#20 clean multiqc output to avoid duplicated name in the general stat…

…istics table, add section in readme to explain how to look at the results

README.md

@@ -21,6 +21,7 @@ AliNe is a pipeline written in Nextflow that aims to efficiently align reads aga
         * [Singularity](#singularity)  
    * [Usage and test](#usage)
    * [Parameters](#parameters)
+   * [Comparing output](#comparing-output)
    * [Contributing](#contributing)
 
 ## Foreword
@@ -350,6 +351,88 @@ On success you should get a message looking like this:
         --sublong_options           additional options for sublong
 ```
 
+## Comparing output
+
+In order to compare several aligner output you should activate the `--fastqc` parameter. AliNe will run the FastQC program for each output file, thereafter all FastQC file will be gathered in an html file using MultiQC. The resulting file called `multiqc_report.html` can be found in <outdir>/MultiQC ( <outdir> by default is called `alignment_results` and can be setup using `--outdir` AliNe parameter).
+
+FastQC collect the following information:  
+  * Sequence Counts
+  * Sequence Quality 
+  * Per Sequence Quality Scores
+  * Per Base Sequence Content
+  * Per Sequence GC Content
+  * Per Base N Content
+  * Sequence Length Distribution
+  * Sequence Duplication Levels 
+  * Overrepresented sequences
+  * Adapter Content
+
+### General Statistics
+
+Sequence Duplication Levels, Per Sequence GC Content and Sequence Counts are reported at the top of the `multiqc_report.html` file in a table called `General Statistics` as % Dups, %GC a,d M Seqs accordingly (see below).
+
+<img src="img/multiqc.png" />
+
+In order to facilitate the reading of this `General Statistics` you can export the table in tsv using the `Export as CSV...` button and execute the following piece of R code on the downloaded `general_stats_table.tsv` file :
+
+```R
+# install packages
+install.packages("dplyr")
+install.packages("stringr")
+install.packages("tidyr")
+
+# Load necessary libraries
+library(dplyr)
+library(stringr)
+library(tidyr)
+
+# Read the TSV file
+file_path <- "general_stats_table.tsv"
+df <- read.delim(file_path, check.names = FALSE)
+
+# sample name as row name
+rownames(df) <- df$Sample
+
+# remove Sample column and clean up the column names
+tmp <- cbind(ID = rownames(df), stack(df[-1])) |> 
+  transform(ind = as.character(ind) |> stringr::str_remove_all("\\.\\d+"))
+
+# remove na values
+tmp <- tmp[!is.na(tmp$values),]
+# remove . values
+tmp$values <- tmp$values |> stringr::str_remove_all("^\\.$")
+
+# pivot data
+tmp <- tmp |> pivot_wider(id_cols = ID , names_from = ind, values_from = values, 
+              values_fn = \(x) paste(unique(x), collapse = ""))
+
+# print only 4 first columns
+tmp[1:4]
+```
+
+You will get a table similar to this one:  
+
+```
+   ID                                       Dups              GC    Seqs                  
+   <chr>                                    <chr>             <chr> <chr>                 
+ 1 yeast_R1                                 73.01             43    0.01                  
+ 2 yeast_R1_seqkit_trim                     69.21661409043114 44    0.007607999999999999  
+ 3 yeast_R1_seqkit_trim_STAR_sorted         69.54090258811289 44    0.007689              
+ 4 yeast_R1_seqkit_trim_bbmap_sorted        69.21661409043114 44    0.007607999999999999  
+ 5 yeast_R1_seqkit_trim_bowtie2_sorted      69.21661409043114 44    0.007607999999999999  
+ 6 yeast_R1_seqkit_trim_bwaaln_sorted       69.21661409043114 44    0.007607999999999999  
+ 7 yeast_R1_seqkit_trim_bwamem_sorted       69.18933123111286 44    0.007611              
+ 8 yeast_R1_seqkit_trim_bwasw_sorted        69.23279033105622 44    0.007612              
+ 9 yeast_R1_seqkit_trim_graphmap2_sorted    69.21661409043114 44    0.007607999999999999  
+10 yeast_R1_seqkit_trim_hisat2_sorted       69.21661409043114 44    0.007607999999999999  
+11 yeast_R1_seqkit_trim_kallisto_sorted     69.21661409043114 44    0.007607999999999999  
+12 yeast_R1_seqkit_trim_minimap2_sorted     69.63058976020739 44    0.007715              
+13 yeast_R1_seqkit_trim_nucmer.fixed_sorted 64.70588235294117 42    0.00016999999999999999
+14 yeast_R1_seqkit_trim_sublong             53.23343848580442 44    0.007607999999999999  
+15 yeast_R1_seqkit_trim_subread             69.21661409043114 44    0.007607999999999999  
+```
+
+
 ## Contributing
 
 Contributions from the community are welcome ! See the [Contributing guidelines](https://github.com/Juke34/aline/blob/main/CONTRIBUTING.md)

aline.nf

@@ -509,7 +509,7 @@ workflow align {
         // ------------------- QC -----------------
         if(params.fastqc){
             fastqc_raw(raw_reads, "fastqc/raw", "raw")
-            logs.mix(fastqc_raw.out)
+            logs.concat(fastqc_raw.out).set{logs} // save log
         }
 
         // ------------------- Standardize Score to Be Phred+33 ----------------
@@ -1088,4 +1088,5 @@ In aline.nf
     Think to convert sam to bam if necessary
     Think to sort bam output if necessary 
 Add info in README.md
+Add tool in multiqc config file (at least for fastqc)
 */

config/multiqc_conf.yml

@@ -1,4 +1,4 @@
-title: "Nextflow Alignment pipeline"
+title: "AliNe - Nextflow Alignment pipeline (https://github.com/Juke34/AliNe)"
 
 run_modules:
     - fastqc
@@ -45,6 +45,10 @@ module_order:
         name: "FastQC (graphmap2)"
         path_filters:
           - "*graphmap2_logs*"
+    - fastqc:
+        name: "FastQC (hisat)"
+        path_filters:
+          - "*hisat_logs*"
     - hisat2
     - fastqc:
         name: "FastQC (hisat2)"
@@ -73,6 +77,12 @@ module_order:
           - "*sublong_logs*"
     - star
     - fastqc:
-        name: "FastQC (star)"
+        name: "FastQC (STAR)"
+        path_filters:
+          - "*star_logs*"
+          - "*STAR_logs*"
+    - fastqc:
+        name: "FastQC (STARlong)"
         path_filters:
-          - "*star_logs*"
+          - "*starlong_logs*"
+          - "*STARlong_logs*"

config/softwares.config

@@ -33,7 +33,8 @@ process {
         container = 'quay.io/biocontainers/minimap2:2.26--he4a0461_1'
     }
     withName: 'multiqc' {
-        container = 'quay.io/biocontainers/multiqc:1.14--pyhdfd78af_0'
+        //container = 'quay.io/biocontainers/multiqc:1.14--pyhdfd78af_0'
+        container = 'quay.io/biocontainers/multiqc:1.27--pyhdfd78af_0'
     }
     withLabel: 'mummer4' {
         container = 'quay.io/biocontainers/mummer4:4.0.0rc1--pl5321hdbdd923_6'

modules/bbmap.nf

@@ -83,7 +83,7 @@ process bbmap {
     }
 
     // set fileName
-    def fileName = fastq[0].baseName.replace('.fastq','')
+    def fileName = fastq[0].baseName.replace('.fastq','') + "_bbmap"
     """
     ${tool} \\
         ref=${genome_index} \\

modules/bowtie2.nf

@@ -30,7 +30,7 @@ process bowtie2 {
 
     output:
         tuple val(sample), path ("*.sam"), emit: tuple_sample_sam
-        path "*bowtie2.log",  emit: bowtie2_summary
+        path "*.log",  emit: bowtie2_summary
 
     script:
 
@@ -48,22 +48,25 @@ process bowtie2 {
                 read_orientation = "--ff"
             }  
         }
+
+        // catch filename
+        filename = reads[0].baseName.replace('.fastq','') + "_bowtie2"
 
         if (params.read_type == "short_paired"){
         """
             bowtie2 ${params.bowtie2_options} ${read_orientation}\\
                 -p ${task.cpus} \\
                 -x ${genome.baseName} \\
-                -S ${reads[0].baseName.replace('.fastq','')}_bowtie2.sam \\
-                -1 ${reads[0]} -2 ${reads[1]}  2> ${reads[0].baseName.replace('.fastq','')}_bowtie2.log
+                -S ${filename}.sam \\
+                -1 ${reads[0]} -2 ${reads[1]}  2> ${filename}_sorted.log
         """
         } else {
         """
             bowtie2 ${params.bowtie2_options} ${read_orientation}\\
                     -p ${task.cpus} \\
                     -x ${genome.baseName} \\
-                    -S ${reads.baseName.replace('.fastq','')}_bowtie2.sam \\
-                    -U ${reads} 2> ${reads.baseName}_bowtie2.log
+                    -S ${filename}.sam \\
+                    -U ${reads} 2> ${filename}_sorted.log
         """
         }
 

modules/hisat2.nf

@@ -34,16 +34,16 @@ process hisat2 {
 
     output:
         tuple val(sample), path ("*.sam"), emit: tuple_sample_sam
-        path "${sample}_splicesite.txt"
-        path "*hisat2-summary.txt",  emit: hisat2_summary
+        path "*_splicesite.txt"
+        path "*_summary.txt",  emit: hisat2_summary
 
     script:
 
         // catch index basename
         index_basename = hisat2_index_files[0].toString() - ~/.\d.ht2l?/
 
         // catch filename
-        filename = reads[0].baseName.replace('.fastq','')
+        filename = reads[0].baseName.replace('.fastq','') + "_hisat2"
 
         // deal with library type - default is unstranded.
         def lib_strand=""
@@ -82,14 +82,14 @@ process hisat2 {
 
         if (params.read_type == "short_paired"){
             """
-            hisat2 ${lib_strand} ${read_orientation} ${params.hisat2_options} --novel-splicesite-outfile ${sample}_splicesite.txt \\
-                --new-summary --summary-file ${sample}.hisat2-summary.txt \\
+            hisat2 ${lib_strand} ${read_orientation} ${params.hisat2_options} --novel-splicesite-outfile ${filename}_splicesite.txt \\
+                --new-summary --summary-file ${filename}_sorted_summary.txt \\
                 -p ${task.cpus} -x $index_basename -1 ${reads[0]} -2 ${reads[1]} > ${filename}.sam
             """
             } else {
             """
-            hisat2 ${lib_strand} ${read_orientation} ${params.hisat2_options} --novel-splicesite-outfile ${sample}_splicesite.txt \\
-                --new-summary --summary-file ${sample}.hisat2-summary.txt \\
+            hisat2 ${lib_strand} ${read_orientation} ${params.hisat2_options} --novel-splicesite-outfile ${filename}_splicesite.txt \\
+                --new-summary --summary-file ${filename}_sorted_summary.txt \\
                 -p ${task.cpus} -x $index_basename -U $reads > ${filename}.sam
             """
         }

modules/kallisto.nf

@@ -26,21 +26,21 @@ process kallisto_index {
 process kallisto {
     label 'kallisto'
     tag "$sample"
-    publishDir "${params.outdir}/${outpath}", pattern: "*.bam", mode: 'copy'
+    publishDir "${params.outdir}/${outpath}", pattern: "${filename}/*.bam", mode: 'copy'
 
     input:
         tuple val(sample), path(reads), val(library), val(read_length)
         path kallisto_index
         val outpath
 
     output:
-        tuple val(sample), path ("*.bam"), emit: tuple_sample_bam
-        path "*kallisto.log",  emit: kallisto_summary
+        tuple val(sample), path ("${filename}/*.bam"), emit: tuple_sample_bam
+        path "*.log",  emit: kallisto_summary
 
     script:
 
         // catch filename
-        filename = reads[0].baseName.replace('.fastq','')
+        filename = reads[0].baseName.replace('.fastq','') + "_kallisto_sorted"
 
         // deal with library type 
         def read_orientation=""
@@ -61,7 +61,11 @@ process kallisto {
                 -t ${task.cpus} \
                 --pseudobam \
                 -i ${kallisto_index} \
-                ${reads[0]} ${reads[1]} -o ${filename}.bam 2> ${filename}_kallisto.log
+                ${reads[0]} ${reads[1]} -o ${filename} 2> ${filename}.log
+            
+            mv ${filename}/pseudoalignments.bam ${filename}/${filename}.bam
+            # in order that the log file contains the name of the output fastq files (MultiQC)
+            sed -i 's/${reads[0]}/${filename}.fastq.gz/' ${filename}.log
             """
         } else {
 
@@ -83,8 +87,11 @@ process kallisto {
                 --pseudobam \
                 -i ${kallisto_index} \
                 --single \
-                ${reads} -o ${filename}.bam 2> ${filename}_kallisto.log
+                ${reads} -o ${filename} 2> ${filename}.log
+
+            mv ${filename}/pseudoalignments.bam ${filename}/${filename}.bam
+            # in order that the log file contains the name of the output fastq files (MultiQC)
+            sed -i 's/${reads[0]}/${filename}.fastq.gz/' ${filename}.log
             """
         }
-
 }

modules/star.nf

@@ -107,7 +107,7 @@ process star {
                 --readFilesIn pipedRead1 pipedRead2 \\
                 --runThreadN ${task.cpus} \\
                 --runMode alignReads \\
-                --outFileNamePrefix ${reads[0].baseName.replace('.fastq','')}  \\
+                --outFileNamePrefix ${reads[0].baseName.replace('.fastq','')}_${star_tool}_sorted \\
                 --outSAMunmapped Within \\
                 --outSAMtype BAM SortedByCoordinate
         """
@@ -121,7 +121,7 @@ process star {
                 --readFilesIn pipedRead  \\
                 --runThreadN ${task.cpus} \\
                 --runMode alignReads \\
-                --outFileNamePrefix ${reads.baseName.replace('.fastq','')}  \\
+                --outFileNamePrefix ${reads.baseName.replace('.fastq','')}_${star_tool}_sorted \\
                 --outSAMunmapped Within \\
                 --outSAMtype BAM SortedByCoordinate
         """
@@ -172,7 +172,7 @@ process star2pass{
                 --readFilesIn pipedRead1 pipedRead2  \\
                 --runThreadN ${task.cpus} \\
                 --runMode alignReads \\
-                --outFileNamePrefix ${reads.baseName.replace('.fastq','')}_2pass  \\
+                --outFileNamePrefix ${reads.baseName.replace('.fastq','')}_${star_tool}_2pass_sorted \\
                 --outSAMunmapped Within \\
                 --outSAMtype BAM SortedByCoordinate \\
                 --sjdbFileChrStartEnd *SJ.out.tab
@@ -195,7 +195,7 @@ process star2pass{
                 --readFilesIn pipedRead  \\
                 --runThreadN ${task.cpus} \\
                 --runMode alignReads \\
-                --outFileNamePrefix ${reads[0].baseName.replace('.fastq','')}_2pass  \\
+                --outFileNamePrefix ${reads[0].baseName.replace('.fastq','')}_${star_tool}_2pass_sorted \\
                 --outSAMunmapped Within \\
                 --outSAMtype BAM SortedByCoordinate \\
                 --sjdbFileChrStartEnd *SJ.out.tab

modules/subread.nf

@@ -53,7 +53,7 @@ process subread {
         }
 
         // remove fastq.gz
-        def fileName = fastq[0].baseName.replace('.fastq','')
+        def fileName = fastq[0].baseName.replace('.fastq','') + "_subread"
 
         // prepare index name
         def index_prefix = genome.baseName + "_index"
@@ -120,7 +120,7 @@ process sublong {
     script:
 
         // remove fastq.gz
-        def fileName = fastq[0].baseName.replace('.fastq','')
+        def fileName = fastq[0].baseName.replace('.fastq','') + "_sublong"
 
         // prepare index name
         def index_prefix = genome.baseName + "_index"