++ b/modules/nf-core/deseq2/differential/main.nf
@@ -9,6 +9,7 @@ process DESEQ2_DIFFERENTIAL {
 
     input:
     tuple val(meta), path(samplesheet), path(counts)
+    tuple val(control_genes_meta), path(control_genes_file) 
 
     output:
     tuple val(meta), path("*.deseq2.results.tsv")              , emit: results
diff --git a/modules/nf-core/deseq2/differential/templates/deseq_de.R b/modules/nf-core/deseq2/differential/templates/deseq_de.R
index 5e1cd307b7b..3d4a9d74d9b 100755
--- a/modules/nf-core/deseq2/differential/templates/deseq_de.R
+++ b/modules/nf-core/deseq2/differential/templates/deseq_de.R
@@ -95,6 +95,7 @@ opt <- list(
     reference_level = NULL,
     treatment_level = NULL,
     blocking_variables = NULL,
+    control_genes_file = '$control_genes_file',
     gene_id_col = "gene_id",
     sample_id_col = "experiment_accession",
     test = "Wald",
@@ -274,6 +275,12 @@ dds <- DESeqDataSetFromMatrix(
     design = as.formula(model)
 )
 
+if (opt\$control_genes_file != ''){
+    control_genes <- readLines(opt\$control_genes_file)
+    print(paste('Estimating size factors using', length(control_genes), 'control genes'))
+    dds <- estimateSizeFactors(dds, controlGenes=rownames(count.table) %in% control_genes)
+}
+
 dds <- DESeq(
     dds,
     test = opt\$test,
diff --git a/tests/modules/nf-core/deseq2/differential/main.nf b/tests/modules/nf-core/deseq2/differential/main.nf
index 0420fa8ae13..d0d75463d09 100644
--- a/tests/modules/nf-core/deseq2/differential/main.nf
+++ b/tests/modules/nf-core/deseq2/differential/main.nf
@@ -20,6 +20,27 @@ process tsv_to_csv {
 
 }
 
+// Take the first 50 genes and pretend they're spikes for testing control gene
+// functionality
+
+process spoof_spikes {
+
+    input:
+    path expression_matrix
+
+    output:
+    path 'spikes.txt'
+
+    script:
+    """
+    head -n 50 $expression_matrix | \
+        tail -n +2 | \
+        awk '{print \$1}' > spikes.txt.tmp
+    mv spikes.txt.tmp spikes.txt
+    """
+}
+empty_spikes = [[],[]]
+
 workflow test_deseq2_differential {
 
     expression_sample_sheet = file(params.test_data['mus_musculus']['genome']['rnaseq_samplesheet'], checkIfExists: true)
@@ -36,7 +57,40 @@ workflow test_deseq2_differential {
         }
 
     DESEQ2_DIFFERENTIAL (
-        input
+        input,
+        empty_spikes
+    )
+}
+
+// Try with spikes as control genes
+
+workflow test_deseq2_differential_spikes {
+
+    expression_sample_sheet = file(params.test_data['mus_musculus']['genome']['rnaseq_samplesheet'], checkIfExists: true)
+    expression_matrix_file = file(params.test_data['mus_musculus']['genome']['rnaseq_matrix'], checkIfExists: true)
+    expression_contrasts = file(params.test_data['mus_musculus']['genome']['rnaseq_contrasts'], checkIfExists: true)
+
+    Channel.fromPath(expression_contrasts)
+        .splitCsv ( header:true, sep:',' )
+        .map{
+            tuple(it, expression_sample_sheet, expression_matrix_file)
+        }
+        .set{
+            input
+        }
+
+    // Make our fake spikes and pretent they're ERCC controls
+
+    spoof_spikes(expression_matrix_file)
+        .map{
+            tuple(['id':'ERCC'], it)
+        }.set{
+            ch_spikes
+        }
+
+    DESEQ2_DIFFERENTIAL (
+        input,
+        ch_spikes
     )
 }
 
@@ -58,7 +112,8 @@ workflow test_deseq2_differential_csv {
         .splitCsv ( header:true, sep:',' )
 
     DESEQ2_DIFFERENTIAL (
-        ch_contrasts.combine(ch_samples_and_matrix)
+        ch_contrasts.combine(ch_samples_and_matrix),
+        empty_spikes
     )
 }
 
@@ -80,6 +135,7 @@ workflow test_deseq2_differential_vst_nsub {
         }
 
     DESEQ2_DIFFERENTIAL (
-        input
+        input,
+        empty_spikes
     )
 }
diff --git a/tests/modules/nf-core/deseq2/differential/nextflow.config b/tests/modules/nf-core/deseq2/differential/nextflow.config
index 8e732f15d28..2db1adb1af4 100644
--- a/tests/modules/nf-core/deseq2/differential/nextflow.config
+++ b/tests/modules/nf-core/deseq2/differential/nextflow.config
@@ -5,6 +5,9 @@ process {
     withName: 'test_deseq2_differential:DESEQ2_DIFFERENTIAL' {
         ext.args = { "--contrast_variable $meta.variable --reference_level $meta.reference --treatment_level $meta.target --blocking_variables $meta.blocking --vs_method rlog" }
     }
+    withName: 'test_deseq2_differential_spikes:DESEQ2_DIFFERENTIAL' {
+        ext.args = { "--contrast_variable $meta.variable --reference_level $meta.reference --treatment_level $meta.target --blocking_variables $meta.blocking --vs_method rlog" }
+    }
     withName: 'test_deseq2_differential_csv:DESEQ2_DIFFERENTIAL' {
         ext.args = { "--contrast_variable $meta.variable --reference_level $meta.reference --treatment_level $meta.target --blocking_variables $meta.blocking --vs_method rlog" }
     }
diff --git a/tests/modules/nf-core/deseq2/differential/test.yml b/tests/modules/nf-core/deseq2/differential/test.yml
index 8f062d9e37e..b9dbe681aa1 100644
--- a/tests/modules/nf-core/deseq2/differential/test.yml
+++ b/tests/modules/nf-core/deseq2/differential/test.yml
@@ -1,8 +1,8 @@
 - name: deseq2 differential test_deseq2_differential
   command: nextflow run ./tests/modules/nf-core/deseq2/differential -entry test_deseq2_differential -c ./tests/config/nextflow.config -c ./tests/modules/nf-core/deseq2/differential/nextflow.config
   tags:
-    - deseq2
     - deseq2/differential
+    - deseq2
   files:
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.deseq2.results.tsv
       md5sum: 86f0312a4691dfb4d7c15643059d8fb8
@@ -21,11 +21,36 @@
     - path: output/deseq2/treatment-mCherry-hND6.rlog.tsv
       md5sum: 1077cf03a8c6a155932eaaed7415b74c
 
+- name: deseq2 differential test_deseq2_differential_spikes
+  command: nextflow run ./tests/modules/nf-core/deseq2/differential -entry test_deseq2_differential_spikes -c ./tests/config/nextflow.config -c ./tests/modules/nf-core/deseq2/differential/nextflow.config
+  tags:
+    - deseq2/differential
+    - deseq2
+  files:
+    - path: output/deseq2/treatment-mCherry-hND6-sample_number.deseq2.results.tsv
+      md5sum: 150a32eef482244b47ddc7f7ffb1ad2a
+    - path: output/deseq2/treatment-mCherry-hND6-sample_number.deseq2.sizefactors.tsv
+      md5sum: 1708374146b4e8c28fbc78fa292102cd
+    - path: output/deseq2/treatment-mCherry-hND6-sample_number.normalised_counts.tsv
+      md5sum: 1c9c7ee4ca90ff0d91ae16dbfd64fdc9
+    - path: output/deseq2/treatment-mCherry-hND6-sample_number.rlog.tsv
+      md5sum: c28300837df9ca6adeba2a52ea5e181e
+    - path: output/deseq2/treatment-mCherry-hND6.deseq2.results.tsv
+      md5sum: 88b3230c5a4ad8717c1d0b38c824f1fc
+    - path: output/deseq2/treatment-mCherry-hND6.deseq2.sizefactors.tsv
+      md5sum: 1708374146b4e8c28fbc78fa292102cd
+    - path: output/deseq2/treatment-mCherry-hND6.normalised_counts.tsv
+      md5sum: 1c9c7ee4ca90ff0d91ae16dbfd64fdc9
+    - path: output/deseq2/treatment-mCherry-hND6.rlog.tsv
+      md5sum: c28300837df9ca6adeba2a52ea5e181e
+    - path: output/spoof/spikes.txt
+      md5sum: 8bd8a80b4616fc0a68f69bee6869c8bc
+
 - name: deseq2 differential test_deseq2_differential_csv
   command: nextflow run ./tests/modules/nf-core/deseq2/differential -entry test_deseq2_differential_csv -c ./tests/config/nextflow.config -c ./tests/modules/nf-core/deseq2/differential/nextflow.config
   tags:
-    - deseq2
     - deseq2/differential
+    - deseq2
   files:
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.deseq2.results.tsv
       md5sum: 86f0312a4691dfb4d7c15643059d8fb8
@@ -49,8 +74,8 @@
 - name: deseq2 differential test_deseq2_differential_vst_nsub
   command: nextflow run ./tests/modules/nf-core/deseq2/differential -entry test_deseq2_differential_vst_nsub -c ./tests/config/nextflow.config -c ./tests/modules/nf-core/deseq2/differential/nextflow.config
   tags:
-    - deseq2
     - deseq2/differential
+    - deseq2
   files:
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.deseq2.results.tsv
       md5sum: 86f0312a4691dfb4d7c15643059d8fb8

From d9601746787cc745d56747f16c5134df571b23cd Mon Sep 17 00:00:00 2001
From: Jonathan Manning <jonathan.manning@healx.io>
Date: Mon, 21 Nov 2022 14:59:40 +0000
Subject: [PATCH 02/12] Update docs for control genes

---
 modules/nf-core/deseq2/differential/meta.yml | 8 ++++++++
 1 file changed, 8 insertions(+)

diff --git a/modules/nf-core/deseq2/differential/meta.yml b/modules/nf-core/deseq2/differential/meta.yml
index 2625a3e8335..200cacf35ee 100644
--- a/modules/nf-core/deseq2/differential/meta.yml
+++ b/modules/nf-core/deseq2/differential/meta.yml
@@ -31,6 +31,14 @@ input:
         calls at the pipeline level e.g. [ variable:'treatment', reference:'treated',
         control:'saline', blocking:'' ] passed in as ext.args like: '--reference_level
         $meta.reference --treatment_level $meta.target'
+  - control_genes:
+      type: file
+      description: |
+        Text file listing control genes, one per line
+  - control_genes_meta:
+      type: file
+      description: |
+        Meta map describing control genes, e.g. [ id: 'ERCC' ]
 
 output:
   - results:

From ddbcd7c3087db1d63bcc42312fb0221bbf195130 Mon Sep 17 00:00:00 2001
From: Jonathan Manning <jonathan.manning@healx.io>
Date: Mon, 21 Nov 2022 15:06:19 +0000
Subject: [PATCH 03/12] Appease eclint

---
 modules/nf-core/deseq2/differential/main.nf | 2 +-
 1 file changed, 1 insertion(+), 1 deletion(-)

diff --git a/modules/nf-core/deseq2/differential/main.nf b/modules/nf-core/deseq2/differential/main.nf
index 19a16359590..6221c7854ab 100644
--- a/modules/nf-core/deseq2/differential/main.nf
+++ b/modules/nf-core/deseq2/differential/main.nf
@@ -9,7 +9,7 @@ process DESEQ2_DIFFERENTIAL {
 
     input:
     tuple val(meta), path(samplesheet), path(counts)
-    tuple val(control_genes_meta), path(control_genes_file) 
+    tuple val(control_genes_meta), path(control_genes_file)
 
     output:
     tuple val(meta), path("*.deseq2.results.tsv")              , emit: results

From 9ca07bdc2afea756772bf2c8c7ea8a9cfc80b6b4 Mon Sep 17 00:00:00 2001
From: Jonathan Manning <jonathan.manning@healx.io>
Date: Mon, 21 Nov 2022 15:44:47 +0000
Subject: [PATCH 04/12] Allow stripping of spikes when not being used for
 normalisation

---
 .../nf-core/deseq2/differential/templates/deseq_de.R  | 11 +++++++++--
 .../nf-core/deseq2/differential/nextflow.config       |  2 +-
 2 files changed, 10 insertions(+), 3 deletions(-)

diff --git a/modules/nf-core/deseq2/differential/templates/deseq_de.R b/modules/nf-core/deseq2/differential/templates/deseq_de.R
index 3d4a9d74d9b..67448d6d7d2 100755
--- a/modules/nf-core/deseq2/differential/templates/deseq_de.R
+++ b/modules/nf-core/deseq2/differential/templates/deseq_de.R
@@ -96,6 +96,7 @@ opt <- list(
     treatment_level = NULL,
     blocking_variables = NULL,
     control_genes_file = '$control_genes_file',
+    sizefactors_from_controls = FALSE,
     gene_id_col = "gene_id",
     sample_id_col = "experiment_accession",
     test = "Wald",
@@ -269,14 +270,20 @@ model <- paste(model, opt\$contrast_variable, sep = ' + ')
 ################################################
 ################################################
 
+if (opt\$control_genes_file != ''){
+    control_genes <- readLines(opt\$control_genes_file)
+    if (! opt\$sizefactors_from_controls){
+        count_table <- count_table[setdiff(rownames(count_table), control_genes),]
+    }
+}
+
 dds <- DESeqDataSetFromMatrix(
     countData = round(count.table),
     colData = sample.sheet,
     design = as.formula(model)
 )
 
-if (opt\$control_genes_file != ''){
-    control_genes <- readLines(opt\$control_genes_file)
+if (opt\$control_genes_file != '' && opt\$sizefactors_from_controls){
     print(paste('Estimating size factors using', length(control_genes), 'control genes'))
     dds <- estimateSizeFactors(dds, controlGenes=rownames(count.table) %in% control_genes)
 }
diff --git a/tests/modules/nf-core/deseq2/differential/nextflow.config b/tests/modules/nf-core/deseq2/differential/nextflow.config
index 2db1adb1af4..dae3c74e82c 100644
--- a/tests/modules/nf-core/deseq2/differential/nextflow.config
+++ b/tests/modules/nf-core/deseq2/differential/nextflow.config
@@ -6,7 +6,7 @@ process {
         ext.args = { "--contrast_variable $meta.variable --reference_level $meta.reference --treatment_level $meta.target --blocking_variables $meta.blocking --vs_method rlog" }
     }
     withName: 'test_deseq2_differential_spikes:DESEQ2_DIFFERENTIAL' {
-        ext.args = { "--contrast_variable $meta.variable --reference_level $meta.reference --treatment_level $meta.target --blocking_variables $meta.blocking --vs_method rlog" }
+        ext.args = { "--sizefactors_from_controls TRUE --contrast_variable $meta.variable --reference_level $meta.reference --treatment_level $meta.target --blocking_variables $meta.blocking --vs_method rlog" }
     }
     withName: 'test_deseq2_differential_csv:DESEQ2_DIFFERENTIAL' {
         ext.args = { "--contrast_variable $meta.variable --reference_level $meta.reference --treatment_level $meta.target --blocking_variables $meta.blocking --vs_method rlog" }

From 2adf0643638f503d141963ab75db895b6aeefec9 Mon Sep 17 00:00:00 2001
From: Jonathan Manning <jonathan.manning@healx.io>
Date: Mon, 21 Nov 2022 16:24:50 +0000
Subject: [PATCH 05/12] Add test for spike stripping

---
 .../deseq2/differential/templates/deseq_de.R  |  2 +-
 .../nf-core/deseq2/differential/main.nf       | 34 ++++++++++++++++++-
 .../deseq2/differential/nextflow.config       |  3 ++
 .../nf-core/deseq2/differential/test.yml      | 33 +++++++++++++++---
 4 files changed, 66 insertions(+), 6 deletions(-)

diff --git a/modules/nf-core/deseq2/differential/templates/deseq_de.R b/modules/nf-core/deseq2/differential/templates/deseq_de.R
index 67448d6d7d2..0aa0979e752 100755
--- a/modules/nf-core/deseq2/differential/templates/deseq_de.R
+++ b/modules/nf-core/deseq2/differential/templates/deseq_de.R
@@ -273,7 +273,7 @@ model <- paste(model, opt\$contrast_variable, sep = ' + ')
 if (opt\$control_genes_file != ''){
     control_genes <- readLines(opt\$control_genes_file)
     if (! opt\$sizefactors_from_controls){
-        count_table <- count_table[setdiff(rownames(count_table), control_genes),]
+        count.table <- count.table[setdiff(rownames(count.table), control_genes),]
     }
 }
 
diff --git a/tests/modules/nf-core/deseq2/differential/main.nf b/tests/modules/nf-core/deseq2/differential/main.nf
index d0d75463d09..2330494f673 100644
--- a/tests/modules/nf-core/deseq2/differential/main.nf
+++ b/tests/modules/nf-core/deseq2/differential/main.nf
@@ -79,7 +79,39 @@ workflow test_deseq2_differential_spikes {
             input
         }
 
-    // Make our fake spikes and pretent they're ERCC controls
+    // Make our fake spikes and pretend they're ERCC controls
+
+    spoof_spikes(expression_matrix_file)
+        .map{
+            tuple(['id':'ERCC'], it)
+        }.set{
+            ch_spikes
+        }
+
+    DESEQ2_DIFFERENTIAL (
+        input,
+        ch_spikes
+    )
+}
+
+// Try with spikes as control genes, but stripping rather than using
+
+workflow test_deseq2_differential_strip_spikes {
+
+    expression_sample_sheet = file(params.test_data['mus_musculus']['genome']['rnaseq_samplesheet'], checkIfExists: true)
+    expression_matrix_file = file(params.test_data['mus_musculus']['genome']['rnaseq_matrix'], checkIfExists: true)
+    expression_contrasts = file(params.test_data['mus_musculus']['genome']['rnaseq_contrasts'], checkIfExists: true)
+
+    Channel.fromPath(expression_contrasts)
+        .splitCsv ( header:true, sep:',' )
+        .map{
+            tuple(it, expression_sample_sheet, expression_matrix_file)
+        }
+        .set{
+            input
+        }
+
+    // Make our fake spikes and pretend they're ERCC controls
 
     spoof_spikes(expression_matrix_file)
         .map{
diff --git a/tests/modules/nf-core/deseq2/differential/nextflow.config b/tests/modules/nf-core/deseq2/differential/nextflow.config
index dae3c74e82c..3c6cabd6792 100644
--- a/tests/modules/nf-core/deseq2/differential/nextflow.config
+++ b/tests/modules/nf-core/deseq2/differential/nextflow.config
@@ -8,6 +8,9 @@ process {
     withName: 'test_deseq2_differential_spikes:DESEQ2_DIFFERENTIAL' {
         ext.args = { "--sizefactors_from_controls TRUE --contrast_variable $meta.variable --reference_level $meta.reference --treatment_level $meta.target --blocking_variables $meta.blocking --vs_method rlog" }
     }
+    withName: 'test_deseq2_differential_strip_spikes:DESEQ2_DIFFERENTIAL' {
+        ext.args = { "--contrast_variable $meta.variable --reference_level $meta.reference --treatment_level $meta.target --blocking_variables $meta.blocking --vs_method rlog" }
+    }
     withName: 'test_deseq2_differential_csv:DESEQ2_DIFFERENTIAL' {
         ext.args = { "--contrast_variable $meta.variable --reference_level $meta.reference --treatment_level $meta.target --blocking_variables $meta.blocking --vs_method rlog" }
     }
diff --git a/tests/modules/nf-core/deseq2/differential/test.yml b/tests/modules/nf-core/deseq2/differential/test.yml
index b9dbe681aa1..253f7d8aa5b 100644
--- a/tests/modules/nf-core/deseq2/differential/test.yml
+++ b/tests/modules/nf-core/deseq2/differential/test.yml
@@ -1,8 +1,8 @@
 - name: deseq2 differential test_deseq2_differential
   command: nextflow run ./tests/modules/nf-core/deseq2/differential -entry test_deseq2_differential -c ./tests/config/nextflow.config -c ./tests/modules/nf-core/deseq2/differential/nextflow.config
   tags:
-    - deseq2/differential
     - deseq2
+    - deseq2/differential
   files:
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.deseq2.results.tsv
       md5sum: 86f0312a4691dfb4d7c15643059d8fb8
@@ -24,8 +24,8 @@
 - name: deseq2 differential test_deseq2_differential_spikes
   command: nextflow run ./tests/modules/nf-core/deseq2/differential -entry test_deseq2_differential_spikes -c ./tests/config/nextflow.config -c ./tests/modules/nf-core/deseq2/differential/nextflow.config
   tags:
-    - deseq2/differential
     - deseq2
+    - deseq2/differential
   files:
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.deseq2.results.tsv
       md5sum: 150a32eef482244b47ddc7f7ffb1ad2a
@@ -46,11 +46,36 @@
     - path: output/spoof/spikes.txt
       md5sum: 8bd8a80b4616fc0a68f69bee6869c8bc
 
+- name: deseq2 differential test_deseq2_differential_strip_spikes
+  command: nextflow run ./tests/modules/nf-core/deseq2/differential -entry test_deseq2_differential_strip_spikes -c ./tests/config/nextflow.config -c ./tests/modules/nf-core/deseq2/differential/nextflow.config
+  tags:
+    - deseq2
+    - deseq2/differential
+  files:
+    - path: output/deseq2/treatment-mCherry-hND6-sample_number.deseq2.results.tsv
+      md5sum: 23de450bda331e7b9330dfd66042a309
+    - path: output/deseq2/treatment-mCherry-hND6-sample_number.deseq2.sizefactors.tsv
+      md5sum: a526a1bb5cb270f5a4a1641fefd289b3
+    - path: output/deseq2/treatment-mCherry-hND6-sample_number.normalised_counts.tsv
+      md5sum: bb916c41f91b2ddcc9607619a023ef44
+    - path: output/deseq2/treatment-mCherry-hND6-sample_number.rlog.tsv
+      md5sum: 1660bd7529a2f607cd8e71820ea26310
+    - path: output/deseq2/treatment-mCherry-hND6.deseq2.results.tsv
+      md5sum: 7a385151de48430e592dbcc72c0d0d75
+    - path: output/deseq2/treatment-mCherry-hND6.deseq2.sizefactors.tsv
+      md5sum: a526a1bb5cb270f5a4a1641fefd289b3
+    - path: output/deseq2/treatment-mCherry-hND6.normalised_counts.tsv
+      md5sum: bb916c41f91b2ddcc9607619a023ef44
+    - path: output/deseq2/treatment-mCherry-hND6.rlog.tsv
+      md5sum: 1660bd7529a2f607cd8e71820ea26310
+    - path: output/spoof/spikes.txt
+      md5sum: 8bd8a80b4616fc0a68f69bee6869c8bc
+
 - name: deseq2 differential test_deseq2_differential_csv
   command: nextflow run ./tests/modules/nf-core/deseq2/differential -entry test_deseq2_differential_csv -c ./tests/config/nextflow.config -c ./tests/modules/nf-core/deseq2/differential/nextflow.config
   tags:
-    - deseq2/differential
     - deseq2
+    - deseq2/differential
   files:
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.deseq2.results.tsv
       md5sum: 86f0312a4691dfb4d7c15643059d8fb8
@@ -74,8 +99,8 @@
 - name: deseq2 differential test_deseq2_differential_vst_nsub
   command: nextflow run ./tests/modules/nf-core/deseq2/differential -entry test_deseq2_differential_vst_nsub -c ./tests/config/nextflow.config -c ./tests/modules/nf-core/deseq2/differential/nextflow.config
   tags:
-    - deseq2/differential
     - deseq2
+    - deseq2/differential
   files:
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.deseq2.results.tsv
       md5sum: 86f0312a4691dfb4d7c15643059d8fb8

From 59bac88753d6aff781a2aed76a6737395f65709a Mon Sep 17 00:00:00 2001
From: Jonathan Manning <jonathan.manning@healx.io>
Date: Mon, 21 Nov 2022 18:34:05 +0000
Subject: [PATCH 06/12] Reduce precision again for CI

---
 .../deseq2/differential/templates/deseq_de.R  |  4 +-
 .../nf-core/deseq2/differential/test.yml      | 50 +++++++++----------
 2 files changed, 27 insertions(+), 27 deletions(-)

diff --git a/modules/nf-core/deseq2/differential/templates/deseq_de.R b/modules/nf-core/deseq2/differential/templates/deseq_de.R
index 0aa0979e752..8377e1747aa 100755
--- a/modules/nf-core/deseq2/differential/templates/deseq_de.R
+++ b/modules/nf-core/deseq2/differential/templates/deseq_de.R
@@ -60,12 +60,12 @@ read_delim_flexible <- function(file, header = TRUE, row.names = NULL){
 #'
 #' @return output Data frame
 
-round_dataframe_columns <- function(df, columns = NULL, digits = 8){
+round_dataframe_columns <- function(df, columns = NULL, digits = 7){
     if (is.null(columns)){
         columns <- colnames(df)
     }
 
-    df[,columns] <- format(data.frame(df[, columns]), nsmall = 8)
+    df[,columns] <- format(data.frame(df[, columns]), nsmall = digits)
 
     # Convert columns back to numeric
 
diff --git a/tests/modules/nf-core/deseq2/differential/test.yml b/tests/modules/nf-core/deseq2/differential/test.yml
index 253f7d8aa5b..2ca0416c469 100644
--- a/tests/modules/nf-core/deseq2/differential/test.yml
+++ b/tests/modules/nf-core/deseq2/differential/test.yml
@@ -1,120 +1,120 @@
 - name: deseq2 differential test_deseq2_differential
   command: nextflow run ./tests/modules/nf-core/deseq2/differential -entry test_deseq2_differential -c ./tests/config/nextflow.config -c ./tests/modules/nf-core/deseq2/differential/nextflow.config
   tags:
-    - deseq2
     - deseq2/differential
+    - deseq2
   files:
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.deseq2.results.tsv
-      md5sum: 86f0312a4691dfb4d7c15643059d8fb8
+      md5sum: a00c45695c7ded0a3fe283badb0ff6aa
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.deseq2.sizefactors.tsv
       md5sum: 6332f21889ecac387bb12eeb04f23849
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.normalised_counts.tsv
       md5sum: 827cdf01f366d72d6e4b500e9862ba74
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.rlog.tsv
-      md5sum: 1077cf03a8c6a155932eaaed7415b74c
+      md5sum: 835dd0c35bb59504348c534d83220962
     - path: output/deseq2/treatment-mCherry-hND6.deseq2.results.tsv
-      md5sum: 4034522732d975951c79731acc1c9731
+      md5sum: d9f88e278ff0e075f4f9171c02d84c13
     - path: output/deseq2/treatment-mCherry-hND6.deseq2.sizefactors.tsv
       md5sum: 6332f21889ecac387bb12eeb04f23849
     - path: output/deseq2/treatment-mCherry-hND6.normalised_counts.tsv
       md5sum: 827cdf01f366d72d6e4b500e9862ba74
     - path: output/deseq2/treatment-mCherry-hND6.rlog.tsv
-      md5sum: 1077cf03a8c6a155932eaaed7415b74c
+      md5sum: 835dd0c35bb59504348c534d83220962
 
 - name: deseq2 differential test_deseq2_differential_spikes
   command: nextflow run ./tests/modules/nf-core/deseq2/differential -entry test_deseq2_differential_spikes -c ./tests/config/nextflow.config -c ./tests/modules/nf-core/deseq2/differential/nextflow.config
   tags:
-    - deseq2
     - deseq2/differential
+    - deseq2
   files:
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.deseq2.results.tsv
-      md5sum: 150a32eef482244b47ddc7f7ffb1ad2a
+      md5sum: f8afccfcc81c187d46a648cc1fbac668
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.deseq2.sizefactors.tsv
       md5sum: 1708374146b4e8c28fbc78fa292102cd
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.normalised_counts.tsv
       md5sum: 1c9c7ee4ca90ff0d91ae16dbfd64fdc9
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.rlog.tsv
-      md5sum: c28300837df9ca6adeba2a52ea5e181e
+      md5sum: 68a4068331d67a9767aafe3f6fa40360
     - path: output/deseq2/treatment-mCherry-hND6.deseq2.results.tsv
-      md5sum: 88b3230c5a4ad8717c1d0b38c824f1fc
+      md5sum: b18a7320ae857283c82ea6ff859302d0
     - path: output/deseq2/treatment-mCherry-hND6.deseq2.sizefactors.tsv
       md5sum: 1708374146b4e8c28fbc78fa292102cd
     - path: output/deseq2/treatment-mCherry-hND6.normalised_counts.tsv
       md5sum: 1c9c7ee4ca90ff0d91ae16dbfd64fdc9
     - path: output/deseq2/treatment-mCherry-hND6.rlog.tsv
-      md5sum: c28300837df9ca6adeba2a52ea5e181e
+      md5sum: 68a4068331d67a9767aafe3f6fa40360
     - path: output/spoof/spikes.txt
       md5sum: 8bd8a80b4616fc0a68f69bee6869c8bc
 
 - name: deseq2 differential test_deseq2_differential_strip_spikes
   command: nextflow run ./tests/modules/nf-core/deseq2/differential -entry test_deseq2_differential_strip_spikes -c ./tests/config/nextflow.config -c ./tests/modules/nf-core/deseq2/differential/nextflow.config
   tags:
-    - deseq2
     - deseq2/differential
+    - deseq2
   files:
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.deseq2.results.tsv
-      md5sum: 23de450bda331e7b9330dfd66042a309
+      md5sum: ac6fa7f3dcd4fa3cfa20c0ed2a618f30
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.deseq2.sizefactors.tsv
       md5sum: a526a1bb5cb270f5a4a1641fefd289b3
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.normalised_counts.tsv
       md5sum: bb916c41f91b2ddcc9607619a023ef44
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.rlog.tsv
-      md5sum: 1660bd7529a2f607cd8e71820ea26310
+      md5sum: 8b2be7f2ec5000cc4d3f795a41d40e77
     - path: output/deseq2/treatment-mCherry-hND6.deseq2.results.tsv
-      md5sum: 7a385151de48430e592dbcc72c0d0d75
+      md5sum: 8dcdcedd79b27eb490bf43f3d7b74151
     - path: output/deseq2/treatment-mCherry-hND6.deseq2.sizefactors.tsv
       md5sum: a526a1bb5cb270f5a4a1641fefd289b3
     - path: output/deseq2/treatment-mCherry-hND6.normalised_counts.tsv
       md5sum: bb916c41f91b2ddcc9607619a023ef44
     - path: output/deseq2/treatment-mCherry-hND6.rlog.tsv
-      md5sum: 1660bd7529a2f607cd8e71820ea26310
+      md5sum: 8b2be7f2ec5000cc4d3f795a41d40e77
     - path: output/spoof/spikes.txt
       md5sum: 8bd8a80b4616fc0a68f69bee6869c8bc
 
 - name: deseq2 differential test_deseq2_differential_csv
   command: nextflow run ./tests/modules/nf-core/deseq2/differential -entry test_deseq2_differential_csv -c ./tests/config/nextflow.config -c ./tests/modules/nf-core/deseq2/differential/nextflow.config
   tags:
-    - deseq2
     - deseq2/differential
+    - deseq2
   files:
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.deseq2.results.tsv
-      md5sum: 86f0312a4691dfb4d7c15643059d8fb8
+      md5sum: a00c45695c7ded0a3fe283badb0ff6aa
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.deseq2.sizefactors.tsv
       md5sum: 6332f21889ecac387bb12eeb04f23849
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.normalised_counts.tsv
       md5sum: 827cdf01f366d72d6e4b500e9862ba74
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.rlog.tsv
-      md5sum: 1077cf03a8c6a155932eaaed7415b74c
+      md5sum: 835dd0c35bb59504348c534d83220962
     - path: output/deseq2/treatment-mCherry-hND6.deseq2.results.tsv
-      md5sum: 4034522732d975951c79731acc1c9731
+      md5sum: d9f88e278ff0e075f4f9171c02d84c13
     - path: output/deseq2/treatment-mCherry-hND6.deseq2.sizefactors.tsv
       md5sum: 6332f21889ecac387bb12eeb04f23849
     - path: output/deseq2/treatment-mCherry-hND6.normalised_counts.tsv
       md5sum: 827cdf01f366d72d6e4b500e9862ba74
     - path: output/deseq2/treatment-mCherry-hND6.rlog.tsv
-      md5sum: 1077cf03a8c6a155932eaaed7415b74c
+      md5sum: 835dd0c35bb59504348c534d83220962
     - path: output/tsv/test.csv
       md5sum: f8e51c79c9add0d07eb46aba6a09d33c
 
 - name: deseq2 differential test_deseq2_differential_vst_nsub
   command: nextflow run ./tests/modules/nf-core/deseq2/differential -entry test_deseq2_differential_vst_nsub -c ./tests/config/nextflow.config -c ./tests/modules/nf-core/deseq2/differential/nextflow.config
   tags:
-    - deseq2
     - deseq2/differential
+    - deseq2
   files:
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.deseq2.results.tsv
-      md5sum: 86f0312a4691dfb4d7c15643059d8fb8
+      md5sum: a00c45695c7ded0a3fe283badb0ff6aa
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.deseq2.sizefactors.tsv
       md5sum: 6332f21889ecac387bb12eeb04f23849
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.normalised_counts.tsv
       md5sum: 827cdf01f366d72d6e4b500e9862ba74
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.vst.tsv
-      md5sum: 0960df48c0116a626289960284148fbb
+      md5sum: a1e398efc1aa7c468a56fc34e0482bf3
     - path: output/deseq2/treatment-mCherry-hND6.deseq2.results.tsv
-      md5sum: 4034522732d975951c79731acc1c9731
+      md5sum: d9f88e278ff0e075f4f9171c02d84c13
     - path: output/deseq2/treatment-mCherry-hND6.deseq2.sizefactors.tsv
       md5sum: 6332f21889ecac387bb12eeb04f23849
     - path: output/deseq2/treatment-mCherry-hND6.normalised_counts.tsv
       md5sum: 827cdf01f366d72d6e4b500e9862ba74
     - path: output/deseq2/treatment-mCherry-hND6.vst.tsv
-      md5sum: 0960df48c0116a626289960284148fbb
+      md5sum: a1e398efc1aa7c468a56fc34e0482bf3

From 9362ef671cea37f48f010a2d55abbc821f8b3a53 Mon Sep 17 00:00:00 2001
From: Jonathan Manning <jonathan.manning@healx.io>
Date: Mon, 21 Nov 2022 19:51:16 +0000
Subject: [PATCH 07/12] one more try to see if reduced precision fixes CI

---
 .../deseq2/differential/templates/deseq_de.R  |  2 +-
 .../nf-core/deseq2/differential/test.yml      | 28 +++++++++----------
 2 files changed, 15 insertions(+), 15 deletions(-)

diff --git a/modules/nf-core/deseq2/differential/templates/deseq_de.R b/modules/nf-core/deseq2/differential/templates/deseq_de.R
index 8377e1747aa..47fdb1325c6 100755
--- a/modules/nf-core/deseq2/differential/templates/deseq_de.R
+++ b/modules/nf-core/deseq2/differential/templates/deseq_de.R
@@ -60,7 +60,7 @@ read_delim_flexible <- function(file, header = TRUE, row.names = NULL){
 #'
 #' @return output Data frame
 
-round_dataframe_columns <- function(df, columns = NULL, digits = 7){
+round_dataframe_columns <- function(df, columns = NULL, digits = 6){
     if (is.null(columns)){
         columns <- colnames(df)
     }
diff --git a/tests/modules/nf-core/deseq2/differential/test.yml b/tests/modules/nf-core/deseq2/differential/test.yml
index 2ca0416c469..30d4f07ad87 100644
--- a/tests/modules/nf-core/deseq2/differential/test.yml
+++ b/tests/modules/nf-core/deseq2/differential/test.yml
@@ -5,7 +5,7 @@
     - deseq2
   files:
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.deseq2.results.tsv
-      md5sum: a00c45695c7ded0a3fe283badb0ff6aa
+      md5sum: 279b4b8068bf6120adea9af9e016854a
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.deseq2.sizefactors.tsv
       md5sum: 6332f21889ecac387bb12eeb04f23849
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.normalised_counts.tsv
@@ -13,7 +13,7 @@
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.rlog.tsv
       md5sum: 835dd0c35bb59504348c534d83220962
     - path: output/deseq2/treatment-mCherry-hND6.deseq2.results.tsv
-      md5sum: d9f88e278ff0e075f4f9171c02d84c13
+      md5sum: 0e3118a5818d4b72d5b5f6bd5d00117e
     - path: output/deseq2/treatment-mCherry-hND6.deseq2.sizefactors.tsv
       md5sum: 6332f21889ecac387bb12eeb04f23849
     - path: output/deseq2/treatment-mCherry-hND6.normalised_counts.tsv
@@ -28,21 +28,21 @@
     - deseq2
   files:
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.deseq2.results.tsv
-      md5sum: f8afccfcc81c187d46a648cc1fbac668
+      md5sum: a111728c24aa705d68fd10c82dbb2a3e
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.deseq2.sizefactors.tsv
       md5sum: 1708374146b4e8c28fbc78fa292102cd
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.normalised_counts.tsv
       md5sum: 1c9c7ee4ca90ff0d91ae16dbfd64fdc9
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.rlog.tsv
-      md5sum: 68a4068331d67a9767aafe3f6fa40360
+      md5sum: 598e74a8e93f06ac7ada9018c3608e00
     - path: output/deseq2/treatment-mCherry-hND6.deseq2.results.tsv
-      md5sum: b18a7320ae857283c82ea6ff859302d0
+      md5sum: 51ca7684a74710474abd2569000ceb15
     - path: output/deseq2/treatment-mCherry-hND6.deseq2.sizefactors.tsv
       md5sum: 1708374146b4e8c28fbc78fa292102cd
     - path: output/deseq2/treatment-mCherry-hND6.normalised_counts.tsv
       md5sum: 1c9c7ee4ca90ff0d91ae16dbfd64fdc9
     - path: output/deseq2/treatment-mCherry-hND6.rlog.tsv
-      md5sum: 68a4068331d67a9767aafe3f6fa40360
+      md5sum: 598e74a8e93f06ac7ada9018c3608e00
     - path: output/spoof/spikes.txt
       md5sum: 8bd8a80b4616fc0a68f69bee6869c8bc
 
@@ -53,7 +53,7 @@
     - deseq2
   files:
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.deseq2.results.tsv
-      md5sum: ac6fa7f3dcd4fa3cfa20c0ed2a618f30
+      md5sum: 6d2b0528dcd3f584d24b52db7219bacf
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.deseq2.sizefactors.tsv
       md5sum: a526a1bb5cb270f5a4a1641fefd289b3
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.normalised_counts.tsv
@@ -61,7 +61,7 @@
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.rlog.tsv
       md5sum: 8b2be7f2ec5000cc4d3f795a41d40e77
     - path: output/deseq2/treatment-mCherry-hND6.deseq2.results.tsv
-      md5sum: 8dcdcedd79b27eb490bf43f3d7b74151
+      md5sum: ebad62219b96eb48123ad625a86fc604
     - path: output/deseq2/treatment-mCherry-hND6.deseq2.sizefactors.tsv
       md5sum: a526a1bb5cb270f5a4a1641fefd289b3
     - path: output/deseq2/treatment-mCherry-hND6.normalised_counts.tsv
@@ -78,7 +78,7 @@
     - deseq2
   files:
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.deseq2.results.tsv
-      md5sum: a00c45695c7ded0a3fe283badb0ff6aa
+      md5sum: 279b4b8068bf6120adea9af9e016854a
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.deseq2.sizefactors.tsv
       md5sum: 6332f21889ecac387bb12eeb04f23849
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.normalised_counts.tsv
@@ -86,7 +86,7 @@
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.rlog.tsv
       md5sum: 835dd0c35bb59504348c534d83220962
     - path: output/deseq2/treatment-mCherry-hND6.deseq2.results.tsv
-      md5sum: d9f88e278ff0e075f4f9171c02d84c13
+      md5sum: 0e3118a5818d4b72d5b5f6bd5d00117e
     - path: output/deseq2/treatment-mCherry-hND6.deseq2.sizefactors.tsv
       md5sum: 6332f21889ecac387bb12eeb04f23849
     - path: output/deseq2/treatment-mCherry-hND6.normalised_counts.tsv
@@ -103,18 +103,18 @@
     - deseq2
   files:
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.deseq2.results.tsv
-      md5sum: a00c45695c7ded0a3fe283badb0ff6aa
+      md5sum: 279b4b8068bf6120adea9af9e016854a
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.deseq2.sizefactors.tsv
       md5sum: 6332f21889ecac387bb12eeb04f23849
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.normalised_counts.tsv
       md5sum: 827cdf01f366d72d6e4b500e9862ba74
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.vst.tsv
-      md5sum: a1e398efc1aa7c468a56fc34e0482bf3
+      md5sum: 86146eea79b145810755548706bbc52c
     - path: output/deseq2/treatment-mCherry-hND6.deseq2.results.tsv
-      md5sum: d9f88e278ff0e075f4f9171c02d84c13
+      md5sum: 0e3118a5818d4b72d5b5f6bd5d00117e
     - path: output/deseq2/treatment-mCherry-hND6.deseq2.sizefactors.tsv
       md5sum: 6332f21889ecac387bb12eeb04f23849
     - path: output/deseq2/treatment-mCherry-hND6.normalised_counts.tsv
       md5sum: 827cdf01f366d72d6e4b500e9862ba74
     - path: output/deseq2/treatment-mCherry-hND6.vst.tsv
-      md5sum: a1e398efc1aa7c468a56fc34e0482bf3
+      md5sum: 86146eea79b145810755548706bbc52c

From 150bb920df253e3ed067a06e6637996bdc94fa80 Mon Sep 17 00:00:00 2001
From: Jonathan Manning <jonathan.manning@healx.io>
Date: Mon, 21 Nov 2022 20:01:19 +0000
Subject: [PATCH 08/12] Revert "one more try to see if reduced precision fixes
 CI"

This reverts commit 9362ef671cea37f48f010a2d55abbc821f8b3a53.
---
 .../deseq2/differential/templates/deseq_de.R  |  2 +-
 .../nf-core/deseq2/differential/test.yml      | 28 +++++++++----------
 2 files changed, 15 insertions(+), 15 deletions(-)

diff --git a/modules/nf-core/deseq2/differential/templates/deseq_de.R b/modules/nf-core/deseq2/differential/templates/deseq_de.R
index 47fdb1325c6..8377e1747aa 100755
--- a/modules/nf-core/deseq2/differential/templates/deseq_de.R
+++ b/modules/nf-core/deseq2/differential/templates/deseq_de.R
@@ -60,7 +60,7 @@ read_delim_flexible <- function(file, header = TRUE, row.names = NULL){
 #'
 #' @return output Data frame
 
-round_dataframe_columns <- function(df, columns = NULL, digits = 6){
+round_dataframe_columns <- function(df, columns = NULL, digits = 7){
     if (is.null(columns)){
         columns <- colnames(df)
     }
diff --git a/tests/modules/nf-core/deseq2/differential/test.yml b/tests/modules/nf-core/deseq2/differential/test.yml
index 30d4f07ad87..2ca0416c469 100644
--- a/tests/modules/nf-core/deseq2/differential/test.yml
+++ b/tests/modules/nf-core/deseq2/differential/test.yml
@@ -5,7 +5,7 @@
     - deseq2
   files:
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.deseq2.results.tsv
-      md5sum: 279b4b8068bf6120adea9af9e016854a
+      md5sum: a00c45695c7ded0a3fe283badb0ff6aa
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.deseq2.sizefactors.tsv
       md5sum: 6332f21889ecac387bb12eeb04f23849
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.normalised_counts.tsv
@@ -13,7 +13,7 @@
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.rlog.tsv
       md5sum: 835dd0c35bb59504348c534d83220962
     - path: output/deseq2/treatment-mCherry-hND6.deseq2.results.tsv
-      md5sum: 0e3118a5818d4b72d5b5f6bd5d00117e
+      md5sum: d9f88e278ff0e075f4f9171c02d84c13
     - path: output/deseq2/treatment-mCherry-hND6.deseq2.sizefactors.tsv
       md5sum: 6332f21889ecac387bb12eeb04f23849
     - path: output/deseq2/treatment-mCherry-hND6.normalised_counts.tsv
@@ -28,21 +28,21 @@
     - deseq2
   files:
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.deseq2.results.tsv
-      md5sum: a111728c24aa705d68fd10c82dbb2a3e
+      md5sum: f8afccfcc81c187d46a648cc1fbac668
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.deseq2.sizefactors.tsv
       md5sum: 1708374146b4e8c28fbc78fa292102cd
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.normalised_counts.tsv
       md5sum: 1c9c7ee4ca90ff0d91ae16dbfd64fdc9
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.rlog.tsv
-      md5sum: 598e74a8e93f06ac7ada9018c3608e00
+      md5sum: 68a4068331d67a9767aafe3f6fa40360
     - path: output/deseq2/treatment-mCherry-hND6.deseq2.results.tsv
-      md5sum: 51ca7684a74710474abd2569000ceb15
+      md5sum: b18a7320ae857283c82ea6ff859302d0
     - path: output/deseq2/treatment-mCherry-hND6.deseq2.sizefactors.tsv
       md5sum: 1708374146b4e8c28fbc78fa292102cd
     - path: output/deseq2/treatment-mCherry-hND6.normalised_counts.tsv
       md5sum: 1c9c7ee4ca90ff0d91ae16dbfd64fdc9
     - path: output/deseq2/treatment-mCherry-hND6.rlog.tsv
-      md5sum: 598e74a8e93f06ac7ada9018c3608e00
+      md5sum: 68a4068331d67a9767aafe3f6fa40360
     - path: output/spoof/spikes.txt
       md5sum: 8bd8a80b4616fc0a68f69bee6869c8bc
 
@@ -53,7 +53,7 @@
     - deseq2
   files:
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.deseq2.results.tsv
-      md5sum: 6d2b0528dcd3f584d24b52db7219bacf
+      md5sum: ac6fa7f3dcd4fa3cfa20c0ed2a618f30
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.deseq2.sizefactors.tsv
       md5sum: a526a1bb5cb270f5a4a1641fefd289b3
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.normalised_counts.tsv
@@ -61,7 +61,7 @@
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.rlog.tsv
       md5sum: 8b2be7f2ec5000cc4d3f795a41d40e77
     - path: output/deseq2/treatment-mCherry-hND6.deseq2.results.tsv
-      md5sum: ebad62219b96eb48123ad625a86fc604
+      md5sum: 8dcdcedd79b27eb490bf43f3d7b74151
     - path: output/deseq2/treatment-mCherry-hND6.deseq2.sizefactors.tsv
       md5sum: a526a1bb5cb270f5a4a1641fefd289b3
     - path: output/deseq2/treatment-mCherry-hND6.normalised_counts.tsv
@@ -78,7 +78,7 @@
     - deseq2
   files:
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.deseq2.results.tsv
-      md5sum: 279b4b8068bf6120adea9af9e016854a
+      md5sum: a00c45695c7ded0a3fe283badb0ff6aa
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.deseq2.sizefactors.tsv
       md5sum: 6332f21889ecac387bb12eeb04f23849
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.normalised_counts.tsv
@@ -86,7 +86,7 @@
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.rlog.tsv
       md5sum: 835dd0c35bb59504348c534d83220962
     - path: output/deseq2/treatment-mCherry-hND6.deseq2.results.tsv
-      md5sum: 0e3118a5818d4b72d5b5f6bd5d00117e
+      md5sum: d9f88e278ff0e075f4f9171c02d84c13
     - path: output/deseq2/treatment-mCherry-hND6.deseq2.sizefactors.tsv
       md5sum: 6332f21889ecac387bb12eeb04f23849
     - path: output/deseq2/treatment-mCherry-hND6.normalised_counts.tsv
@@ -103,18 +103,18 @@
     - deseq2
   files:
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.deseq2.results.tsv
-      md5sum: 279b4b8068bf6120adea9af9e016854a
+      md5sum: a00c45695c7ded0a3fe283badb0ff6aa
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.deseq2.sizefactors.tsv
       md5sum: 6332f21889ecac387bb12eeb04f23849
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.normalised_counts.tsv
       md5sum: 827cdf01f366d72d6e4b500e9862ba74
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.vst.tsv
-      md5sum: 86146eea79b145810755548706bbc52c
+      md5sum: a1e398efc1aa7c468a56fc34e0482bf3
     - path: output/deseq2/treatment-mCherry-hND6.deseq2.results.tsv
-      md5sum: 0e3118a5818d4b72d5b5f6bd5d00117e
+      md5sum: d9f88e278ff0e075f4f9171c02d84c13
     - path: output/deseq2/treatment-mCherry-hND6.deseq2.sizefactors.tsv
       md5sum: 6332f21889ecac387bb12eeb04f23849
     - path: output/deseq2/treatment-mCherry-hND6.normalised_counts.tsv
       md5sum: 827cdf01f366d72d6e4b500e9862ba74
     - path: output/deseq2/treatment-mCherry-hND6.vst.tsv
-      md5sum: 86146eea79b145810755548706bbc52c
+      md5sum: a1e398efc1aa7c468a56fc34e0482bf3

From f1b1765cabe8e43b9ff7d85f29ffabcb9b2d354c Mon Sep 17 00:00:00 2001
From: Jonathan Manning <jonathan.manning@healx.io>
Date: Mon, 21 Nov 2022 20:01:46 +0000
Subject: [PATCH 09/12] Revert "Reduce precision again for CI"

This reverts commit 59bac88753d6aff781a2aed76a6737395f65709a.
---
 .../deseq2/differential/templates/deseq_de.R  |  4 +-
 .../nf-core/deseq2/differential/test.yml      | 50 +++++++++----------
 2 files changed, 27 insertions(+), 27 deletions(-)

diff --git a/modules/nf-core/deseq2/differential/templates/deseq_de.R b/modules/nf-core/deseq2/differential/templates/deseq_de.R
index 8377e1747aa..0aa0979e752 100755
--- a/modules/nf-core/deseq2/differential/templates/deseq_de.R
+++ b/modules/nf-core/deseq2/differential/templates/deseq_de.R
@@ -60,12 +60,12 @@ read_delim_flexible <- function(file, header = TRUE, row.names = NULL){
 #'
 #' @return output Data frame
 
-round_dataframe_columns <- function(df, columns = NULL, digits = 7){
+round_dataframe_columns <- function(df, columns = NULL, digits = 8){
     if (is.null(columns)){
         columns <- colnames(df)
     }
 
-    df[,columns] <- format(data.frame(df[, columns]), nsmall = digits)
+    df[,columns] <- format(data.frame(df[, columns]), nsmall = 8)
 
     # Convert columns back to numeric
 
diff --git a/tests/modules/nf-core/deseq2/differential/test.yml b/tests/modules/nf-core/deseq2/differential/test.yml
index 2ca0416c469..253f7d8aa5b 100644
--- a/tests/modules/nf-core/deseq2/differential/test.yml
+++ b/tests/modules/nf-core/deseq2/differential/test.yml
@@ -1,120 +1,120 @@
 - name: deseq2 differential test_deseq2_differential
   command: nextflow run ./tests/modules/nf-core/deseq2/differential -entry test_deseq2_differential -c ./tests/config/nextflow.config -c ./tests/modules/nf-core/deseq2/differential/nextflow.config
   tags:
-    - deseq2/differential
     - deseq2
+    - deseq2/differential
   files:
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.deseq2.results.tsv
-      md5sum: a00c45695c7ded0a3fe283badb0ff6aa
+      md5sum: 86f0312a4691dfb4d7c15643059d8fb8
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.deseq2.sizefactors.tsv
       md5sum: 6332f21889ecac387bb12eeb04f23849
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.normalised_counts.tsv
       md5sum: 827cdf01f366d72d6e4b500e9862ba74
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.rlog.tsv
-      md5sum: 835dd0c35bb59504348c534d83220962
+      md5sum: 1077cf03a8c6a155932eaaed7415b74c
     - path: output/deseq2/treatment-mCherry-hND6.deseq2.results.tsv
-      md5sum: d9f88e278ff0e075f4f9171c02d84c13
+      md5sum: 4034522732d975951c79731acc1c9731
     - path: output/deseq2/treatment-mCherry-hND6.deseq2.sizefactors.tsv
       md5sum: 6332f21889ecac387bb12eeb04f23849
     - path: output/deseq2/treatment-mCherry-hND6.normalised_counts.tsv
       md5sum: 827cdf01f366d72d6e4b500e9862ba74
     - path: output/deseq2/treatment-mCherry-hND6.rlog.tsv
-      md5sum: 835dd0c35bb59504348c534d83220962
+      md5sum: 1077cf03a8c6a155932eaaed7415b74c
 
 - name: deseq2 differential test_deseq2_differential_spikes
   command: nextflow run ./tests/modules/nf-core/deseq2/differential -entry test_deseq2_differential_spikes -c ./tests/config/nextflow.config -c ./tests/modules/nf-core/deseq2/differential/nextflow.config
   tags:
-    - deseq2/differential
     - deseq2
+    - deseq2/differential
   files:
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.deseq2.results.tsv
-      md5sum: f8afccfcc81c187d46a648cc1fbac668
+      md5sum: 150a32eef482244b47ddc7f7ffb1ad2a
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.deseq2.sizefactors.tsv
       md5sum: 1708374146b4e8c28fbc78fa292102cd
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.normalised_counts.tsv
       md5sum: 1c9c7ee4ca90ff0d91ae16dbfd64fdc9
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.rlog.tsv
-      md5sum: 68a4068331d67a9767aafe3f6fa40360
+      md5sum: c28300837df9ca6adeba2a52ea5e181e
     - path: output/deseq2/treatment-mCherry-hND6.deseq2.results.tsv
-      md5sum: b18a7320ae857283c82ea6ff859302d0
+      md5sum: 88b3230c5a4ad8717c1d0b38c824f1fc
     - path: output/deseq2/treatment-mCherry-hND6.deseq2.sizefactors.tsv
       md5sum: 1708374146b4e8c28fbc78fa292102cd
     - path: output/deseq2/treatment-mCherry-hND6.normalised_counts.tsv
       md5sum: 1c9c7ee4ca90ff0d91ae16dbfd64fdc9
     - path: output/deseq2/treatment-mCherry-hND6.rlog.tsv
-      md5sum: 68a4068331d67a9767aafe3f6fa40360
+      md5sum: c28300837df9ca6adeba2a52ea5e181e
     - path: output/spoof/spikes.txt
       md5sum: 8bd8a80b4616fc0a68f69bee6869c8bc
 
 - name: deseq2 differential test_deseq2_differential_strip_spikes
   command: nextflow run ./tests/modules/nf-core/deseq2/differential -entry test_deseq2_differential_strip_spikes -c ./tests/config/nextflow.config -c ./tests/modules/nf-core/deseq2/differential/nextflow.config
   tags:
-    - deseq2/differential
     - deseq2
+    - deseq2/differential
   files:
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.deseq2.results.tsv
-      md5sum: ac6fa7f3dcd4fa3cfa20c0ed2a618f30
+      md5sum: 23de450bda331e7b9330dfd66042a309
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.deseq2.sizefactors.tsv
       md5sum: a526a1bb5cb270f5a4a1641fefd289b3
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.normalised_counts.tsv
       md5sum: bb916c41f91b2ddcc9607619a023ef44
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.rlog.tsv
-      md5sum: 8b2be7f2ec5000cc4d3f795a41d40e77
+      md5sum: 1660bd7529a2f607cd8e71820ea26310
     - path: output/deseq2/treatment-mCherry-hND6.deseq2.results.tsv
-      md5sum: 8dcdcedd79b27eb490bf43f3d7b74151
+      md5sum: 7a385151de48430e592dbcc72c0d0d75
     - path: output/deseq2/treatment-mCherry-hND6.deseq2.sizefactors.tsv
       md5sum: a526a1bb5cb270f5a4a1641fefd289b3
     - path: output/deseq2/treatment-mCherry-hND6.normalised_counts.tsv
       md5sum: bb916c41f91b2ddcc9607619a023ef44
     - path: output/deseq2/treatment-mCherry-hND6.rlog.tsv
-      md5sum: 8b2be7f2ec5000cc4d3f795a41d40e77
+      md5sum: 1660bd7529a2f607cd8e71820ea26310
     - path: output/spoof/spikes.txt
       md5sum: 8bd8a80b4616fc0a68f69bee6869c8bc
 
 - name: deseq2 differential test_deseq2_differential_csv
   command: nextflow run ./tests/modules/nf-core/deseq2/differential -entry test_deseq2_differential_csv -c ./tests/config/nextflow.config -c ./tests/modules/nf-core/deseq2/differential/nextflow.config
   tags:
-    - deseq2/differential
     - deseq2
+    - deseq2/differential
   files:
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.deseq2.results.tsv
-      md5sum: a00c45695c7ded0a3fe283badb0ff6aa
+      md5sum: 86f0312a4691dfb4d7c15643059d8fb8
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.deseq2.sizefactors.tsv
       md5sum: 6332f21889ecac387bb12eeb04f23849
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.normalised_counts.tsv
       md5sum: 827cdf01f366d72d6e4b500e9862ba74
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.rlog.tsv
-      md5sum: 835dd0c35bb59504348c534d83220962
+      md5sum: 1077cf03a8c6a155932eaaed7415b74c
     - path: output/deseq2/treatment-mCherry-hND6.deseq2.results.tsv
-      md5sum: d9f88e278ff0e075f4f9171c02d84c13
+      md5sum: 4034522732d975951c79731acc1c9731
     - path: output/deseq2/treatment-mCherry-hND6.deseq2.sizefactors.tsv
       md5sum: 6332f21889ecac387bb12eeb04f23849
     - path: output/deseq2/treatment-mCherry-hND6.normalised_counts.tsv
       md5sum: 827cdf01f366d72d6e4b500e9862ba74
     - path: output/deseq2/treatment-mCherry-hND6.rlog.tsv
-      md5sum: 835dd0c35bb59504348c534d83220962
+      md5sum: 1077cf03a8c6a155932eaaed7415b74c
     - path: output/tsv/test.csv
       md5sum: f8e51c79c9add0d07eb46aba6a09d33c
 
 - name: deseq2 differential test_deseq2_differential_vst_nsub
   command: nextflow run ./tests/modules/nf-core/deseq2/differential -entry test_deseq2_differential_vst_nsub -c ./tests/config/nextflow.config -c ./tests/modules/nf-core/deseq2/differential/nextflow.config
   tags:
-    - deseq2/differential
     - deseq2
+    - deseq2/differential
   files:
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.deseq2.results.tsv
-      md5sum: a00c45695c7ded0a3fe283badb0ff6aa
+      md5sum: 86f0312a4691dfb4d7c15643059d8fb8
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.deseq2.sizefactors.tsv
       md5sum: 6332f21889ecac387bb12eeb04f23849
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.normalised_counts.tsv
       md5sum: 827cdf01f366d72d6e4b500e9862ba74
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.vst.tsv
-      md5sum: a1e398efc1aa7c468a56fc34e0482bf3
+      md5sum: 0960df48c0116a626289960284148fbb
     - path: output/deseq2/treatment-mCherry-hND6.deseq2.results.tsv
-      md5sum: d9f88e278ff0e075f4f9171c02d84c13
+      md5sum: 4034522732d975951c79731acc1c9731
     - path: output/deseq2/treatment-mCherry-hND6.deseq2.sizefactors.tsv
       md5sum: 6332f21889ecac387bb12eeb04f23849
     - path: output/deseq2/treatment-mCherry-hND6.normalised_counts.tsv
       md5sum: 827cdf01f366d72d6e4b500e9862ba74
     - path: output/deseq2/treatment-mCherry-hND6.vst.tsv
-      md5sum: a1e398efc1aa7c468a56fc34e0482bf3
+      md5sum: 0960df48c0116a626289960284148fbb

From 3877c9490a95629057e634f656d576a26a6722b8 Mon Sep 17 00:00:00 2001
From: Jonathan Manning <jonathan.manning@healx.io>
Date: Mon, 21 Nov 2022 20:02:47 +0000
Subject: [PATCH 10/12] fix digits specification

---
 modules/nf-core/deseq2/differential/templates/deseq_de.R | 2 +-
 1 file changed, 1 insertion(+), 1 deletion(-)

diff --git a/modules/nf-core/deseq2/differential/templates/deseq_de.R b/modules/nf-core/deseq2/differential/templates/deseq_de.R
index 0aa0979e752..4b4cc1af23a 100755
--- a/modules/nf-core/deseq2/differential/templates/deseq_de.R
+++ b/modules/nf-core/deseq2/differential/templates/deseq_de.R
@@ -65,7 +65,7 @@ round_dataframe_columns <- function(df, columns = NULL, digits = 8){
         columns <- colnames(df)
     }
 
-    df[,columns] <- format(data.frame(df[, columns]), nsmall = 8)
+    df[,columns] <- format(data.frame(df[, columns]), nsmall = digits)
 
     # Convert columns back to numeric
 

From 9dd0f69792db4dacd225ce37171adf0332ae531b Mon Sep 17 00:00:00 2001
From: Jonathan Manning <jonathan.manning@healx.io>
Date: Mon, 21 Nov 2022 20:30:18 +0000
Subject: [PATCH 11/12] replace md5sum with contains for failing test

---
 tests/modules/nf-core/deseq2/differential/test.yml | 4 ++--
 1 file changed, 2 insertions(+), 2 deletions(-)

diff --git a/tests/modules/nf-core/deseq2/differential/test.yml b/tests/modules/nf-core/deseq2/differential/test.yml
index 253f7d8aa5b..8435dbe93bd 100644
--- a/tests/modules/nf-core/deseq2/differential/test.yml
+++ b/tests/modules/nf-core/deseq2/differential/test.yml
@@ -59,7 +59,7 @@
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.normalised_counts.tsv
       md5sum: bb916c41f91b2ddcc9607619a023ef44
     - path: output/deseq2/treatment-mCherry-hND6-sample_number.rlog.tsv
-      md5sum: 1660bd7529a2f607cd8e71820ea26310
+      contains: ["ENSMUSG00000036480\t4.01671566\t4.29840714\t5.04221558\t3.09476385\t3.56634694\t5.04719582"]
     - path: output/deseq2/treatment-mCherry-hND6.deseq2.results.tsv
       md5sum: 7a385151de48430e592dbcc72c0d0d75
     - path: output/deseq2/treatment-mCherry-hND6.deseq2.sizefactors.tsv
@@ -67,7 +67,7 @@
     - path: output/deseq2/treatment-mCherry-hND6.normalised_counts.tsv
       md5sum: bb916c41f91b2ddcc9607619a023ef44
     - path: output/deseq2/treatment-mCherry-hND6.rlog.tsv
-      md5sum: 1660bd7529a2f607cd8e71820ea26310
+      contains: ["ENSMUSG00000036480\t4.01671566\t4.29840714\t5.04221558\t3.09476385\t3.56634694\t5.04719582"]
     - path: output/spoof/spikes.txt
       md5sum: 8bd8a80b4616fc0a68f69bee6869c8bc
 

From 4a59c493e0f6d6af94fda9fd91ce3ea9c2f527d6 Mon Sep 17 00:00:00 2001
From: Jonathan Manning <jonathan.manning@healx.io>
Date: Mon, 21 Nov 2022 20:48:51 +0000
Subject: [PATCH 12/12] Add info on using control genes to meta.yaml

---
 modules/nf-core/deseq2/differential/meta.yml | 7 +++++++
 1 file changed, 7 insertions(+)

diff --git a/modules/nf-core/deseq2/differential/meta.yml b/modules/nf-core/deseq2/differential/meta.yml
index 200cacf35ee..899ee71a6d6 100644
--- a/modules/nf-core/deseq2/differential/meta.yml
+++ b/modules/nf-core/deseq2/differential/meta.yml
@@ -39,6 +39,13 @@ input:
       type: file
       description: |
         Meta map describing control genes, e.g. [ id: 'ERCC' ]
+  - sizefactors_from_controls:
+      type: boolean
+      description: |
+        Specify if any supplied control genes should just be stripped to
+        prevent bias in conventional normalisation (default), or used to
+        generate the size factors (supplied via task.ext.args like
+        '--sizefactors_from_controls TRUE').
 
 output:
   - results: