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prophage_tracer-WGS.sh
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prophage_tracer-WGS.sh
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#!/bin/bash -e
set -eo pipefail
##########################################################
# Prophage Tracer V1.0.0
#########################################################
## usage
usage() {
echo "
Prophage Tracer V1.0.0 07/05/2021
usage: bash prophage_tracer-WGS.sh [options] -m <in.sam> -r <in.fasta> -p <prefix>
requirement：locally installed BLAST+ software
options:
-m FILE a full SAM file (required)
-r FILE a reference genome sequence (required)
-p STRING prefix of output files (required; usually a strain name or a sample name)
-x INT maximal size of prophage (default: 150000)
-n INT minimal size of prophage (default: 5000)
-a INT minimal length of attchment site (default: 10)
-t INT number of threads used for BlastN (default: 1)
-s INT minimal event of split reads required for supporting a prophage candidate (default: 1)
-d INT minimal event of discordant read pairs required for supporting a prophage candidate (default: 1)
"
}
while getopts ":hm:r:p:x:n:a:t:s:d:" opt
do
case $opt in
h) usage
exit 0
;;
m) sam_file=$OPTARG ;;
r) fasta=$OPTARG ;;
p) prefix=$OPTARG ;;
x) prophage_size_max=$OPTARG ;;
n) prophage_size_min=$OPTARG ;;
a) att_length_min=$OPTARG ;;
t) threads=$OPTARG ;;
s) SR_COUNT=$OPTARG ;;
d) DRP_COUNT=$OPTARG ;;
:)
echo "Option -$OPTARG requires an argument."
usage
exit 1
;;
?)
echo "Invalid option: -$OPTARG"
usage
exit 1
;;
esac
done
#If options of 'x', 'n', 'a' and 't' are not defined, setting to default values
prophage_size_max=${prophage_size_max:-150000}
prophage_size_min=${prophage_size_min:-5000}
att_length_min=${att_length_min:-3}
threads=${threads:-1}
SR_COUNT=${SR_COUNT:-1}
DRP_COUNT=${DRP_COUNT:-1}
#Check locally installed BLAST+ software
BLASTN=`which blastn || true`
if [[ -z "$BLASTN" ]]
then
usage
echo "blastn not found"
exit 1
fi
###########统计contig的长度
cat $fasta | awk '$0 ~ ">" {print c; c=0;split($0,a,"[> ]"); printf a[2] "\t"; } $0 !~ ">" {c+=length($0);} END { print c; }' > contiglength.file
#Get read length and insert size
read_length=`head -n 10000 $sam_file | gawk 'BEGIN { max=0 } { if (length($10)>max) max=length($10) } END { print max }'`
insert_size=`head -n 10000 $sam_file | awk '($2 ~ /163|83|99|147/ )' | cut -f9 | awk '{print sqrt($0^2)}' | awk '$0<10000'| awk '{ sum += $0;i++ } END { print int(sum/i) }'`
#Step 1: Extracting split reads
echo -e "Step 1: Extracting split reads"
awk '($1 ~ /^@/)||($2 ~ /145|81|99|163/ && $6 ~ /^...?S...?M$/) ||($2 ~ /113|177/ && $6 ~ /^...?S...?M$/ && $9 == 0 ) || ($2 ~ /97|161|147|83/ && $6 ~ /^...?M...?S$/ ) || ($2 ~ /65|129/ && $6 ~ /^...?M...?S$/ && $9 == 0 )' $sam_file > $prefix.sr.temp.1
#Extracting split reads.If paried reads are overlapping, only one kept.
awk '!a[$2"_"$3"_"$4"_"$5"_"$6"_"$7"_"$8]++' $prefix.sr.temp.1 | awk '($1 !~ /^@/)'| awk '!a[$1]++'| awk '{printf ">"$1"_"$2"\n"$10"\n"}' > $prefix.reads.fasta
#Blastn searching reads against the reference genome.
makeblastdb -in $fasta -dbtype nucl -out $prefix.nuclDB > makeblastdb.log 2>&1
blastn -query $prefix.reads.fasta -db $prefix.nuclDB -out $prefix.blastn.result -evalue 1e-3 -outfmt 6 -word_size 11 -num_threads $threads > blastn.log 2>&1
#Manipulate blastn result ######################################################################################下面筛选到2条后再加一个按照reads起始位点排序的命令
awk 'BEGIN{FS="\t";OFS="\t"} {if ($3 > 90) a[$1,++b[$1]]=$0}END{for(i in b) if (b[i] == 2) print a[i,1]"\n"a[i,2]}' $prefix.blastn.result| awk '{if($0!="") print}' |sort -k1,1 -k7n,7 > $prefix.sr.temp.2
#Classify reads containing attB or attP
awk '
BEGIN {FS="\t";OFS="\t"}
function calculation1() {
attL_start=send1-qend1+qstart2
attL_end=send1
attR_start=sstart2
attR_end=sstart2+qend1-qstart2
prophage_size=attR_end-attL_end
print SR_type,SR_DRP_type,evidence,attL_start,attL_end,attR_start,attR_end,prophage_size,$0
}
function calculation2() {
attL_start=send2-qend2+qstart1
attL_end=send2
attR_start=sstart1
attR_end=sstart1+qend2-qstart1
prophage_size=attR_end-attL_end
print SR_type,SR_DRP_type,evidence,attL_start,attL_end,attR_start,attR_end,prophage_size,$0
}
function calculation3() {
attL_start=sstart2
attL_end=sstart2+qend1-qstart2
attR_start=send1-qend1+qstart2
attR_end=send1
prophage_size=attR_end-attL_end
print SR_type,SR_DRP_type,evidence,attL_start,attL_end,attR_start,attR_end,prophage_size,$0
}
function calculation4() {
attL_start=sstart1
attL_end=sstart1+qend2-qstart1
attR_start=send2-qend2+qstart1
attR_end=send2
prophage_size=attR_end-attL_end
print SR_type,SR_DRP_type,evidence,attL_start,attL_end,attR_start,attR_end,prophage_size,$0
}
{
if (NR==1)
{
query = $1; contig=$2; qstart1 = $7; qend1 = $8; sstart1 = $9; send1 = $10; next
}
##make sure to extract correct flag value if "_" was used in read names
Flag_pos=split ($1,flag,"_")
if ($1 == query && $2 == contig)
{ qstart2 = $7; qend2 = $8; sstart2 = $9; send2 = $10
if (flag[Flag_pos] == 83)
{
#attB
if ( (qend2 > qend1 && qend1 > qstart2 && qstart2 > qstart1 && qstart1 < 10) && (send2 > sstart2 && sstart2 > send1 && send1 > sstart1) )
{
SR_type="SR"
SR_DRP_type="SR_PR_1"
evidence="attB"
calculation1()
}
else if ( (qend1 > qend2 && qend2 > qstart1 && qstart1 > qstart2 && qstart2 < 10) && (send1 > sstart1 && sstart1 > send2 && send2 > sstart2) )
{
SR_type="SR"
SR_DRP_type="SR_PR_1"
evidence="attB"
calculation2()
}
#attP
if ( (qend2 > qend1 && qend1 > qstart2 && qstart2 > qstart1 && qstart1 < 10) && (send1 > sstart1 && sstart1 > send2 && send2 > sstart2) )
{
SR_type="SR"
SR_DRP_type="SR_PR_5"
evidence="attP"
calculation3()
}
else if ( (qend1 > qend2 && qend2 > qstart1 && qstart1 > qstart2 && qstart2 < 10) && (send2 > sstart2 && sstart2 > send1 && send1 > sstart1) )
{
SR_type="SR"
SR_DRP_type="SR_PR_5"
evidence="attP"
calculation4()
}
}
if (flag[Flag_pos] == 147)
{
#attB
if ( (qend2 > qend1 && qend1 > qstart2 && qstart2 > qstart1 && qstart1 < 10) && (send2 > sstart2 && sstart2 > send1 && send1 > sstart1) )
{
SR_type="SR"
SR_DRP_type="SR_PR_2"
evidence="attB"
calculation1()
}
else if ( (qend1 > qend2 && qend2 > qstart1 && qstart1 > qstart2 && qstart2 < 10) && (send1 > sstart1 && sstart1 > send2 && send2 > sstart2) )
{
SR_type="SR"
SR_DRP_type="SR_PR_2"
evidence="attB"
calculation2()
}
#attP
if ( (qend2 > qend1 && qend1 > qstart2 && qstart2 > qstart1 && qstart1 < 10) && (send1 > sstart1 && sstart1 > send2 && send2 > sstart2) )
{
SR_type="SR"
SR_DRP_type="SR_PR_6"
evidence="attP"
calculation3()
}
else if ( (qend1 > qend2 && qend2 > qstart1 && qstart1 > qstart2 && qstart2 < 10) && (send2 > sstart2 && sstart2 > send1 && send1 > sstart1) )
{
SR_type="SR"
SR_DRP_type="SR_PR_6"
evidence="attP"
calculation4()
}
}
if (flag[Flag_pos] == 97)
{
#attB
if ( (qend2 > qend1 && qend1 > qstart2 && qstart2 > qstart1 && qstart1 < 10) && (send2 > sstart2 && sstart2 > send1 && send1 > sstart1) )
{
SR_type="SR"
SR_DRP_type="SR_DRP_1"
evidence="attB"
calculation1()
}
else if ( (qend1 > qend2 && qend2 > qstart1 && qstart1 > qstart2 && qstart2< 10) && (send1 > sstart1 && sstart1 > send2 && send2 > sstart2) )
{
SR_type="SR"
SR_DRP_type="SR_DRP_1"
evidence="attB"
calculation2()
}
#attP
if ( (qend2 > qend1 && qend1 > qstart2 && qstart2 > qstart1 && qstart1 < 10) && (send1 > sstart1 && sstart1 > send2 && send2 > sstart2) )
{
SR_type="SR"
SR_DRP_type="SR_DRP_5"
evidence="attP"
calculation3()
}
else if ( (qend1 > qend2 && qend2 > qstart1 && qstart1 > qstart2 && qstart2 < 10) && (send2 > sstart2 && sstart2 > send1 && send1 > sstart1) )
{
SR_type="SR"
SR_DRP_type="SR_DRP_5"
evidence="attP"
calculation4()
}
}
if (flag[Flag_pos] == 161)
{
#attB
if ( (qend2 > qend1 && qend1 > qstart2 && qstart2 > qstart1 && qstart1 < 10) && (send2 > sstart2 && sstart2 > send1 && send1 > sstart1) )
{
SR_type="SR"
SR_DRP_type="SR_DRP_2"
evidence="attB"
calculation1()
}
else if ( (qend1 > qend2 && qend2 > qstart1 && qstart1 > qstart2 && qstart2 < 10) && (send1 > sstart1 && sstart1 > send2 && send2 > sstart2) )
{
SR_type="SR"
SR_DRP_type="SR_DRP_2"
evidence="attB"
calculation2()
}
#attP
if ( (qend2 > qend1 && qend1 > qstart2 && qstart2 > qstart1 && qstart1 < 10) && (send1 > sstart1 && sstart1 > send2 && send2 > sstart2) )
{
SR_type="SR"
SR_DRP_type="SR_DRP_6"
evidence="attP"
calculation3()
}
else if ( (qend1 > qend2 && qend2 > qstart1 && qstart1 > qstart2 && qstart2 < 10) && (send2 > sstart2 && sstart2 > send1 && send1 > sstart1) )
{
SR_type="SR"
SR_DRP_type="SR_DRP_6"
evidence="attP"
calculation4()
}
}
####Second
if (flag[Flag_pos] == 99)
{
#attB
if ( (qend2 > qend1 && qend1 > qstart2 && qstart2 > qstart1 && qstart1 < 10) && (send2 > sstart2 && sstart2 > send1 && send1 > sstart1) )
{
SR_type="SR"
SR_DRP_type="SR_PR_7"
evidence="attB"
calculation1()
}
else if ( (qend1 > qend2 && qend2 > qstart1 && qstart1 > qstart2 && qstart2 < 10) && (send1 > sstart1 && sstart1 > send2 && send2 > sstart2) )
{
SR_type="SR"
SR_DRP_type="SR_PR_7"
evidence="attB"
calculation2()
}
#attP
if ( (qend2 > qend1 && qend1 > qstart2 && qstart2 > qstart1 && qstart1 < 10) && (send1 > sstart1 && sstart1 > send2 && send2 > sstart2) )
{
SR_type="SR"
SR_DRP_type="SR_PR_3"
evidence="attP"
calculation3()
}
else if ( (qend1 > qend2 && qend2 > qstart1 && qstart1 > qstart2 && qstart2 < 10) && (send2 > sstart2 && sstart2 > send1 && send1 > sstart1) )
{
SR_type="SR"
SR_DRP_type="SR_PR_3"
evidence="attP"
calculation4()
}
}
if (flag[Flag_pos] == 163)
{
#attB
if ( (qend2 > qend1 && qend1 > qstart2 && qstart2 > qstart1 && qstart1 < 10) && (send2 > sstart2 && sstart2 > send1 && send1 > sstart1) )
{
SR_type="SR"
SR_DRP_type="SR_PR_8"
evidence="attB"
calculation1()
}
else if ( (qend1 > qend2 && qend2 > qstart1 && qstart1 > qstart2 && qstart2 < 10) && (send1 > sstart1 && sstart1 > send2 && send2 > sstart2) )
{
SR_type="SR"
SR_DRP_type="SR_PR_8"
evidence="attB"
calculation2()
}
#attP
if ( (qend2 > qend1 && qend1 > qstart2 && qstart2 > qstart1 && qstart1 < 10) && (send1 > sstart1 && sstart1 > send2 && send2 > sstart2) )
{
SR_type="SR"
SR_DRP_type="SR_PR_4"
evidence="attP"
calculation3()
}
else if ( (qend1 > qend2 && qend2 > qstart1 && qstart1 > qstart2 && qstart2 < 10) && (send2 > sstart2 && sstart2 > send1 && send1 > sstart1) )
{
SR_type="SR"
SR_DRP_type="SR_PR_4"
evidence="attP"
calculation4()
}
}
if (flag[Flag_pos] == 81)
{
#attB
if ( (qend2 > qend1 && qend1 > qstart2 && qstart2 > qstart1 && qstart1 < 10) && (send2 > sstart2 && sstart2 > send1 && send1 > sstart1) )
{
SR_type="SR"
SR_DRP_type="SR_DRP_7"
evidence="attB"
calculation1()
}
else if ( (qend1 > qend2 && qend2 > qstart1 && qstart1 > qstart2 && qstart2 < 10) && (send1 > sstart1 && sstart1 > send2 && send2 > sstart2) )
{
SR_type="SR"
SR_DRP_type="SR_DRP_7"
evidence="attB"
calculation2()
}
#attP
if ( (qend2 > qend1 && qend1 > qstart2 && qstart2 > qstart1 && qstart1 < 10) && (send1 > sstart1 && sstart1 > send2 && send2 > sstart2) )
{
SR_type="SR"
SR_DRP_type="SR_DRP_3"
evidence="attP"
calculation3()
}
else if ( (qend1 > qend2 && qend2 > qstart1 && qstart1 > qstart2 && qstart2 < 10) && (send2 > sstart2 && sstart2 > send1 && send1 > sstart1) )
{
SR_type="SR"
SR_DRP_type="SR_DRP_3"
evidence="attP"
calculation4()
}
}
if (flag[Flag_pos] == 145)
{
#attB
if ( (qend2 > qend1 && qend1 > qstart2 && qstart2 > qstart1 && qstart1 < 10) && (send2 > sstart2 && sstart2 > send1 && send1 > sstart1) )
{
SR_type="SR"
SR_DRP_type="SR_DRP_8"
evidence="attB"
calculation1()
}
else if ( (qend1 > qend2 && qend2 > qstart1 && qstart1 > qstart2 && qstart2 < 10) && (send1 > sstart1 && sstart1 > send2 && send2 > sstart2) )
{
SR_type="SR"
SR_DRP_type="SR_DRP_8"
evidence="attB"
calculation2()
}
#attP
if ( (qend2 > qend1 && qend1 > qstart2 && qstart2 > qstart1 && qstart1 < 10) && (send1 > sstart1 && sstart1 > send2 && send2 > sstart2) )
{
SR_type="SR"
SR_DRP_type="SR_DRP_4"
evidence="attP"
calculation3()
}
else if ( (qend1 > qend2 && qend2 > qstart1 && qstart1 > qstart2 && qstart2 < 10) && (send2 > sstart2 && sstart2 > send1 && send1 > sstart1) )
{
SR_type="SR"
SR_DRP_type="SR_DRP_4"
evidence="attP"
calculation4()
}
}
}
else if ($1 != query)
{
query = $1; contig = $2; qstart1 = $7; qend1 = $8; sstart1 = $9; send1 = $10
}
}' $prefix.sr.temp.2 | awk '
#Cluster split reads
BEGIN{FS="\t";OFS="\t"}
{
if( $8 > '"$prophage_size_min"' && $8 < '"$prophage_size_max"' ) a[$9]=$3"\t"$4"\t"$5"\t"$6"\t"$7"\t"$8"\t"$10
}
END{
count=1
for (i in a)
{
split(a[i],b,"\t")
print i,b[1],b[2],b[3],b[4],b[5],b[6],b[7],"candidate_"count
for (j in a)
{
split(a[j],c,"\t")
if ( i != j && c[7] == b[7])
{
if (b[7]==c[7] && b[2]-c[2] > -5 && b[2]-c[2] < 5 && b[4]-c[4] > -5 && b[4]-c[4] < 5 && b[6]-c[6] > -10 && b[6]-c[6] < 10)
{
print j,c[1],c[2],c[3],c[4],c[5],c[6],c[7],"candidate_"count
delete a[j]
}
}
}
count++
delete a[i]
}
}' |awk '{if (!($3 == "")) print $0}' >$prefix.sr.temp.3
awk '
BEGIN{FS="\t";OFS="\t"}
{
if ( !(a[$NF,1] >0) )
{
a[$NF,1]=0
}
if ( !(a[$NF,2] >0) )
{
a[$NF,2]=0
}
if ($2 == "attB")
{
a[$NF,1]+=1
sum[$NF,3]+=$3
sum[$NF,4]+=$4
sum[$NF,5]+=$5
sum[$NF,6]+=$6
sum[$NF,7]+=$7
a[$NF,8]=$8
}
else if ($2 == "attP")
{
a[$NF,2]+=1
sum[$NF,3]+=$3
sum[$NF,4]+=$4
sum[$NF,5]+=$5
sum[$NF,6]+=$6
sum[$NF,7]+=$7
a[$NF,8]=$8
}
}
END{
for(i in a)
{
split(i,idx,SUBSEP)
a[idx[1],3]=int(sum[idx[1],3]/(a[idx[1],1]+a[idx[1],2]))
a[idx[1],4]=int(sum[idx[1],4]/(a[idx[1],1]+a[idx[1],2]))
a[idx[1],5]=int(sum[idx[1],5]/(a[idx[1],1]+a[idx[1],2]))
a[idx[1],6]=int(sum[idx[1],6]/(a[idx[1],1]+a[idx[1],2]))
a[idx[1],7]=int(sum[idx[1],7]/(a[idx[1],1]+a[idx[1],2]))
print idx[1],a[idx[1],1],a[idx[1],2],a[idx[1],1]+a[idx[1],2],a[idx[1],3],a[idx[1],4],a[idx[1],5],a[idx[1],6],a[idx[1],7],a[idx[1],8]
}
}
' $prefix.sr.temp.3 | awk '!a[$0]++' > $prefix.sr.temp.out
if [ $? -eq 0 ]
then
echo -e "Step 2: Extracting discordant read pairs "
else
echo "!!!Unsuccessfully summarizing informations of split reads!!!"
exit
fi
#Step 2: Extracting discordant read pairs
awk --re-interval '$1 ~ /^@/ || ($2 ~ /97|145|81|161/ && ($6 ~ /^[1][0-5][0-9]M/ || $6 ~ /^[6-9][0-9]M/) && $7 == "=" )' $sam_file | awk '!a[$2"_"$3"_"$4"_"$5"_"$6"_"$7"_"$8]++' | sort -k1,1 -k2n,2 | awk '
BEGIN {FS="\t";OFS="\t"}
{
if (NR==1)
{
read = $1; read_flag = $2; len=$9; a[$1] = $0; next
}
if ($1 == read)
{
if ((read_flag == 97 && len < 0 && $2 ==145 && $9 >0) || (read_flag == 145 && len > 0 && $2 == 97 && $9 <0) || (read_flag == 161 && len < 0 && $2 == 81 && $9 >0) || (read_flag == 81 && len > 0 && $2 == 161 && $9 <0))
{
print "DRP","attP",a[$1]"\n""DRP","attP",$0
}
else if ((read_flag == 145 && len < 0 && $2 ==97 && $9 >0) || (read_flag == 97 && len > 0 && $2 == 145 && $9 <0) || (read_flag == 81 && len < 0 && $2 == 161 && $9 >0) || (read_flag == 161 && len > 0 && $2 == 81 && $9 <0))
{
print "DRP","attB",a[$1]"\n""DRP","attB",$0
}
}
else if ($1 != read)
{
read = $1; read_flag = $2; len=$9; a[$1] = $0; next
}
}' | awk '($4 ~ /97|161|81|145/ && $11 > 0)' | awk 'BEGIN {FS="\t";OFS="\t"} NR==FNR {split($1,readname,"_"); a[readname[1]]=readname[1]} NR>FNR { if(!($3 in a)) print $0}' $prefix.sr.temp.2 - >$prefix.drp.temp.1
##First round merging DRP reads accroding to the SR position. Not merged DRP reads collected in the '$prefix'.drp.temp.left file
awk ' BEGIN {FS="\t";OFS="\t"}
FILENAME==ARGV[1] { a[$3]=$0; next}
FILENAME==ARGV[2] {
T_L=int('"$insert_size"'*1.6) #threshold low for clsutering
T_H=int('"$insert_size"'*3.5) #threshold low for clsutering
attP_count = 0
attB_count = 0
for (i in a)
{
split(a[i],b,"\t")
if ($6-b[6] > -T_L && $6-b[6] <T_L && $7-b[10] >-T_L && $7-b[10] <T_L && $9-b[11] > -T_H && $9-b[11] < T_H && b[5] == $10)
{
if (b[2] == "attP" && b[6] >= $5 && $8-b[10]+10 > '"$read_length"' )
{
attP_count++
delete a[i]
}
else if (b[2] == "attB" && $6-b[6]+10 > '"$read_length"' && b[10] >= $7)
{
attB_count++
delete a[i]
}
}
}
c[$1]=$0"\t"attB_count"\t"attP_count"\t"attB_count+attP_count
}
END {
for (i in c)
{
print c[i]
}
for (i in a)
{
print a[i] >> "'$prefix'.drp.temp.left"
}
}
' $prefix.drp.temp.1 $prefix.sr.temp.out > $prefix.sr-drp.temp.out
##Cluster the left DRP reads independent of SR reads: first candidate assign
if test -s $prefix.drp.temp.left; then
awk '
BEGIN{FS="\t";OFS="\t"}
{
if($11 > '"$prophage_size_min"' && $11 < '"$prophage_size_max"') a[$3]=$2"\t"$4"\t"$6"\t"$10"\t"$11"\t"$5
}
END{
count=1
T_L=int('"$insert_size"'*2.2) #threshold low for clsutering
T_H=int('"$insert_size"'*3.5) #threshold low for clsutering
for (i in a)
{
split(a[i],b,"\t")
print i,b[1],b[2],b[3],b[4],b[5],b[6],"DRP_candidate_"count
for (j in a)
{
split(a[j],c,"\t")
if ( i != j )
{
if (b[6]==c[6] && b[3]-c[3] > -T_L && b[3]-c[3] < T_L && b[4]-c[4] > -T_L && b[4]-c[4] < T_L && b[5]-c[5] > -T_H && b[5]-c[5] < T_H)
{
if ((b[1] == "attB" && c[1] == "attB")||(b[1] == "attP" && c[1] == "attP")||(b[1] == "attB" && c[1] == "attP" && b[3] < c[3] && b[4] > c[4])||(b[1] == "attP" && c[1] == "attB" && b[3] > c[3] && b[4] < c[4]))
{
print j,c[1],c[2],c[3],c[4],c[5],c[6],"DRP_candidate_"count
delete a[j]
}
}
}
}
count++
delete a[i]
}
}' $prefix.drp.temp.left >$prefix.drp.temp.2
else
touch $prefix.drp.temp.2
fi
##Cluster the left DRP reads independent of SR reads: merging assigned candidate
if test -s $prefix.drp.temp.2; then
awk '{if (!($3 == "")) print $0}' $prefix.drp.temp.2| awk '
BEGIN{FS="\t";OFS="\t"}
{
#if (NR==1)
#{
# a[$NF,3] = $4+0; a[$NF,4] = $5+0
#}
a[$NF,4]=10000000000
if ( !(a[$NF,1] >0) )
{
a[$NF,1]=0
}
if ( !(a[$NF,2] >0) )
{
a[$NF,2]=0
}
if ($2 == "attB")
{
a[$NF,1]+=1
a[$NF,3]=a[$NF,3]>=$4?a[$NF,3]:$4
a[$NF,4]=a[$NF,4]<=$5?a[$NF,4]:$5
a[$NF,5]=$7
}
else if ($2 == "attP")
{
a[$NF,2]+=1
a[$NF,3]=a[$NF,3]>=$4?a[$NF,3]:$4
a[$NF,4]=a[$NF,4]<=$5?a[$NF,4]:$5
a[$NF,5]=$7
}
}
END{
for(i in a)
{
split(i,idx,SUBSEP)
print idx[1],a[idx[1],1],a[idx[1],2],a[idx[1],1]+a[idx[1],2],a[idx[1],3],a[idx[1],4],a[idx[1],4]-a[idx[1],3],a[idx[1],5]
}
}
' | awk '!a[$0]++' > $prefix.drp.temp.out
else
touch $prefix.drp.temp.out
fi
if [ $? -eq 0 ]
then
echo -e "Step 3: Combine split reads and discordant read pairs"
else
echo "!!!Unsuccessfully extracting discordant read pairs!!!"
exit
fi
#Classify reads containing attB or attP in WGS (only contigs)
awk '
BEGIN {FS="\t";OFS="\t"}
function calculation1() {
attL_start=send1-qend1+qstart2
attL_end=send1
attR_start=sstart2
attR_end=sstart2+qend1-qstart2
prophage_size=CONTIGLEN[contig]-send1+sstart2+qend1-qstart2
CONTIGPOSITION=contig"=::="$2
print SR_type,contig"::"$2,evidence,attL_start,attL_end,attR_start,attR_end,prophage_size,$1,CONTIGPOSITION
}
function calculation2() {
attL_start=sstart2
attL_end=sstart2+qend1-qstart2
attR_start=send1
attR_end=send1+qend1-qstart2
prophage_size=send1+sstart2+qend1-qstart2-1
CONTIGPOSITION="="$2"::="contig
print SR_type,$2"::"contig,evidence,attL_start,attL_end,attR_start,attR_end,prophage_size,$1,CONTIGPOSITION
}
function calculation3() {
attL_start=sstart2
attL_end=sstart2+qend1-qstart2
attR_start=send1-qend1+qstart2
attR_end=send1
prophage_size=CONTIGLEN[$2]-sstart2-qend1+qstart2+send1
CONTIGPOSITION=$2"=::="contig
print SR_type,$2"::"contig,evidence,attL_start,attL_end,attR_start,attR_end,prophage_size,$1,CONTIGPOSITION
}
function calculation4() {
attL_start=send1
attL_end=send1+qend1-qstart2
attR_start=sstart2
attR_end=sstart2+qend1-qstart2
prophage_size=send1+sstart2+qend1-qstart2-1
CONTIGPOSITION="="contig"::="$2
print SR_type,contig"::"$2,evidence,attL_start,attL_end,attR_start,attR_end,prophage_size,$1,CONTIGPOSITION
}
function calculation5() {
attL_start=sstart2-qend1+qstart2
attL_end=sstart2
attR_start=send1-qend1+qstart2
attR_end=send1
prophage_size=send1+sstart2-qend1+qstart2-1
CONTIGPOSITION="="$2"::="contig
print SR_type,$2"::"contig,evidence,attL_start,attL_end,attR_start,attR_end,prophage_size,$1,CONTIGPOSITION
}
function calculation6() {
attL_start=send1-qend1+qstart2
attL_end=send1
attR_start=sstart2-qend1+qstart2
attR_end=sstart2
prophage_size=send1+sstart2-qend1+qstart2-1
CONTIGPOSITION="="contig"::="$2
print SR_type,contig"::"$2,evidence,attL_start,attL_end,attR_start,attR_end,prophage_size,$1,CONTIGPOSITION
}
function calculation7() {
attL_start=sstart2
attL_end=sstart2+qend1-qstart2
attR_start=send1
attR_end=send1+qend1-qstart2
prophage_size=CONTIGLEN[$2]-sstart2+CONTIGLEN[contig]-send1-qend1+qstart2+1
CONTIGPOSITION=$2"=::"contig"="
print SR_type,$2"::"contig,evidence,attL_start,attL_end,attR_start,attR_end,prophage_size,$1,CONTIGPOSITION
}
function calculation8() {
attL_start=send1
attL_end=send1+qend1-qstart2
attR_start=sstart2
attR_end=sstart2+qend1-qstart2
prophage_size=CONTIGLEN[$2]-sstart2+CONTIGLEN[contig]-send1-qend1+qstart2+1
CONTIGPOSITION=contig"=::"$2"="
print SR_type,contig"::"$2,evidence,attL_start,attL_end,attR_start,attR_end,prophage_size,$1,CONTIGPOSITION
}
function calculation9() {
attL_start=sstart2-qend1+qstart2
attL_end=sstart2
attR_start=send1-qend1+qstart2
attR_end=send1
prophage_size=CONTIGLEN[$2]-sstart2+CONTIGLEN[contig]-send1+qend1-qstart2+1
CONTIGPOSITION=$2"=::"contig"="
print SR_type,$2"::"contig,evidence,attL_start,attL_end,attR_start,attR_end,prophage_size,$1,CONTIGPOSITION
}
function calculation10() {
attL_start=send1-qend1+qstart2
attL_end=send1
attR_start=sstart2-qend1+qstart2
attR_end=sstart2
prophage_size=CONTIGLEN[$2]-sstart2+CONTIGLEN[contig]-send1+qend1-qstart2+1
CONTIGPOSITION=contig"=::"$2"="
print SR_type,contig"::"$2,evidence,attL_start,attL_end,attR_start,attR_end,prophage_size,$1,CONTIGPOSITION
}
###########contig length
FILENAME==ARGV[1] {CONTIGLEN[$1]=$2}
FILENAME==ARGV[2] {
if (FNR==1)
{
query = $1; contig=$2; qstart1 = $7; qend1 = $8; sstart1 = $9; send1 = $10; next
}
##make sure to extract correct flag value if "_" was used in read names
Flag_pos=split ($1,flag,"_")
if ($1 == query && $2 != contig)
{ qstart2 = $7; qend2 = $8; sstart2 = $9; send2 = $10
#Plus-Plus or Minus-Minus
if (flag[Flag_pos] ~ /83|97|147|161|81|99|145|163/ && (qend2 > qend1 && qend1 > qstart2 && qstart2 > qstart1 && qstart1 < 10))
{
if ( (send2 > sstart2 && send1 > sstart1) && CONTIGLEN[contig]-send1+send2 > '"$prophage_size_min"' && CONTIGLEN[contig]-send1+send2 < '"$prophage_size_max"' )
{
SR_type="SR"
evidence="attB"
calculation1()
}
else if ( (send2 > sstart2 && send1 > sstart1) && CONTIGLEN[$2]-send2+send1 > '"$prophage_size_min"' && CONTIGLEN[$2]-send2+send1 < '"$prophage_size_max"' )
{
SR_type="SR"
evidence="attP"
calculation3()
}
}
#Plus-Minus or Minus-Plus
if (flag[Flag_pos] ~ /65|83|129|147|99|113|163|177/ && (qend2 > qend1 && qend1 > qstart2 && qstart2 > qstart1 && qstart1 < 10))
{
if ( flag[Flag_pos] ~ /99|113|163|177/ && (send2 > sstart2 && send1 < sstart1) && send1+send2 > '"$prophage_size_min"' && send1+send2 < '"$prophage_size_max"' )
{
SR_type="SR"
evidence="attB"
if($2 < contig)
{ calculation2() }
else if($2 > contig)
{ calculation4() }
}
else if ( flag[Flag_pos] ~ /99|113|163|177/ && (send2 > sstart2 && send1 < sstart1) && (CONTIGLEN[$2]+CONTIGLEN[contig]-send2-send1 > '"$prophage_size_min"') && (CONTIGLEN[$2]+CONTIGLEN[contig]-send2-send1 < '"$prophage_size_max"') )
{
SR_type="SR"
evidence="attP"
if($2 < contig)
{ calculation7() }
else if($2 > contig)
{ calculation8() }
}
else if ( flag[Flag_pos] ~ /65|83|129|147/ && (send2 < sstart2 && send1 > sstart1) && (send1+send2 > '"$prophage_size_min"') && (send1+send2 < '"$prophage_size_max"') )
{
SR_type="SR"
evidence="attP"
if($2 < contig)
{ calculation5() }
else if($2 > contig)
{ calculation6() }
}
else if ( flag[Flag_pos] ~ /65|83|129|147/ && (send2 < sstart2 && send1 > sstart1) && (CONTIGLEN[$2]+CONTIGLEN[contig]-send2-send1 > '"$prophage_size_min"') && (CONTIGLEN[$2]+CONTIGLEN[contig]-send2-send1 < '"$prophage_size_max"'))
{
SR_type="SR"
evidence="attB"
if($2 < contig)
{ calculation9() }
else if($2 > contig)
{ calculation10() }
}
}
}
else if ($1 != query)
{
query = $1; contig = $2; qstart1 = $7; qend1 = $8; sstart1 = $9; send1 = $10
}
}' contiglength.file $prefix.sr.temp.2 >$prefix.WGS.sr.temp.3
awk '
#Cluster split reads
BEGIN{FS="\t";OFS="\t"}
{
if( $8 > '"$prophage_size_min"' && $8 < '"$prophage_size_max"' ) a[$9]=$3"\t"$4"\t"$5"\t"$6"\t"$7"\t"$8"\t"$10
}
END{
count=1
T_L=int('"$read_length"'/3) #threshold low for clsutering
T_H=int('"$read_length"'*0.7) #threshold low for clsutering
for (i in a)
{
split(a[i],b,"\t")
print i,b[1],b[2],b[3],b[4],b[5],b[6],b[7],"WGS_candidate_"count
for (j in a)
{
split(a[j],c,"\t")
if ( i != j && c[7] == b[7])
{
if (b[7]==c[7] && b[2]-c[2] > -T_L && b[2]-c[2] < T_L && b[4]-c[4] > -T_L && b[4]-c[4] < T_L && b[6]-c[6] > -T_H && b[6]-c[6] < T_H)
{
print j,c[1],c[2],c[3],c[4],c[5],c[6],c[7],"WGS_candidate_"count
delete a[j]
}
}
}
count++
delete a[i]
}
}' $prefix.WGS.sr.temp.3|awk '{if (!($3 == "")) print $0}' >$prefix.WGS.sr.temp.4
awk '
BEGIN{FS="\t";OFS="\t"}
{
if ( !(a[$NF,1] >0) )
{
a[$NF,1]=0
}
if ( !(a[$NF,2] >0) )
{
a[$NF,2]=0
}
if ($2 == "attB")
{
a[$NF,1]+=1
sum[$NF,3]+=$3
sum[$NF,4]+=$4
sum[$NF,5]+=$5
sum[$NF,6]+=$6
sum[$NF,7]+=$7
a[$NF,8]=$8
}
else if ($2 == "attP")
{
a[$NF,2]+=1
sum[$NF,3]+=$3
sum[$NF,4]+=$4
sum[$NF,5]+=$5
sum[$NF,6]+=$6
sum[$NF,7]+=$7
a[$NF,8]=$8
}
}
END{
for(i in a)
{
split(i,idx,SUBSEP)
a[idx[1],3]=int(sum[idx[1],3]/(a[idx[1],1]+a[idx[1],2]))
a[idx[1],4]=int(sum[idx[1],4]/(a[idx[1],1]+a[idx[1],2]))
a[idx[1],5]=int(sum[idx[1],5]/(a[idx[1],1]+a[idx[1],2]))
a[idx[1],6]=int(sum[idx[1],6]/(a[idx[1],1]+a[idx[1],2]))
a[idx[1],7]=int(sum[idx[1],7]/(a[idx[1],1]+a[idx[1],2]))
print idx[1],a[idx[1],1],a[idx[1],2],a[idx[1],1]+a[idx[1],2],a[idx[1],3],a[idx[1],4],a[idx[1],5],a[idx[1],6],a[idx[1],7],a[idx[1],8]
}
}