diff summarize_unique_barcodes.sh @ 24:431aebd93843 draft default tip

Fixed a bug in k2n.R where the function k2n_calc() would result in an error for single-end read files.

--- /dev/null	Thu Jan 01 00:00:00 1970 +0000
+++ b/summarize_unique_barcodes.sh	Wed Aug 05 09:21:02 2015 -0400
@@ -0,0 +1,231 @@
+#!/bin/bash
+
+####################################################################################################
+#Copyright (C) 2014 Lukasz Kielpinski, Nikos Sidiropoulos
+
+#This program is free software: you can redistribute it and/or modify it under the terms of the
+#GNU General Public License as published by the Free Software Foundation, either version 3 of the
+#License, or (at your option) any later version.
+
+#This program is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without
+#even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the
+#GNU General Public License for more details (http://www.gnu.org/licenses/).
+####################################################################################################
+
+function print_help {
+cat <<End-of-message
+Summarize Unique Barcodes.
+Counts the number of unique random barcodes and reads associated with each sequenced fragment.
+-------------------------------------
+Input arguments:
+-h: Help
+-f: Aligned reads in BAM format.
+-b: Barcode file (optional).
+-p: Set priming position to a fixed value.
+-t: Trim untemplated nucleotides.
+-k: Produce k2n file. Warning: Can be sloooow!
+-r: Rscript path
+-o: Output folder (default: "output_dir")
+-------------------------------------
+Usage : summarize_unique_barcodes.sh -f <BAM_file> -b <BARCODES> -p <PRIMING_POSITION> -t -k -r <R_SCRIPT_PATH>
+End-of-message
+exit
+}
+
+#defaults
+output_dir="output_dir"
+trim_flag="False"
+
+#parse input
+while getopts hf:b::p:o:ktr: myarg
+do  case "$myarg" in
+    h)  print_help
+        exit ;;
+    f)  bamfile="$OPTARG" ;; #required
+    b)  barcodes="$OPTARG" ;; #optional
+    t)  trim_flag="True" ;;
+    k)  k2n="True" ;;
+    p)  priming_pos="$OPTARG" ;;
+    o)  output_dir="$OPTARG" ;;
+    r)  R_SCRIPT_PATH="$OPTARG" ;; #required
+    [?])  echo "ERROR: Unknown parameter"
+        print_help
+        exit 1 ;;
+    esac
+done
+
+###### Sanity checks ######
+if [ -z $bamfile ]; then
+    echo "Error: Aligned reads file is missing!"
+    print_help
+    exit 1
+fi
+
+if [ "$barcodes" == "None" ] && [ "$k2n" == "True" ]; then
+    echo "Error: k2n file cannot be produced without a barcode file!"
+    exit 1
+fi
+
+
+mkdir -p $output_dir
+
+#Check if bamfile contains single or paired-end reads
+samtools view -f 0x1 $bamfile | head -n 1 > paired
+
+if [ -s paired ]; then
+    #paired-end
+    samtools view $bamfile | awk 'BEGIN{OFS="\t"}{if(substr($0,1,1)!="@"){print}}' - | awk -v out="${output_dir}/trimming_stats.txt" -v flag="${trim_flag}" 'BEGIN{OFS="\t";counter[0]=0;counter[1]=0;counter[2]=0;counter[3]=0;counter[4]=0}
+    function abs(value){return(value<0?-value:value)}
+    function return_offset(local_offset){print($1, $3, $4+local_offset, $4+abs($9)-1);counter[local_offset]++}
+    ($2 != 99) {next};
+    (flag == "False") {return_offset(0);next};
+    (/[\s\t]MD:Z:/ && !/MD:Z:([012][ACGT])/)  {return_offset(0);next};
+    (/[\s\t]MD:Z:0[ACGT]/ && !/MD:Z:0[ACGT][01][ACGT]/ && substr($10,1,1)=="N") {return_offset(0);next};
+    (/[\s\t]MD:Z:0[ACGT]/ && !/MD:Z:0[ACGT][01][ACGT]/) {return_offset(1);next};
+
+    (/[\s\t]MD:Z:1[ACGT]/ && !/MD:Z:1[ACGT]0[ACGT]/ && substr($10,2,1)=="N") {return_offset(0);next};
+    (/[\s\t]MD:Z:1[ACGT]/ && !/MD:Z:1[ACGT]0[ACGT]/) {return_offset(2);next};
+
+    (/MD:Z:0[ACGT]0[ACGT]/ && !/MD:Z:0[ACGT]0[ACGT]0[ACGT]/ && substr($10,1,2)=="NN") {return_offset(0);next};
+    (/MD:Z:0[ACGT]0[ACGT]/ && !/MD:Z:0[ACGT]0[ACGT]0[ACGT]/ && substr($10,1,1)=="N") {return_offset(2);next};
+    (/MD:Z:0[ACGT]0[ACGT]/ && !/MD:Z:0[ACGT]0[ACGT]0[ACGT]/ && substr($10,2,1)=="N") {return_offset(1);next};
+    (/MD:Z:0[ACGT]0[ACGT]/ && !/MD:Z:0[ACGT]0[ACGT]0[ACGT]/) {return_offset(2);next};
+
+    (/[\s\t]MD:Z:1[ACGT]0[ACGT]/ && substr($10,2,2)=="NN") {return_offset(0);next};
+    (/[\s\t]MD:Z:1[ACGT]0[ACGT]/ && substr($10,2,1)=="N") {return_offset(3);next};
+    (/[\s\t]MD:Z:1[ACGT]0[ACGT]/ && substr($10,3,1)=="N") {return_offset(2);next};
+    (/[\s\t]MD:Z:1[ACGT]0[ACGT]/) {return_offset(3);next};
+
+    (/MD:Z:0[ACGT]1[ACGT]/ && substr($10,3,1)=="N" && substr($10,1,1)=="N") {return_offset(0);next};
+    (/MD:Z:0[ACGT]1[ACGT]/ && substr($10,3,1)=="N") {return_offset(1);next};
+    (/MD:Z:0[ACGT]1[ACGT]/ && substr($10,1,1)=="N") {return_offset(3);next};
+    (/MD:Z:0[ACGT]1[ACGT]/) {return_offset(3);next};
+
+    (/MD:Z:2[ACGT]/ && substr($10,3,1)=="N")  {return_offset(0);next};
+    (/MD:Z:2[ACGT]/)  {return_offset(3);next};
+
+    (substr($10,3,1)!="N" && /MD:Z:0[ACGT]0[ACGT]0[ACGT]/) {return_offset(3);next};
+    (substr($10,2,1)!="N" && /MD:Z:0[ACGT]0[ACGT]0[ACGT]/) {return_offset(2);next};
+    (substr($10,1,1)!="N" && /MD:Z:0[ACGT]0[ACGT]0[ACGT]/) {return_offset(1);next};
+
+    {return_offset(0);counter[4]++}
+    END{print("No trimming:",counter[0],", out of which not recognized MD field for:",counter[4],"; 1 nt trimmed:", counter[1],"; 2 nt trimmed:", counter[2],"; 3 nt trimmed:",counter[3]) > out}' | sort -S1G -k1,1 | gzip > positions_temp_sorted.gz
+
+else
+    #single-end
+    samtools view $bamfile | awk 'BEGIN{OFS="\t"}{if(substr($0,1,1)!="@"){print}}' - | awk -v out="${output_dir}/trimming_stats.txt" -v flag="${trim_flag}" 'BEGIN{OFS="\t";counter[0]=0;counter[1]=0;counter[2]=0;counter[3]=0;counter[4]=0}
+    function abs(value){return(value<0?-value:value)}
+    function return_offset(local_offset){print($1, $3, $4+local_offset, $4+abs($9)-1);counter[local_offset]++}
+    ($2 != 0) {next};
+    (flag == "False") {return_offset(0);next};
+    (/[\s\t]MD:Z:/ && !/MD:Z:([012][ACGT])/)  {return_offset(0);next};
+    (/[\s\t]MD:Z:0[ACGT]/ && !/MD:Z:0[ACGT][01][ACGT]/ && substr($10,1,1)=="N") {return_offset(0);next};
+    (/[\s\t]MD:Z:0[ACGT]/ && !/MD:Z:0[ACGT][01][ACGT]/) {return_offset(1);next};
+
+    (/[\s\t]MD:Z:1[ACGT]/ && !/MD:Z:1[ACGT]0[ACGT]/ && substr($10,2,1)=="N") {return_offset(0);next};
+    (/[\s\t]MD:Z:1[ACGT]/ && !/MD:Z:1[ACGT]0[ACGT]/) {return_offset(2);next};
+
+    (/MD:Z:0[ACGT]0[ACGT]/ && !/MD:Z:0[ACGT]0[ACGT]0[ACGT]/ && substr($10,1,2)=="NN") {return_offset(0);next};
+    (/MD:Z:0[ACGT]0[ACGT]/ && !/MD:Z:0[ACGT]0[ACGT]0[ACGT]/ && substr($10,1,1)=="N") {return_offset(2);next};
+    (/MD:Z:0[ACGT]0[ACGT]/ && !/MD:Z:0[ACGT]0[ACGT]0[ACGT]/ && substr($10,2,1)=="N") {return_offset(1);next};
+    (/MD:Z:0[ACGT]0[ACGT]/ && !/MD:Z:0[ACGT]0[ACGT]0[ACGT]/) {return_offset(2);next};
+
+    (/[\s\t]MD:Z:1[ACGT]0[ACGT]/ && substr($10,2,2)=="NN") {return_offset(0);next};
+    (/[\s\t]MD:Z:1[ACGT]0[ACGT]/ && substr($10,2,1)=="N") {return_offset(3);next};
+    (/[\s\t]MD:Z:1[ACGT]0[ACGT]/ && substr($10,3,1)=="N") {return_offset(2);next};
+    (/[\s\t]MD:Z:1[ACGT]0[ACGT]/) {return_offset(3);next};
+
+    (/MD:Z:0[ACGT]1[ACGT]/ && substr($10,3,1)=="N" && substr($10,1,1)=="N") {return_offset(0);next};
+    (/MD:Z:0[ACGT]1[ACGT]/ && substr($10,3,1)=="N") {return_offset(1);next};
+    (/MD:Z:0[ACGT]1[ACGT]/ && substr($10,1,1)=="N") {return_offset(3);next};
+    (/MD:Z:0[ACGT]1[ACGT]/) {return_offset(3);next};
+
+    (/MD:Z:2[ACGT]/ && substr($10,3,1)=="N")  {return_offset(0);next};
+    (/MD:Z:2[ACGT]/)  {return_offset(3);next};
+
+    (substr($10,3,1)!="N" && /MD:Z:0[ACGT]0[ACGT]0[ACGT]/) {return_offset(3);next};
+    (substr($10,2,1)!="N" && /MD:Z:0[ACGT]0[ACGT]0[ACGT]/) {return_offset(2);next};
+    (substr($10,1,1)!="N" && /MD:Z:0[ACGT]0[ACGT]0[ACGT]/) {return_offset(1);next};
+
+    {return_offset(0);counter[4]++}
+    END{print("No trimming:",counter[0],", out of which not recognized MD field for:",counter[4],"; 1 nt trimmed:", counter[1],"; 2 nt trimmed:", counter[2],"; 3 nt trimmed:",counter[3]) > out}' | sort -S1G -k1,1 | gzip > positions_temp_sorted.gz
+
+fi
+
+#Computing barcode length (Use the first line and compute the string length of the second column)
+if [ "$barcodes" != "None" ]; then
+
+    TMP=$(for line in `cut -f 2 $barcodes`; do if [ ! -z "$line" ]; then echo $line; break; fi; done)
+    BAR_LEN=`echo ${#TMP}`
+
+    #Remove "@" from barcodes and sort them
+    sed 's/^.//' $barcodes | sort -k1,1 -S1G | gzip > barcodes_temp_sorted.gz
+
+    #Merge poistions and barcodes
+    join -1 1 <(zcat positions_temp_sorted.gz) <(zcat barcodes_temp_sorted.gz) | cut -f 2,3,4,5 -d " " | awk '{if($4 !~ /N/){print}}' | awk -v bar_len="${BAR_LEN}" '{if(length($4)==bar_len){print}}' | gzip > merged_temp.gz
+
+    rm barcodes_temp_sorted.gz
+
+else
+    zcat positions_temp_sorted.gz | cut -f 2,3,4 | gzip > merged_temp.gz
+fi
+
+#If the experiment is single-end set NA values to the priming column.
+if [ ! -s paired ]; then
+    zcat merged_temp.gz | awk '{print $1, $2, "NA", $4, $5}' - > merged_temp2
+    gzip -c merged_temp2 > merged_temp.gz
+    rm merged_temp2
+fi
+
+#Fix priming position
+if [ ! -z $priming_pos ]; then
+    zcat merged_temp.gz | awk -v pos="${priming_pos}" '{print $1, $2, pos, $4, $5}' - > merged_temp2
+    gzip -c merged_temp2 > merged_temp.gz
+    rm merged_temp2
+fi
+
+#File summary.txt columns: RNA_ID, Start, End, barcode sequence, sequenced_count[=number of sequenced fragments fulfilling previous requiremnts]
+
+zcat merged_temp.gz | awk '{barcode[$1][$2][$3][$4]++}END{
+for(RNA in barcode){
+for(start_position in barcode[RNA]){
+for(end_position in barcode[RNA][start_position]){
+for(barseq in barcode[RNA][start_position][end_position]){print RNA,start_position,end_position,barseq,barcode[RNA][start_position][end_position][barseq]}}}}}' > $output_dir/summary.txt
+
+#File unique_barcodes.txt columns: RNA_ID, Start, End, number of unique barcodes observed for this fragment.
+
+awk '{barcode[$1][$2][$3]++}END{
+for(RNA in barcode){
+for(start_position in barcode[RNA]){
+for(end_position in barcode[RNA][start_position]){print RNA "\t" start_position "\t" end_position "\t" barcode[RNA][start_position][end_position]}}}}' $output_dir/summary.txt > $output_dir/unique_barcodes.txt &
+
+#File read_counts.txt colums: RNA_ID, Start, End, sequenced_count
+
+zcat merged_temp.gz | awk '{barcode[$1][$2][$3]++}END{
+for(RNA in barcode){
+for(start_position in barcode[RNA]){
+for(end_position in barcode[RNA][start_position]){print RNA "\t" start_position "\t" end_position "\t" barcode[RNA][start_position][end_position]}}}}' > $output_dir/read_counts.txt &
+
+wait
+
+#Print the maximum observed barcodes value. Usefull to assess the necessity of producing the k2n file.
+if [ "$barcodes" != "None" ]; then
+
+    cut -f 4 $output_dir/unique_barcodes.txt | sort -S1G -rn > sorted_bars
+    max_observed_barcodes=`head -n 1 sorted_bars`
+
+    echo "Maximum observed Barcodes = ${max_observed_barcodes}"
+    rm sorted_bars
+fi
+
+#Produce k2n file
+if [ "$k2n" == "True" ]; then
+    if [ ! -s paired ]; then
+        Rscript $R_SCRIPT_PATH/k2n.R merged_temp.gz $output_dir/unique_barcodes.txt $output_dir/k2n.txt FALSE
+    else
+	Rscript $R_SCRIPT_PATH/k2n.R merged_temp.gz $output_dir/unique_barcodes.txt $output_dir/k2n.txt TRUE
+    fi
+fi
+
+#Remove temp files
+rm merged_temp.gz positions_temp_sorted.gz paired