Merge pull request 'Pulling from system_genetics' (#1) from GRIAC/system_genetics:master into master

Reviewed-on: P300299/system_genetics#1

rnaseq/step1_fastqc/snippet.sh

@@ -1,5 +1,12 @@
-# The command to do a FastQC on a fastq file is
-file="file_to_analyse.fq.gz"
-fastqc_out="./path/to/fastqc/output/dir"
+#!/bin/bash
+R1="sample1_R1.fastq.gz"
+R2="sample1_R2.fastq.gz"
 
-fastqc -o "$fastqc_out" "$file"
+PROJECT_DIRECTORY="/groups/umcg-griac/tmp01/rawdata/$(whoami)/rnaseq"
+FASTQC_OUT="${PROJECT_DIRECTORY}/step1/"
+mkdir -p "${FASTQC_OUT}"
+
+# Run FastQC on paired-end data.
+fastqc \
+    -o "${FASTQC_OUT}" \
+    "${R1}" "${R2}"

rnaseq/step2_trim/snippet.sh

@@ -1,9 +1,12 @@
 #!/bin/bash
-
-# reference: http://www.usadellab.org/cms/?page=trimmomatic
+#
+# Reference: http://www.usadellab.org/cms/?page=trimmomatic
 
 
 module load Trimmomatic
+PROJECT_DIRECTORY="/groups/umcg-griac/tmp01/rawdata/$(whoami)/rnaseq"
+FASTQ_OUT="${PROJECT_DIRECTORY}/step2/"
+mkdir -p "${FASTQ_OUT}"
 
 
 # Adapters can be found at
@@ -11,15 +14,25 @@ module load Trimmomatic
 # But should be verified with FastQC, or in another way.
 
 
-# (Example) Paired end
+# Trimmomatic example Paired end data.
+#
+# Flags:
+# - ILLUMINACLIP: Cut adapter and other illumina-specific sequences from the
+#                 read.
+# - SLIDINGWINDOW: Perform a sliding window trimming, cutting once the average
+#                  quality within the window falls below a threshold.
+# - LEADING: Cut bases off the start of a read, if below a threshold quality.
+# - TRAILING: Cut bases off the end of a read, if below a threshold quality.
+# - HEADCROP: Cut the specified number of bases from the start of the read.
+# - MINLEN: Drop the read if it is below a specified length.
 java -jar $EBROOTTRIMMOMATIC/trimmomatic.jar PE \
   -phred33 \
-  input_forward.fq.gz \
-  input_reverse.fq.gz \
-  output_forward_paired.fq.gz \
-  output_forward_unpaired.fq.gz \
-  output_reverse_paired.fq.gz \
-  output_reverse_unpaired.fq.gz \
+  sample1_R1.fastq.gz \
+  sample1_R2.fastq.gz \
+  "${FASTQ_OUT}/sample1_R1_paired.fastq.gz" \
+  "${FASTQ_OUT}/sample1_R1_unpaired.fastq.gz" \
+  "${FASTQ_OUT}/sample1_R2_paired.fastq.gz" \
+  "${FASTQ_OUT}/sample1_R2_unpaired.fastq.gz" \
   ILLUMINACLIP: TruSeq3-PE.fa:2:30:10 \
   LEADING:3 \
   TRAILING:3 \
@@ -28,11 +41,11 @@ java -jar $EBROOTTRIMMOMATIC/trimmomatic.jar PE \
   MINLEN:50
 
 
-# (Example) Single end
+# Example single end data.
 java -jar $EBROOTTRIMMOMATIC/trimmomatic.jar SE \
   -phred33 \
-  input.fq.gz \
-  output.fq.gz \
+  sample1.fastq.gz \
+  output.fastq.gz \
   ILLUMINACLIP:TruSeq3-SE:2:30:10 \
   LEADING:3 \
   TRAILING:3 \

rnaseq/step3_align/snippet.sh

@@ -0,0 +1,54 @@
+#!/bin/bash
+#
+# Align reads against reference genome.
+
+PROJECT_DIRECTORY="/groups/umcg-griac/tmp01/rawdata/$(whoami)/rnaseq"
+# Store genome index in this location:.
+GENOME_INDEX="${PROJECT_DIRECTORY}/step3/genome_index"
+mkdir -p "${GENOME_INDEX}"
+# Store the generated `Aligned.sortedByCoord.out.bam` in this dir.
+ALIGNMENT_OUTPUT="${PROJECT_DIRECTORY}/step3/alignment/"
+mkdir -p "${ALIGNMENT_OUTPUT}"
+
+# 1) Generate genome index.
+#
+# N.B.:
+# - We're assuming a read size of 100 bp (--sjdbOverhang 100). An alternative
+#   cut-off is 150, for low-input methods. In general, refer back to the
+#   previous quality control steps if you are unsure about the size. In case of
+#   reads of varying length, the ideal value is max(ReadLength)-1.
+# - We're using gzip compressed reference data (--readFilesCommand zcat), i.e.,
+#   .gtf.gz and fa.gz. If not, you can remove the `zcat` flag.
+
+# Storage location reference data (in this case on Gearshift).
+REFERENCE_DATA="/groups/umcg-griac/prm03/rawdata/reference/genome"
+GTF_FILE="${REFERENCE_DATA}/Homo_sapiens.GRCh38.100.gtf.gz"
+FASTA_FILE="${REFERENCE_DATA}/Homo_sapiens.GRCh38.dna.primary_assembly.fa.gz"
+
+STAR \
+    --runThreadN 8 \
+    --runMode genomeGenerate \
+    --readFilesCommand zcat \
+    --sjdbOverhang 100 \
+    --genomeFastaFiles ${FASTA_FILE} \
+    --sjdbGTFfile ${GTF_FILE} \
+    --genomeDir ${GENOME_INDEX}
+
+
+# 2) Do the actual alignment.
+#
+# N.B.:
+# - We are assuming paired-end, gzip compressed (--readFilesCommand zcat)  FastQ
+#   files.
+
+# The compressed, paired-end, FastQ's after trimming (step 2).
+R1="${PROJECT_DIRECTORY}/step2/sample1_R1_paired.fastq.gz"
+R2="${PROJECT_DIRECTORY}/step2/sample1_R2_paired.fastq.gz"
+
+STAR \
+    --runThreadN 8 \
+    --readFilesCommand zcat \
+    --readFilesIn "${R1}" "${R2}" \
+    --outSAMtype BAM SortedByCoordinate \
+    --genomeDir ${GENOME_INDEX} \
+    --outFileNamePrefix "${ALIGNMENT_OUTPUT}"

rnaseq/step4_multiqc/snippet.sh

@@ -0,0 +1,5 @@
+#!/bin/bash
+module load multiqc
+
+PROJECT_DIRECTORY="/groups/umcg-griac/tmp01/rawdata/$(whoami)/rnaseq"
+multiqc "${PROJECT_DIRECTORY}"

rnaseq/step6_overall_QC/01 - Principle Component Analysis.R

@@ -18,7 +18,7 @@ do.scale = FALSE
 
 
 # The analysis
-# We ise prcomp to calculate the PCAs. Afterwards you should plot the results.
+# We use prcomp to calculate the PCAs. Afterwards you should plot the results.
 norm.expr.data <- expression.data %>%
 	tibble::column_to_rownames("Gene")
 norm.expr.data <- norm.expr.data[rowSums(norm.expr.data) >= 10,] %>%
@@ -37,8 +37,8 @@ norm.expr.data.pcs <- norm.expr.data %>%
   )
 
 # Write summary of PCAs to files
-pcs.summery <- summary(norm.expr.data.pcs)
-pcs.summery$importance %>%
+pcs.summary <- summary(norm.expr.data.pcs)
+pcs.summary$importance %>%
 	t() %>%
 	as.data.frame() %>%
 	tibble::rownames_to_column("PC.name") %>%
@@ -46,7 +46,7 @@ pcs.summery$importance %>%
 		file.path(results.dir.pca, "importance.csv")
 	)
 
-pcs.summery$x %>%
+pcs.summary$x %>%
 	t() %>%
 	as.data.frame() %>%
 	tibble::rownames_to_column("ensembl.id") %>%
@@ -54,7 +54,7 @@ pcs.summery$x %>%
 		file.path(results.dir.pca, "values.csv")
 	)
 
-pcs.summery$rotation %>%
+pcs.summary$rotation %>%
 	t() %>%
 	as.data.frame() %>%
 	tibble::rownames_to_column("sample.id") %>%
@@ -63,15 +63,15 @@ pcs.summery$rotation %>%
 	)
 
 data.frame(
-		rownames = names(pcs.summery$center),
-		center = pcs.summery$center,
-		scale = pcs.summery$scale
+		rownames = names(pcs.summary$center),
+		center = pcs.summary$center,
+		scale = pcs.summary$scale
 	) %>%
 	readr::write_csv(
 		file.path(results.dir.pca, "rest.csv")
 	)
 
-# Not saved: pcs.summery$sdev,
+# Not saved: pcs.summary$sdev,
 
 
 # Next thing to do:

rnaseq/umi/07_remove_dups.pl

@@ -0,0 +1,60 @@
+#!/usr/bin/perl -w
+use strict;
+use Parallel::ForkManager;
+# this script creats one file with UMI unique reads and one with UMI duplicated reads
+my @torun = ();
+foreach my $file ( <*Aligned.sortedByCoord.out.bam> ) {
+    push @torun, $file;
+}
+
+my $pm = Parallel::ForkManager->new( 12 );
+foreach my $file ( @torun ) {
+    my $sample = $file;
+    $sample =~ s/Aligned\.sortedByCoord\.out\.bam$//;
+    next if -s $sample.'_uniq.bam';
+    warn "Parsing $sample\n";
+    $pm->start and next;
+    my %seen = ();
+    my %duplicates = ();
+    my ( $uniqs, $dups ) = ( 0, 0 );
+    open F, 'samtools view -h '.$file.' |';
+    open F1, '| samtools view -bS - >'.$sample.'_uniq.bam';
+    open F2, '| samtools view -bS - >'.$sample.'_dups.bam';
+    while ( <F> ) {
+        if ( m/^\@/ ) {
+            print F1;
+            print F2;
+            next;
+        }
+        my ( $id, $flag, $chr, $pos, $mapq, $cigar, $chr2, $pos2, $tlen ) = split /\t/;
+        next if $flag & 256 or $flag & 512 or $flag & 1024; #skip if the read is not primary alignment/read fails platform/vendor quality checks/read is PCR or optical duplicate
+#        foreach ( 256, 512, 1024 ) { $flag-=$_ if $flag&$_ }
+        my ( $bc ) = $id =~ m/\:([GATCN\d]+)$/; #extract UMI barcode
+        my $uniq = join( ':', $chr, $pos, $flag, $tlen, $bc );
+        my $pos_ = $pos-1;
+        while ( $cigar =~ m/(\d+)([SHMDIN=])/g ) {
+            $pos_+=$1 if $2 eq 'M' or $2 eq '=' or $2 eq 'D' or $2 eq 'N'; 
+        }
+        my $uniq2 = join( ':', $chr, $pos_, $flag, $tlen, $bc );
+        if ( exists($duplicates{$id}) or # already marked as duplicate
+            ( not($flag&16) and ++$seen{ $uniq } > 1 ) or # plus strand
+            ( $flag&16 and ++$seen{ $uniq2 } > 1 )
+            ) {
+            print F2;
+            $dups++;
+            $duplicates{$id} = 1;
+        }
+        else {
+            print F1;
+            $uniqs++;
+        }
+    }
+    close F;
+    close F1;
+    close F2;
+    warn "Unique: $uniqs (", 100*$uniqs/($uniqs+$dups), ")\n";
+    system( 'samtools', 'index', $sample.'_uniq.bam' );
+#    last;
+    $pm->finish;
+}
+$pm->wait_all_children;