From ac05d5f8d1128084675104611dd795d982a42dad Mon Sep 17 00:00:00 2001
From: Joon-Klaps <joon.klaps@kuleuven.be>
Date: Wed, 17 Jan 2024 13:37:36 +0000
Subject: [PATCH 1/7] Add Freyja module to multiqc_config files

---
 assets/multiqc_config_illumina.yml | 6 ++++++
 assets/multiqc_config_nanopore.yml | 4 ++++
 modules/local/multiqc_illumina.nf  | 7 ++++---
 modules/local/multiqc_nanopore.nf  | 7 ++++---
 workflows/illumina.nf              | 6 ++++--
 workflows/nanopore.nf              | 6 ++++--
 6 files changed, 26 insertions(+), 10 deletions(-)

diff --git a/assets/multiqc_config_illumina.yml b/assets/multiqc_config_illumina.yml
index 2f7b84c2..545bdf44 100644
--- a/assets/multiqc_config_illumina.yml
+++ b/assets/multiqc_config_illumina.yml
@@ -20,6 +20,7 @@ run_modules:
   - quast
   - pangolin
   - cutadapt
+  - freyja
 
 module_order:
   - fastqc:
@@ -62,6 +63,11 @@ module_order:
       info: "This section of the report shows Pangolin lineage analysis results for the called variants."
       path_filters:
         - "./variants/*.pangolin.csv"
+  - freyja:
+      name: "VARIANTS: Freyja"
+      info: "This section of the report shows relative lineage abundances from mixed SARS-CoV-2 samples from Freyja demix."
+      path_filters:
+        - "./freyja_demix/*.tsv"
   - bcftools:
       name: "VARIANTS: BCFTools"
       info: "This section of the report shows BCFTools stats results for the called variants."
diff --git a/assets/multiqc_config_nanopore.yml b/assets/multiqc_config_nanopore.yml
index f4851ee2..ad155d94 100644
--- a/assets/multiqc_config_nanopore.yml
+++ b/assets/multiqc_config_nanopore.yml
@@ -16,6 +16,7 @@ run_modules:
   - snpeff
   - quast
   - pangolin
+  - freyja
 
 module_order:
   - pangolin:
@@ -26,6 +27,9 @@ module_order:
       path_filters:
         - "./samtools_stats/*"
   - mosdepth
+  - freyja:
+    path_filters:
+      - "./freyja_demix/*.tsv"
   - bcftools:
       path_filters:
         - "./bcftools_stats/*.txt"
diff --git a/modules/local/multiqc_illumina.nf b/modules/local/multiqc_illumina.nf
index bcfc37f1..e87b45a7 100644
--- a/modules/local/multiqc_illumina.nf
+++ b/modules/local/multiqc_illumina.nf
@@ -1,10 +1,10 @@
 process MULTIQC {
     label 'process_medium'
 
-    conda "bioconda::multiqc=1.14"
+    conda "bioconda::multiqc=1.19"
     container "${ workflow.containerEngine == 'singularity' && !task.ext.singularity_pull_docker_container ?
-        'https://depot.galaxyproject.org/singularity/multiqc:1.14--pyhdfd78af_0' :
-        'quay.io/biocontainers/multiqc:1.14--pyhdfd78af_0' }"
+        'https://depot.galaxyproject.org/singularity/multiqc:1.19--pyhdfd78af_0' :
+        'quay.io/biocontainers/multiqc:1.19--pyhdfd78af_0' }"
 
     input:
     path 'multiqc_config.yaml'
@@ -32,6 +32,7 @@ process MULTIQC {
     path ('assembly_spades/*')
     path ('assembly_unicycler/*')
     path ('assembly_minia/*')
+    path ('freyja_demix/*')
 
     output:
     path "*multiqc_report.html"     , emit: report
diff --git a/modules/local/multiqc_nanopore.nf b/modules/local/multiqc_nanopore.nf
index c6ffbb4d..92ec1f8a 100644
--- a/modules/local/multiqc_nanopore.nf
+++ b/modules/local/multiqc_nanopore.nf
@@ -1,10 +1,10 @@
 process MULTIQC {
     label 'process_medium'
 
-    conda "bioconda::multiqc=1.14"
+    conda "bioconda::multiqc=1.19"
     container "${ workflow.containerEngine == 'singularity' && !task.ext.singularity_pull_docker_container ?
-        'https://depot.galaxyproject.org/singularity/multiqc:1.14--pyhdfd78af_0' :
-        'quay.io/biocontainers/multiqc:1.14--pyhdfd78af_0' }"
+        'https://depot.galaxyproject.org/singularity/multiqc:1.19--pyhdfd78af_0' :
+        'quay.io/biocontainers/multiqc:1.19--pyhdfd78af_0' }"
 
     input:
     path 'multiqc_config.yaml'
@@ -25,6 +25,7 @@ process MULTIQC {
     path ('snpeff/*')
     path pangolin_lineage
     path nextclade_clade
+    path ('freyja_demix/*')
 
     output:
     path "*multiqc_report.html", emit: report
diff --git a/workflows/illumina.nf b/workflows/illumina.nf
index 1c369a78..3c53281b 100644
--- a/workflows/illumina.nf
+++ b/workflows/illumina.nf
@@ -486,7 +486,8 @@ workflow ILLUMINA {
             params.freyja_barcodes,
             params.freyja_lineages,
         )
-        ch_versions= ch_versions.mix(BAM_VARIANT_DEMIX_BOOT_FREYJA.out.versions)
+        ch_versions       = ch_versions.mix(BAM_VARIANT_DEMIX_BOOT_FREYJA.out.versions)
+        ch_freyja_multiqc = BAM_VARIANT_DEMIX_BOOT_FREYJA.out.demix
     }
 
     //
@@ -670,7 +671,8 @@ workflow ILLUMINA {
             ch_cutadapt_multiqc.collect{it[1]}.ifEmpty([]),
             ch_spades_quast_multiqc.collect{it[1]}.ifEmpty([]),
             ch_unicycler_quast_multiqc.collect{it[1]}.ifEmpty([]),
-            ch_minia_quast_multiqc.collect{it[1]}.ifEmpty([])
+            ch_minia_quast_multiqc.collect{it[1]}.ifEmpty([]),
+            ch_freyja_multiqc.collect{it[1]}.ifEmpty([]),
         )
         multiqc_report = MULTIQC.out.report.toList()
     }
diff --git a/workflows/nanopore.nf b/workflows/nanopore.nf
index 698220cd..8a83e30e 100644
--- a/workflows/nanopore.nf
+++ b/workflows/nanopore.nf
@@ -453,7 +453,8 @@ workflow NANOPORE {
             params.freyja_barcodes,
             params.freyja_lineages,
         )
-        ch_versions= ch_versions.mix(BAM_VARIANT_DEMIX_BOOT_FREYJA.out.versions)
+        ch_versions       = ch_versions.mix(BAM_VARIANT_DEMIX_BOOT_FREYJA.out.versions)
+        ch_freyja_multiqc = BAM_VARIANT_DEMIX_BOOT_FREYJA.out.demix
     }
 
     //
@@ -564,7 +565,8 @@ workflow NANOPORE {
             ch_quast_multiqc.collect{it[1]}.ifEmpty([]),
             ch_snpeff_multiqc.collect{it[1]}.ifEmpty([]),
             ch_pangolin_multiqc.collect{it[1]}.ifEmpty([]),
-            ch_nextclade_multiqc.collectFile(name: 'nextclade_clade_mqc.tsv').ifEmpty([])
+            ch_nextclade_multiqc.collectFile(name: 'nextclade_clade_mqc.tsv').ifEmpty([]),
+            ch_freyja_multiqc.collect{it[1]}.ifEmpty([]),
         )
         multiqc_report = MULTIQC.out.report.toList()
     }

From f0ecb40c3d17ac30949efce039546d1637930ea0 Mon Sep 17 00:00:00 2001
From: Joon-Klaps <joon.klaps@kuleuven.be>
Date: Wed, 17 Jan 2024 13:43:53 +0000
Subject: [PATCH 2/7] Declare empty freyja_mqc channel don't pubslih freyja
 update version

---
 conf/modules_illumina.config | 1 +
 conf/modules_nanopore.config | 1 +
 workflows/illumina.nf        | 1 +
 workflows/nanopore.nf        | 1 +
 4 files changed, 4 insertions(+)

diff --git a/conf/modules_illumina.config b/conf/modules_illumina.config
index 2cc48bea..8bb72cea 100644
--- a/conf/modules_illumina.config
+++ b/conf/modules_illumina.config
@@ -219,6 +219,7 @@ if (!params.skip_variants) {
                 publishDir = [
                     path: { "${params.outdir}/variants/freyja/" },
                     mode: params.publish_dir_mode,
+                    saveAs: { filename -> filename.equals('versions.yml') ? null : filename },
                 ]
             }
         }
diff --git a/conf/modules_nanopore.config b/conf/modules_nanopore.config
index 31ca44fb..0297911f 100644
--- a/conf/modules_nanopore.config
+++ b/conf/modules_nanopore.config
@@ -260,6 +260,7 @@ if (!params.skip_freyja) {
             publishDir = [
                 path: { "${params.outdir}/${params.artic_minion_caller}/freyja/" },
                 mode: params.publish_dir_mode,
+                saveAs: { filename -> filename.equals('versions.yml') ? null : filename },
             ]
         }
     }
diff --git a/workflows/illumina.nf b/workflows/illumina.nf
index 3c53281b..38abdf56 100644
--- a/workflows/illumina.nf
+++ b/workflows/illumina.nf
@@ -477,6 +477,7 @@ workflow ILLUMINA {
     //
     // SUBWORKFLOW: Determine variants with Freyja
     //
+    ch_freyja_multiqc = Channel.empty()
     if (!params.skip_variants && !params.skip_freyja) {
         BAM_VARIANT_DEMIX_BOOT_FREYJA(
             ch_bam,
diff --git a/workflows/nanopore.nf b/workflows/nanopore.nf
index 8a83e30e..87afd075 100644
--- a/workflows/nanopore.nf
+++ b/workflows/nanopore.nf
@@ -444,6 +444,7 @@ workflow NANOPORE {
     //
     // SUBWORKFLOW: Determine variants with Freyja
     //
+    ch_freyja_multiqc = Channel.empty()
     if (!params.skip_freyja) {
         BAM_VARIANT_DEMIX_BOOT_FREYJA(
             ARTIC_MINION.out.bam_primertrimmed,

From 4bb74c8337b074912552ca612fe606415677746f Mon Sep 17 00:00:00 2001
From: nf-core-bot <core@nf-co.re>
Date: Wed, 17 Jan 2024 19:45:14 +0000
Subject: [PATCH 3/7] [automated] Fix linting with Prettier

---
 .devcontainer/devcontainer.json | 8 ++++----
 1 file changed, 4 insertions(+), 4 deletions(-)

diff --git a/.devcontainer/devcontainer.json b/.devcontainer/devcontainer.json
index 4ecfbfe3..4a9bc5c7 100644
--- a/.devcontainer/devcontainer.json
+++ b/.devcontainer/devcontainer.json
@@ -18,11 +18,11 @@
                 "python.linting.flake8Path": "/opt/conda/bin/flake8",
                 "python.linting.pycodestylePath": "/opt/conda/bin/pycodestyle",
                 "python.linting.pydocstylePath": "/opt/conda/bin/pydocstyle",
-                "python.linting.pylintPath": "/opt/conda/bin/pylint"
+                "python.linting.pylintPath": "/opt/conda/bin/pylint",
             },
 
             // Add the IDs of extensions you want installed when the container is created.
-            "extensions": ["ms-python.python", "ms-python.vscode-pylance", "nf-core.nf-core-extensionpack"]
-        }
-    }
+            "extensions": ["ms-python.python", "ms-python.vscode-pylance", "nf-core.nf-core-extensionpack"],
+        },
+    },
 }

From 7e65e9b4c268b2f977b313dc2c38e9f8e529519e Mon Sep 17 00:00:00 2001
From: Joon-Klaps <joon.klaps@kuleuven.be>
Date: Thu, 25 Jan 2024 11:01:11 +0000
Subject: [PATCH 4/7] Update multiqc module & include freyja in report

---
 CHANGELOG.md | 2 ++
 1 file changed, 2 insertions(+)

diff --git a/CHANGELOG.md b/CHANGELOG.md
index 0f0676a3..01fbca92 100644
--- a/CHANGELOG.md
+++ b/CHANGELOG.md
@@ -20,6 +20,7 @@ Thank you to everyone else that has contributed by reporting bugs, enhancements
 - [[PR #387](https://github.com/nf-core/viralrecon/pull/387)] - Software closes gracefully when encountering an error
 - [[PR #395](https://github.com/nf-core/viralrecon/pull/395)] - Remove minia from default assemblers because it is unreliable
 - [[PR #393](https://github.com/nf-core/viralrecon/pull/393)] - Changed primer set to params
+- [[PR #413](https://github.com/nf-core/viralrecon/pull/413)] - Update multiqc module & include freyja in report
 
 ### Parameters
 
@@ -42,6 +43,7 @@ Note, since the pipeline is now using Nextflow DSL2, each process will be run wi
 | Dependency | Old version | New version |
 | ---------- | ----------- | ----------- |
 | `freyja`   |             | 1.3.12      |
+| `multiqc`  | 1.14        | 1.19        |
 
 > **NB:** Dependency has been **updated** if both old and new version information is present.
 >

From 4366e62e0618df472e870be6cd8a8ca581fdb912 Mon Sep 17 00:00:00 2001
From: Joon-Klaps <joon.klaps@kuleuven.be>
Date: Thu, 25 Jan 2024 15:14:37 +0000
Subject: [PATCH 5/7] add freyja screenshot to output.md

---
 docs/images/freyja_screenshot.png | Bin 0 -> 55926 bytes
 docs/output.md                    | 170 +++++++++++++++++++++---------
 2 files changed, 120 insertions(+), 50 deletions(-)
 create mode 100644 docs/images/freyja_screenshot.png

diff --git a/docs/images/freyja_screenshot.png b/docs/images/freyja_screenshot.png
new file mode 100644
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diff --git a/docs/output.md b/docs/output.md
index 320f845e..5de294c5 100644
--- a/docs/output.md
+++ b/docs/output.md
@@ -6,25 +6,64 @@ The directories listed below will be created in the results directory after the
 
 # Nanopore: Pipeline overview
 
-- [Preprocessing](#nanopore-preprocessing)
-  - [pycoQC](#nanopore-pycoqc) - Sequencing QC
-  - [artic guppyplex](#nanopore-artic-guppyplex) - Aggregate pre-demultiplexed reads from MinKNOW/Guppy
-  - [NanoPlot](#nanopore-nanoplot) - Read QC
-- [Variant calling](#nanopore-variant-calling)
-  - [artic minion](#nanopore-artic-minion) - Align reads, call variants and generate consensus sequence
-- [Downstream analysis](#nanopore-downstream-analysis)
-  - [SAMtools](#nanopore-samtools) - Remove unmapped reads and obtain alignment metrics
-  - [mosdepth](#nanopore-mosdepth) - Genome-wide and amplicon coverage QC plots
-  - [BCFTools](#nanopore-bcftools) - Variant count metrics
-  - [SnpEff and SnpSift](#nanopore-snpeff-and-snpsift) - Genetic variant annotation and functional effect prediction
-  - [QUAST](#nanopore-quast) - Consensus assessment report
-  - [Pangolin](#nanopore-pangolin) - Lineage analysis
-  - [Nextclade](#nanopore-nextclade) - Clade assignment, mutation calling and sequence quality checks
-  - [Freyja](#nanopore-freyja) - Relative lineage abundance analysis from mixed SARS-CoV-2 samples (typically wastewater)
-  - [ASCIIGenome](#nanopore-asciigenome) - Individual variant screenshots with annotation tracks
-  - [Variants long table](#nanopore-variants-long-table) - Collate per-sample information for individual variants, functional effect prediction and lineage analysis
-- [Workflow reporting](#nanopore-workflow-reporting)
-  - [MultiQC](#nanopore-multiqc) - Present QC, visualisation and custom reporting for sequencing, raw reads, alignment and variant calling results
+- [Introduction](#introduction)
+- [Nanopore: Pipeline overview](#nanopore-pipeline-overview)
+  - [Nanopore: Preprocessing](#nanopore-preprocessing)
+    - [Nanopore: pycoQC](#nanopore-pycoqc)
+    - [Nanopore: artic guppyplex](#nanopore-artic-guppyplex)
+    - [Nanopore: NanoPlot](#nanopore-nanoplot)
+  - [Nanopore: Variant calling](#nanopore-variant-calling)
+    - [Nanopore: artic minion](#nanopore-artic-minion)
+  - [Nanopore: Downstream analysis](#nanopore-downstream-analysis)
+    - [Nanopore: SAMtools](#nanopore-samtools)
+    - [Nanopore: mosdepth](#nanopore-mosdepth)
+    - [Nanopore: BCFTools](#nanopore-bcftools)
+    - [Nanopore: SnpEff and SnpSift](#nanopore-snpeff-and-snpsift)
+    - [Nanopore: QUAST](#nanopore-quast)
+    - [Nanopore: Pangolin](#nanopore-pangolin)
+    - [Nanopore: Nextclade](#nanopore-nextclade)
+    - [Nanopore: Freyja](#nanopore-freyja)
+    - [Nanopore: ASCIIGenome](#nanopore-asciigenome)
+    - [Nanopore: Variants long table](#nanopore-variants-long-table)
+  - [Nanopore: Workflow reporting](#nanopore-workflow-reporting)
+    - [Nanopore: MultiQC](#nanopore-multiqc)
+- [Illumina: Pipeline overview](#illumina-pipeline-overview)
+  - [Illumina: Preprocessing](#illumina-preprocessing)
+    - [cat](#cat)
+    - [FastQC](#fastqc)
+    - [fastp](#fastp)
+    - [Kraken 2](#kraken-2)
+  - [Illumina: Variant calling](#illumina-variant-calling)
+    - [Bowtie 2](#bowtie-2)
+    - [SAMtools](#samtools)
+    - [iVar trim](#ivar-trim)
+    - [picard MarkDuplicates](#picard-markduplicates)
+    - [picard CollectMultipleMetrics](#picard-collectmultiplemetrics)
+    - [mosdepth](#mosdepth)
+    - [iVar variants](#ivar-variants)
+    - [BCFTools call](#bcftools-call)
+    - [SnpEff and SnpSift](#snpeff-and-snpsift)
+    - [Freyja](#freyja)
+    - [ASCIIGenome](#asciigenome)
+    - [iVar consensus](#ivar-consensus)
+    - [BCFTools and BEDTools](#bcftools-and-bedtools)
+    - [QUAST](#quast)
+    - [Pangolin](#pangolin)
+    - [Nextclade](#nextclade)
+    - [Variants long table](#variants-long-table)
+  - [Illumina: De novo assembly](#illumina-de-novo-assembly)
+    - [Cutadapt](#cutadapt)
+    - [SPAdes](#spades)
+    - [Unicycler](#unicycler)
+    - [minia](#minia)
+    - [BLAST](#blast)
+    - [ABACAS](#abacas)
+    - [PlasmidID](#plasmidid)
+    - [Assembly QUAST](#assembly-quast)
+  - [Illumina: Workflow reporting and genomes](#illumina-workflow-reporting-and-genomes)
+    - [MultiQC](#multiqc)
+    - [Reference genome files](#reference-genome-files)
+- [Pipeline information](#pipeline-information)
 
 ## Nanopore: Preprocessing
 
@@ -266,6 +305,8 @@ Phylogenetic Assignment of Named Global Outbreak LINeages ([Pangolin](https://gi
 
 [Freyja](https://github.com/andersen-lab/Freyja) is a tool to recover relative lineage abundances from mixed SARS-CoV-2 samples from a sequencing dataset (BAM aligned to the Hu-1 reference). The method uses lineage-determining mutational "barcodes" derived from the [UShER](https://usher-wiki.readthedocs.io/en/latest/#) global phylogenetic tree as a basis set to solve the constrained (unit sum, non-negative) de-mixing problem.
 
+<p align="center"><img src="images/freyja_screenshot.png" alt="Freyja screenshot"></p>
+
 ### Nanopore: ASCIIGenome
 
 <details markdown="1">
@@ -332,37 +373,64 @@ An example MultiQC report generated from a full-sized dataset can be viewed on t
 
 # Illumina: Pipeline overview
 
-- [Preprocessing](#illumina-preprocessing)
-  - [cat](#cat) - Merge re-sequenced FastQ files
-  - [FastQC](#fastqc) - Raw read QC
-  - [fastp](#fastp) - Adapter and quality trimming
-  - [Kraken 2](#kraken-2) - Removal/QC for host reads
-- [Variant calling](#illumina-variant-calling)
-  - [Bowtie 2](#bowtie-2) - Read alignment relative to reference genome
-  - [SAMtools](#samtools) - Sort, index and generate metrics for alignments
-  - [iVar trim](#ivar-trim) - Primer sequence removal for amplicon data
-  - [picard MarkDuplicates](#picard-markduplicates) - Duplicate read marking and removal
-  - [picard CollectMultipleMetrics](#picard-collectmultiplemetrics) - Alignment metrics
-  - [mosdepth](#mosdepth) - Whole-genome and amplicon coverage metrics
-  - [iVar variants](#ivar-variants) _||_ [BCFTools call](#bcftools-call) - Variant calling
-    - [SnpEff and SnpSift](#snpeff-and-snpsift) - Genetic variant annotation and functional effect prediction
-    - [Freyja](#freyja) - Relative lineage abundance analysis from mixed SARS-CoV-2 samples (typically wastewater)
-    - [ASCIIGenome](#asciigenome) - Individual variant screenshots with annotation tracks
-  - [iVar consensus](#ivar-consensus) _||_ [BCFTools and BEDTools](#bcftools-and-bedtools) - Consensus sequence generation
-    - [QUAST](#quast) - Consensus assessment report
-    - [Pangolin](#pangolin) - Lineage analysis
-    - [Nextclade](#nextclade) - Clade assignment, mutation calling and sequence quality checks
-  - [Variants long table](#variants-long-table) - Collate per-sample information for individual variants, functional effect prediction and lineage analysis
-- [De novo assembly](#illumina-de-novo-assembly)
-  - [Cutadapt](#cutadapt) - Primer trimming for amplicon data
-  - [SPAdes](#spades) _||_ [Unicycler](#unicycler) _||_ [minia](#minia) - Viral genome assembly
-    - [BLAST](#blast) - Blast to reference assembly
-    - [ABACAS](#abacas) - Order contigs according to reference genome
-    - [PlasmidID](#plasmidid) - Assembly report and visualisation
-    - [Assembly QUAST](#assembly-quast) - Assembly quality assessment
-- [Workflow reporting and genomes](#illumina-workflow-reporting-and-genomes)
-  - [MultiQC](#multiqc) - Present QC for raw reads, alignment, assembly and variant calling
-  - [Reference genome files](#reference-genome-files) - Save reference genome indices/files
+- [Introduction](#introduction)
+- [Nanopore: Pipeline overview](#nanopore-pipeline-overview)
+  - [Nanopore: Preprocessing](#nanopore-preprocessing)
+    - [Nanopore: pycoQC](#nanopore-pycoqc)
+    - [Nanopore: artic guppyplex](#nanopore-artic-guppyplex)
+    - [Nanopore: NanoPlot](#nanopore-nanoplot)
+  - [Nanopore: Variant calling](#nanopore-variant-calling)
+    - [Nanopore: artic minion](#nanopore-artic-minion)
+  - [Nanopore: Downstream analysis](#nanopore-downstream-analysis)
+    - [Nanopore: SAMtools](#nanopore-samtools)
+    - [Nanopore: mosdepth](#nanopore-mosdepth)
+    - [Nanopore: BCFTools](#nanopore-bcftools)
+    - [Nanopore: SnpEff and SnpSift](#nanopore-snpeff-and-snpsift)
+    - [Nanopore: QUAST](#nanopore-quast)
+    - [Nanopore: Pangolin](#nanopore-pangolin)
+    - [Nanopore: Nextclade](#nanopore-nextclade)
+    - [Nanopore: Freyja](#nanopore-freyja)
+    - [Nanopore: ASCIIGenome](#nanopore-asciigenome)
+    - [Nanopore: Variants long table](#nanopore-variants-long-table)
+  - [Nanopore: Workflow reporting](#nanopore-workflow-reporting)
+    - [Nanopore: MultiQC](#nanopore-multiqc)
+- [Illumina: Pipeline overview](#illumina-pipeline-overview)
+  - [Illumina: Preprocessing](#illumina-preprocessing)
+    - [cat](#cat)
+    - [FastQC](#fastqc)
+    - [fastp](#fastp)
+    - [Kraken 2](#kraken-2)
+  - [Illumina: Variant calling](#illumina-variant-calling)
+    - [Bowtie 2](#bowtie-2)
+    - [SAMtools](#samtools)
+    - [iVar trim](#ivar-trim)
+    - [picard MarkDuplicates](#picard-markduplicates)
+    - [picard CollectMultipleMetrics](#picard-collectmultiplemetrics)
+    - [mosdepth](#mosdepth)
+    - [iVar variants](#ivar-variants)
+    - [BCFTools call](#bcftools-call)
+    - [SnpEff and SnpSift](#snpeff-and-snpsift)
+    - [Freyja](#freyja)
+    - [ASCIIGenome](#asciigenome)
+    - [iVar consensus](#ivar-consensus)
+    - [BCFTools and BEDTools](#bcftools-and-bedtools)
+    - [QUAST](#quast)
+    - [Pangolin](#pangolin)
+    - [Nextclade](#nextclade)
+    - [Variants long table](#variants-long-table)
+  - [Illumina: De novo assembly](#illumina-de-novo-assembly)
+    - [Cutadapt](#cutadapt)
+    - [SPAdes](#spades)
+    - [Unicycler](#unicycler)
+    - [minia](#minia)
+    - [BLAST](#blast)
+    - [ABACAS](#abacas)
+    - [PlasmidID](#plasmidid)
+    - [Assembly QUAST](#assembly-quast)
+  - [Illumina: Workflow reporting and genomes](#illumina-workflow-reporting-and-genomes)
+    - [MultiQC](#multiqc)
+    - [Reference genome files](#reference-genome-files)
+- [Pipeline information](#pipeline-information)
 
 ## Illumina: Preprocessing
 
@@ -641,6 +709,8 @@ iVar outputs a tsv format which is not compatible with downstream analysis such
 
 [Freyja](https://github.com/andersen-lab/Freyja) is a tool to recover relative lineage abundances from mixed SARS-CoV-2 samples from a sequencing dataset (BAM aligned to the Hu-1 reference). The method uses lineage-determining mutational "barcodes" derived from the [UShER](https://usher-wiki.readthedocs.io/en/latest/#) global phylogenetic tree as a basis set to solve the constrained (unit sum, non-negative) de-mixing problem.
 
+<p align="center"><img src="images/freyja_screenshot.png" alt="Freyja screenshot"></p>
+
 ### ASCIIGenome
 
 <details markdown="1">

From cda40f2ed08ad70ed68901e81ddeeded4a98ce95 Mon Sep 17 00:00:00 2001
From: Joon Klaps <61584065+Joon-Klaps@users.noreply.github.com>
Date: Thu, 25 Jan 2024 16:23:08 +0100
Subject: [PATCH 6/7] Weird table of content auto edit  undo output.md

---
 docs/output.md | 166 +++++++++++++++----------------------------------
 1 file changed, 50 insertions(+), 116 deletions(-)

diff --git a/docs/output.md b/docs/output.md
index 5de294c5..d60ce8c5 100644
--- a/docs/output.md
+++ b/docs/output.md
@@ -6,64 +6,25 @@ The directories listed below will be created in the results directory after the
 
 # Nanopore: Pipeline overview
 
-- [Introduction](#introduction)
-- [Nanopore: Pipeline overview](#nanopore-pipeline-overview)
-  - [Nanopore: Preprocessing](#nanopore-preprocessing)
-    - [Nanopore: pycoQC](#nanopore-pycoqc)
-    - [Nanopore: artic guppyplex](#nanopore-artic-guppyplex)
-    - [Nanopore: NanoPlot](#nanopore-nanoplot)
-  - [Nanopore: Variant calling](#nanopore-variant-calling)
-    - [Nanopore: artic minion](#nanopore-artic-minion)
-  - [Nanopore: Downstream analysis](#nanopore-downstream-analysis)
-    - [Nanopore: SAMtools](#nanopore-samtools)
-    - [Nanopore: mosdepth](#nanopore-mosdepth)
-    - [Nanopore: BCFTools](#nanopore-bcftools)
-    - [Nanopore: SnpEff and SnpSift](#nanopore-snpeff-and-snpsift)
-    - [Nanopore: QUAST](#nanopore-quast)
-    - [Nanopore: Pangolin](#nanopore-pangolin)
-    - [Nanopore: Nextclade](#nanopore-nextclade)
-    - [Nanopore: Freyja](#nanopore-freyja)
-    - [Nanopore: ASCIIGenome](#nanopore-asciigenome)
-    - [Nanopore: Variants long table](#nanopore-variants-long-table)
-  - [Nanopore: Workflow reporting](#nanopore-workflow-reporting)
-    - [Nanopore: MultiQC](#nanopore-multiqc)
-- [Illumina: Pipeline overview](#illumina-pipeline-overview)
-  - [Illumina: Preprocessing](#illumina-preprocessing)
-    - [cat](#cat)
-    - [FastQC](#fastqc)
-    - [fastp](#fastp)
-    - [Kraken 2](#kraken-2)
-  - [Illumina: Variant calling](#illumina-variant-calling)
-    - [Bowtie 2](#bowtie-2)
-    - [SAMtools](#samtools)
-    - [iVar trim](#ivar-trim)
-    - [picard MarkDuplicates](#picard-markduplicates)
-    - [picard CollectMultipleMetrics](#picard-collectmultiplemetrics)
-    - [mosdepth](#mosdepth)
-    - [iVar variants](#ivar-variants)
-    - [BCFTools call](#bcftools-call)
-    - [SnpEff and SnpSift](#snpeff-and-snpsift)
-    - [Freyja](#freyja)
-    - [ASCIIGenome](#asciigenome)
-    - [iVar consensus](#ivar-consensus)
-    - [BCFTools and BEDTools](#bcftools-and-bedtools)
-    - [QUAST](#quast)
-    - [Pangolin](#pangolin)
-    - [Nextclade](#nextclade)
-    - [Variants long table](#variants-long-table)
-  - [Illumina: De novo assembly](#illumina-de-novo-assembly)
-    - [Cutadapt](#cutadapt)
-    - [SPAdes](#spades)
-    - [Unicycler](#unicycler)
-    - [minia](#minia)
-    - [BLAST](#blast)
-    - [ABACAS](#abacas)
-    - [PlasmidID](#plasmidid)
-    - [Assembly QUAST](#assembly-quast)
-  - [Illumina: Workflow reporting and genomes](#illumina-workflow-reporting-and-genomes)
-    - [MultiQC](#multiqc)
-    - [Reference genome files](#reference-genome-files)
-- [Pipeline information](#pipeline-information)
+- [Preprocessing](#nanopore-preprocessing)
+  - [pycoQC](#nanopore-pycoqc) - Sequencing QC
+  - [artic guppyplex](#nanopore-artic-guppyplex) - Aggregate pre-demultiplexed reads from MinKNOW/Guppy
+  - [NanoPlot](#nanopore-nanoplot) - Read QC
+- [Variant calling](#nanopore-variant-calling)
+  - [artic minion](#nanopore-artic-minion) - Align reads, call variants and generate consensus sequence
+- [Downstream analysis](#nanopore-downstream-analysis)
+  - [SAMtools](#nanopore-samtools) - Remove unmapped reads and obtain alignment metrics
+  - [mosdepth](#nanopore-mosdepth) - Genome-wide and amplicon coverage QC plots
+  - [BCFTools](#nanopore-bcftools) - Variant count metrics
+  - [SnpEff and SnpSift](#nanopore-snpeff-and-snpsift) - Genetic variant annotation and functional effect prediction
+  - [QUAST](#nanopore-quast) - Consensus assessment report
+  - [Pangolin](#nanopore-pangolin) - Lineage analysis
+  - [Nextclade](#nanopore-nextclade) - Clade assignment, mutation calling and sequence quality checks
+  - [Freyja](#nanopore-freyja) - Relative lineage abundance analysis from mixed SARS-CoV-2 samples (typically wastewater)
+  - [ASCIIGenome](#nanopore-asciigenome) - Individual variant screenshots with annotation tracks
+  - [Variants long table](#nanopore-variants-long-table) - Collate per-sample information for individual variants, functional effect prediction and lineage analysis
+- [Workflow reporting](#nanopore-workflow-reporting)
+  - [MultiQC](#nanopore-multiqc) - Present QC, visualisation and custom reporting for sequencing, raw reads, alignment and variant calling results
 
 ## Nanopore: Preprocessing
 
@@ -373,64 +334,37 @@ An example MultiQC report generated from a full-sized dataset can be viewed on t
 
 # Illumina: Pipeline overview
 
-- [Introduction](#introduction)
-- [Nanopore: Pipeline overview](#nanopore-pipeline-overview)
-  - [Nanopore: Preprocessing](#nanopore-preprocessing)
-    - [Nanopore: pycoQC](#nanopore-pycoqc)
-    - [Nanopore: artic guppyplex](#nanopore-artic-guppyplex)
-    - [Nanopore: NanoPlot](#nanopore-nanoplot)
-  - [Nanopore: Variant calling](#nanopore-variant-calling)
-    - [Nanopore: artic minion](#nanopore-artic-minion)
-  - [Nanopore: Downstream analysis](#nanopore-downstream-analysis)
-    - [Nanopore: SAMtools](#nanopore-samtools)
-    - [Nanopore: mosdepth](#nanopore-mosdepth)
-    - [Nanopore: BCFTools](#nanopore-bcftools)
-    - [Nanopore: SnpEff and SnpSift](#nanopore-snpeff-and-snpsift)
-    - [Nanopore: QUAST](#nanopore-quast)
-    - [Nanopore: Pangolin](#nanopore-pangolin)
-    - [Nanopore: Nextclade](#nanopore-nextclade)
-    - [Nanopore: Freyja](#nanopore-freyja)
-    - [Nanopore: ASCIIGenome](#nanopore-asciigenome)
-    - [Nanopore: Variants long table](#nanopore-variants-long-table)
-  - [Nanopore: Workflow reporting](#nanopore-workflow-reporting)
-    - [Nanopore: MultiQC](#nanopore-multiqc)
-- [Illumina: Pipeline overview](#illumina-pipeline-overview)
-  - [Illumina: Preprocessing](#illumina-preprocessing)
-    - [cat](#cat)
-    - [FastQC](#fastqc)
-    - [fastp](#fastp)
-    - [Kraken 2](#kraken-2)
-  - [Illumina: Variant calling](#illumina-variant-calling)
-    - [Bowtie 2](#bowtie-2)
-    - [SAMtools](#samtools)
-    - [iVar trim](#ivar-trim)
-    - [picard MarkDuplicates](#picard-markduplicates)
-    - [picard CollectMultipleMetrics](#picard-collectmultiplemetrics)
-    - [mosdepth](#mosdepth)
-    - [iVar variants](#ivar-variants)
-    - [BCFTools call](#bcftools-call)
-    - [SnpEff and SnpSift](#snpeff-and-snpsift)
-    - [Freyja](#freyja)
-    - [ASCIIGenome](#asciigenome)
-    - [iVar consensus](#ivar-consensus)
-    - [BCFTools and BEDTools](#bcftools-and-bedtools)
-    - [QUAST](#quast)
-    - [Pangolin](#pangolin)
-    - [Nextclade](#nextclade)
-    - [Variants long table](#variants-long-table)
-  - [Illumina: De novo assembly](#illumina-de-novo-assembly)
-    - [Cutadapt](#cutadapt)
-    - [SPAdes](#spades)
-    - [Unicycler](#unicycler)
-    - [minia](#minia)
-    - [BLAST](#blast)
-    - [ABACAS](#abacas)
-    - [PlasmidID](#plasmidid)
-    - [Assembly QUAST](#assembly-quast)
-  - [Illumina: Workflow reporting and genomes](#illumina-workflow-reporting-and-genomes)
-    - [MultiQC](#multiqc)
-    - [Reference genome files](#reference-genome-files)
-- [Pipeline information](#pipeline-information)
+- [Preprocessing](#illumina-preprocessing)
+  - [cat](#cat) - Merge re-sequenced FastQ files
+  - [FastQC](#fastqc) - Raw read QC
+  - [fastp](#fastp) - Adapter and quality trimming
+  - [Kraken 2](#kraken-2) - Removal/QC for host reads
+- [Variant calling](#illumina-variant-calling)
+  - [Bowtie 2](#bowtie-2) - Read alignment relative to reference genome
+  - [SAMtools](#samtools) - Sort, index and generate metrics for alignments
+  - [iVar trim](#ivar-trim) - Primer sequence removal for amplicon data
+  - [picard MarkDuplicates](#picard-markduplicates) - Duplicate read marking and removal
+  - [picard CollectMultipleMetrics](#picard-collectmultiplemetrics) - Alignment metrics
+  - [mosdepth](#mosdepth) - Whole-genome and amplicon coverage metrics
+  - [iVar variants](#ivar-variants) _||_ [BCFTools call](#bcftools-call) - Variant calling
+    - [SnpEff and SnpSift](#snpeff-and-snpsift) - Genetic variant annotation and functional effect prediction
+    - [Freyja](#freyja) - Relative lineage abundance analysis from mixed SARS-CoV-2 samples (typically wastewater)
+    - [ASCIIGenome](#asciigenome) - Individual variant screenshots with annotation tracks
+  - [iVar consensus](#ivar-consensus) _||_ [BCFTools and BEDTools](#bcftools-and-bedtools) - Consensus sequence generation
+    - [QUAST](#quast) - Consensus assessment report
+    - [Pangolin](#pangolin) - Lineage analysis
+    - [Nextclade](#nextclade) - Clade assignment, mutation calling and sequence quality checks
+  - [Variants long table](#variants-long-table) - Collate per-sample information for individual variants, functional effect prediction and lineage analysis
+- [De novo assembly](#illumina-de-novo-assembly)
+  - [Cutadapt](#cutadapt) - Primer trimming for amplicon data
+  - [SPAdes](#spades) _||_ [Unicycler](#unicycler) _||_ [minia](#minia) - Viral genome assembly
+    - [BLAST](#blast) - Blast to reference assembly
+    - [ABACAS](#abacas) - Order contigs according to reference genome
+    - [PlasmidID](#plasmidid) - Assembly report and visualisation
+    - [Assembly QUAST](#assembly-quast) - Assembly quality assessment
+- [Workflow reporting and genomes](#illumina-workflow-reporting-and-genomes)
+  - [MultiQC](#multiqc) - Present QC for raw reads, alignment, assembly and variant calling
+  - [Reference genome files](#reference-genome-files) - Save reference genome indices/files
 
 ## Illumina: Preprocessing
 

From 33e72562a2a231384c6e22b58bcea7649079d980 Mon Sep 17 00:00:00 2001
From: Joon Klaps <61584065+Joon-Klaps@users.noreply.github.com>
Date: Thu, 25 Jan 2024 16:25:36 +0100
Subject: [PATCH 7/7] Update CHANGELOG.md

---
 CHANGELOG.md | 2 +-
 1 file changed, 1 insertion(+), 1 deletion(-)

diff --git a/CHANGELOG.md b/CHANGELOG.md
index 274a1e37..6e8dd803 100644
--- a/CHANGELOG.md
+++ b/CHANGELOG.md
@@ -20,7 +20,7 @@ Thank you to everyone else that has contributed by reporting bugs, enhancements
 - [[PR #387](https://github.com/nf-core/viralrecon/pull/387)] - Software closes gracefully when encountering an error
 - [[PR #395](https://github.com/nf-core/viralrecon/pull/395)] - Remove minia from default assemblers because it is unreliable
 - [[PR #393](https://github.com/nf-core/viralrecon/pull/393)] - Changed primer set to params
-- [[PR [#405](https://github.com/nf-core/viralrecon/issues/405)]] - Including parameter `depthcutoff` to freyja demix and boot
+- [[PR #405](https://github.com/nf-core/viralrecon/pull/412)] - Including parameter `depthcutoff` to freyja demix and boot
 - [[PR #413](https://github.com/nf-core/viralrecon/pull/413)] - Update multiqc module & include freyja in report
 
 ### Parameters