Added description and some comments for UMi deduplication

rnaseq/step3_align/snippet.sh

@@ -1,53 +0,0 @@
-#!/bin/bash
-#
-# Align reads against reference genome.
-
-STORAGE="/groups/umcg-griac/tmp04/rawdata/$(whoami)/step3"
-# Store genome index in this location:.
-GENOME_INDEX="${STORAGE}/genome_index"
-mkdir -p "${GENOME_INDEX}"
-# Store the generated `Aligned.sortedByCoord.out.bam` in this dir.
-ALIGNMENT_OUTPUT="${STORAGE}/alignment"
-mkdir -p "${OUTPUT}"
-
-# 1) Generate genome index.
-#
-# N.B.:
-# - We're assuming a read size of 100 bp (--sjdbOverhang 100). Refer back to the
-#   previous quality control steps if you are unsure about the size. In case of
-#   reads of varying length, the ideal value is max(ReadLength)-1.
-# - We're using gzip compressed reference data (--readFilesCommand zcat), i.e.,
-#   .gtf.gz and fa.gz. If not, you can remove the `zcat` flag.
-
-# Storage location reference data (in this case on calculon).
-REFERENCE_DATA="/groups/umcg-griac/prm02/rawdata/reference/genome"
-GTF_FILE="${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 ${GENOME_INDEX}
-
-
-# 2) Do the actual alignment.
-#
-# N.B.:
-# - We are assuming paired-end, gzip compressed (--readFilesCommand zcat)  FastQ
-#   files.
-
-# THe compressed paired-end FastQ's that we are aligning.
-R1="sample1_R1.fastq.gz"
-R2="sample1_R2.fastq.gz"
-
-STAR \
-    --runThreadN 8 \
-    --readFilesCommand zcat \
-    --readFilesIn "${R1}" "${R2}" \
-    --outSAMtype BAM SortedByCoordinate \
-    --outFileNamePrefix "${ALIGNMENT_OUTPUT}"
-

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;