annotate TEfinder @ 0:b81a83c743d3 draft default tip

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#!/bin/bash
##
##
## Authors: Vista Sohrab & Dilay Hazal Ayhan
## Date: January 15, 2021
## Description: TEfinder uses discordant reads to detect novel transposable element insertion events in short read paired-end sample sequencing data. 
##              Software dependencies include bedtools 2.28.0 or later, samtools 1.3 or later, picard 2.0.1 or later
##              Required inputs include sample alignment file (.bam|.sam), reference genome FASTA (.fa), reference TE annotation in GFF/GTF or GFF3 (.gff|.gtf), and TEs of interest (.txt)
##
## University of Massachusetts Amherst
##
##
##
##

set -e

margs=4

# Functions
function example {
     echo -e "example: TEfinder -alignment sample.bam -fa reference.fa -gtf TEs.gtf -te List_of_TEs.txt"
}

function help {
     echo -e "REQUIRED:"
     echo -e "  -alignment,  --alignmentFile       STR  sample reads aligned to reference genome (BAM/SAM file)"
     echo -e "  -fa,         --FastaFile           STR  reference genome FASTA index (FA file)"
     echo -e "  -gtf,        --TransposonsInGenome STR  reference genome TE annotation (GFF2/GTF file)"
     echo -e "  -te,         --TransposonsToSearch STR  TE names (single column text file)\n"
     echo -e "OPTIONAL:"
     echo -e "  -bamo,       --DiscordantReads     STR  BAM output\n"
     echo -e "  -bedo,       --bTEinsertions       STR  TEinsertions BED output\n"
     echo -e "  -gtfo,       --gTEinsertions       STR  TEinsertions GTF output\n"
     echo -e "  -fis,        --FragmentInsertSize  INT  short-read sequencing fragment insert size [400]"
     echo -e "  -picard,     --pathToPicardjar     STR  path to picard tools .jar file [picard.jar]"
     echo -e "  -md,         --MaxDistanceForMerge INT  maximum distance between reads for bedtools merge [150]"
     echo -e "  -k,          --MaxTSDLength        INT  maximum TE target site duplication (TSD) length [20]"
     echo -e "  -maxHeapMem, --MaxHeapMemory       INT  java maximum heap memory allocation for picard in Mb [2000]" 
     echo -e "  -workingdir, --WorkingDirectory    STR  working directory name [TEfinder_<Date>]"
     echo -e "  -out,        --OutputFormat        STR  output format as GTF [BED]"
     echo -e "  -outname,    --OutputName          STR  output name prefix added to file names [null]"
     echo -e "  -threads,    --Threads             INT  number of threads for samtools multi-threading [1]"
     echo -e "  -intermed    --IntermediateFiles   STR  keep intermediate files created by pipeline [no]"  
     echo -e "  -h,          --help                     prints help\n"
     example
}

# check if mandatory args are empty
function margs_check {
     if [ $# -lt $margs ]; then
          echo -e "One or more required parameters are missing."
          example
          exit 1 # error
     fi
}

# main workflow
 #### : comment out
function pipeline() {
     mkdir ${workingdir}/${line}
     currdir=${workingdir}/${line}
     echo -e $(date) " Transposon analysis for "${line}" has started\n"

     grep -P '[^(\w|\d|\-|\_|\#|\.)]'${line}'[^(\w|\d|\-|\_|\#|\.)]' $gtf > ${currdir}/${line}_TE.gff
     echo -e $(date) " Individual TE GFF has been created for "${line}"\n" ####
     
     bedtools intersect -abam ${workingdir}/${outname}Alignments.bam -b ${currdir}/${line}_TE.gff -wa > ${currdir}/${line}_MappedReadsToTE.bam
     echo -e $(date) " Mapped reads to TE via bedtools intersect has been completed for "${line}"\n" ####
     samtools view -@ $threads ${currdir}/${line}_MappedReadsToTE.bam | \
          awk -v Ins=`expr $fis \* 10` '{if (($7 != "=") || ($9 > Ins) || ($9 < -Ins)) print $1}' > ${currdir}/${line}_ReadID.txt
     echo -e $(date) " Identifying discordant read IDs has been completed for "${line}"\n" ####
     
	 # if discordant readID file exists, then continue with remainder of TE analysis
	 if  [[ -s  ${currdir}/${line}_ReadID.txt ]]
	 then
		  #java $maxHeapMem -jar $picard FilterSamReads I=${workingdir}/${outname}Alignments.bam O=${currdir}/${line}_DiscordantPairs.bam READ_LIST_FILE=${currdir}/${line}_ReadID.txt FILTER=includeReadList WRITE_READS_FILES=false
          $picard $maxHeapMem FilterSamReads -I ${workingdir}/${outname}Alignments.bam -O ${currdir}/${line}_DiscordantPairs.bam -READ_LIST_FILE ${currdir}/${line}_ReadID.txt -FILTER includeReadList -WRITE_READS_FILES false &>/dev/null
          echo -e $(date) " Filtering original alignment based on discordant reads IDs is complete for "${line}"\n"  ####
          
          bedtools merge -d $md -S + -c 1 -o count -i ${currdir}/${line}_DiscordantPairs.bam | \
               awk '{if ($4 > 3) print $0}' > ${currdir}/${line}_plusCluster.bed 
          echo -e $(date) " Primary reads from the + strand have been merged if read count greater than 3 for "${line}"\n" ####
          
          bedtools merge -d $md -S - -c 1 -o count -i ${currdir}/${line}_DiscordantPairs.bam | \
               awk '{if ($4 > 3) print $0}' > ${currdir}/${line}_minusCluster.bed
          echo -e $(date) " Primary reads from the - strand have been merged if read count greater than 3 for "${line}"\n" ####
          
          # filtering edges piped into bedtools merge (keeping read counts greater than 3 in the line above)
          ## find the closest minus strand to the plus strand in the cluster
          ## filter by the distance between the plus and minus clusters - only retain pairs if reads are 0-100 bases away
          ## if plus strand start is less than minus strand start and plus strand end is less than minus strand end then in proper orientation 
          bedtools closest -d -g ${workingdir}/reference.fa.fai -t first -a ${currdir}/${line}_plusCluster.bed -b ${currdir}/${line}_minusCluster.bed | \
               awk -v TSD=$k '{if ($9 <= TSD && $9 >= 0) print $0}' | \
               awk '{if ($2 < $6 && $3 < $7) print $0}' > ${currdir}/${line}_plusminus.bed
          echo -e $(date) " Filtration of clusters in proper orientation using bedtools closest has been completed for "${line}"\n" ####
          
          # if plus strand end is greater than minus strand start, then report the pair 
          awk '{if ($3 > $6) print $1"\t"$6"\t"$3"\t"$0}' ${currdir}/${line}_plusminus.bed > ${currdir}/${line}_plusminus_1.bed 
          echo -e $(date) " Overlapping reads TE insertions reported for "${line}"\n" ####
          
          #if plus  strand end is less than or equal to minus strand start and the region in between is less than a user-defined value k, report the pair
          awk -v TSD=$k '{if ($3 <= $6 && $6 - $3 < TSD) print $1"\t"$3 - 1"\t"$6 + 1"\t"$0}' ${currdir}/${line}_plusminus.bed  > \
               ${currdir}/${line}_plusminus_2.bed
          echo -e $(date) " Non-overlapping reads TE insertions reported for "${line}"\n" ####
          
          #combine reported TE insertions
          cat ${currdir}/${line}_plusminus_1.bed ${currdir}/${line}_plusminus_2.bed | \
               awk -v TEname=$line '{$0=TEname"\t"$0}1' | sort -k 1 | sort -k 2 > \
               ${currdir}/${line}_insertionRegion.txt
          
          
          cat ${currdir}/${line}_insertionRegion.txt >> ${workingdir}/insertions.txt
          echo -e $(date) " TE insertions for "${line}" have been reported.\n" ####
          
          
          echo -e $(date) " Transposon named "${line}" is processed.\n"
	 else
	      echo -e $(date) " Transposon named "${line}" is processed. No discordant reads found.\n"
	      rm -r ${currdir}
	 fi
}

# functions end

# get arguments
fa=
alignment=
gtf=
te=
out=
intermed=
bamo="DiscordantReads.bam"
bedo="TEinsertions.bed"
gtfo="TEinsertions.gtf"
outname=""
fis=400
picard="picard"
maxHeapMem=-Xmx2000m
md=150
k=20
d=$(date +%Y%m%d%H%M%S)
# workingdir=TEfinder_${d}
workingdir="TEfinder"
threads=1
 
while [ "$1" != "" ];
do
     case $1 in
     -fa | --FastaFile )
          shift
          fa=$1 ;;
     -alignment | --alignmentFile )
	      shift
          alignment=$1 ;;
     -gtf | --TransposonsInGenome )
	      shift
          gtf=$1 ;;
     -te | --TransposonsToSearch )
	      shift
          te=$1  ;;
     -fis | --FragmentInsertSize )
	      shift
          fis=$1 ;;
     -picard | --pathToPicardjar )
	      shift
          picard=$1 ;;
     -md | --MaxDistanceForMerge )
	      shift
          md=$1  ;;
     -k | --MaxTSDLength )
	      shift
          k=$1  ;;
     -bamo | --DiscordantReads )
	      shift
          bamo=$1 ;;
     -bedo | --bTEinsertions )
	      shift
          bedo=$1 ;;
     -gtfo | --gTEinsertions )
           shift
          gtfo=$1 ;;
     -maxHeapMem | --MaxHeapMemory )
	      shift
          maxHeapMem="-Xmx"$1"m" ;;
     -workingdir | --WorkingDirectory )
	      shift
          workingdir=$1 ;;
     -out | --OutputFormat )
	      shift
	   out=$1  ;;
     -outname | --OutputName )
              shift
           outname=$1 ;;
     -threads | --Threads )
              shift
           threads=$1 ;;
     -intermed | --IntermediateFiles )
              shift
           intermed=$1 ;;
     -h   | --help )
	      help
          exit;;
     *) # error
          echo "TEfinder: illegal option $1"
          example
          exit 1 ;;
     esac
     shift
done
margs_check $fa $alignment $gtf $te

# main

mkdir ${workingdir}

# remove empty lines from user provided TE list if present
sed '/^$/d' $te > ${workingdir}"/userTE_noEmptyLines.txt"

# create output file
printf "%s\t"  "track name=TEfinder" "type=bedDetail" "description=FR:forward read, RR:reverse read, InsRegion:insertion region start and end positions, FILTER:comma separated filters" > ${workingdir}/${outname}TEinsertions.bed
printf "\n" >> ${workingdir}/${outname}TEinsertions.bed

# create fasta index (fai) file
cp $fa ${workingdir}/reference.fa
samtools faidx ${workingdir}/reference.fa

# sort alignment input
samtools sort -@ $threads -o ${workingdir}/alignmentInput.sorted.bam ${alignment}
echo -e $(date) " Alignment file sorted successfully.\n"

# remove secondary and supplementary alignments from sorted bam
samtools view -F 2304 -@ $threads -o ${workingdir}/${outname}Alignments.bam ${workingdir}/alignmentInput.sorted.bam
echo -e $(date) " Alignments are filtered - secondary and supplementary alignments have been removed. \n"

# run pipeline for each TE
while IFS="" read -r line || [ -n "$line" ]
do
     pipeline &
done < ${workingdir}/userTE_noEmptyLines.txt
wait
echo -e $(date) " All transposons are processed. Finalizing...\n"

# combine discordant bam files
samtools merge -@ $threads -r ${workingdir}/${outname}DiscordantReads.bam ${workingdir}/*/*_DiscordantPairs.bam
echo -e $(date) " BAM Output: Discordant pair alignment file is now available.\n"
# Sorting by position 
samtools sort -@ $threads ${workingdir}/${outname}DiscordantReads.bam | samtools view -h -o ${workingdir}/${outname}DiscordantReads.sam
grep -v '^@PG' ${workingdir}/${outname}DiscordantReads.sam > ${workingdir}/${outname}DiscordantReadsNoPG.sam
rm ${workingdir}/${outname}DiscordantReads.sam
samtools view -hb -x "PG" --no-PG --remove-flags "PG" -O BAM ${workingdir}/${outname}DiscordantReadsNoPG.sam -o ${bamo}
rm ${workingdir}/${outname}DiscordantReadsNoPG.sam

# update output BED file with TEfinder results: organize the starting file
awk '{print $2"\t"$3"\t"$4"\t"$1"\t"$8+$12"\t.\tFR="$8";RR="$12";InsRegion="$6"-"$11";FILTER="}' ${workingdir}/insertions.txt > ${workingdir}/TEinsertions_putative.bed
# find the entries in repeat regions for filtering
bedtools intersect -wa -u -a ${workingdir}/TEinsertions_putative.bed -b $gtf > ${workingdir}/TEinsertions_putative_inrepeat.bed
# filtering process
while IFS="" read -r line || [ -n "$line" ]
do
	#located in repeat region
	if (grep -Fxq "$line" "${workingdir}/TEinsertions_putative_inrepeat.bed")
	then
        line=$line"in_repeat,"
	fi
	
	#weak evidence
	readc=$(echo $line | awk '{print $5}')
	if (( $readc < 10 ))
	then
	line=$line"weak_evidence,"
	fi
	
	#strand-biased	
	FR=$(echo $line | grep -o 'FR=[[:digit:]]*' | cut -f2 -d'=')
	RR=$(echo $line | grep -o 'RR=[[:digit:]]*' | cut -f2 -d'=')
	var1=$(echo 'e(l('$FR')*1.25)' | bc -l)
	var2=$(echo 'e(l('$FR')*0.8)' | bc -l)
	
	if [ $(echo "$RR > $var1" | bc) -eq 1 ] || [ $(echo "$RR < $var2" | bc) -eq 1 ]
	then
	line=$line"strand_bias,"
	fi
	
	#pass
	lastchar=${line: -1}
	if [ $lastchar == "," ]
	then
	line=${line::${#line}-1}
	else
	line=$line"PASS"
	fi
	
	#write to final output
	printf "%s\n" "$line" >> ${workingdir}/${outname}TEinsertions.bed
     
done < ${workingdir}/TEinsertions_putative.bed
wait
echo -e $(date) " BED Output: TEfinder output BED file is now available.\n"
# Sorting
# cp ${workingdir}/${outname}TEinsertions.bed ${outo}
bedtools sort -chrThenSizeA -i ${workingdir}/${outname}TEinsertions.bed > ${bedo}
# cat ${bedo}

# gtf option - create output GTF files with TEfinder results
if [ ! -z "$out" ]
then
  awk 'FNR > 1 {print $1"\tTEfinder\tTIP\t"$2 + 1"\t"$3"\t"$5"\t.\t.\tte_name \""$4"\"; tags \""$7"\""}' ${workingdir}/${outname}TEinsertions.bed > ${workingdir}/${outname}TEinsertions.gtf
  bedtools sort -chrThenSizeA -i ${workingdir}/${outname}TEinsertions.gtf > ${gtfo}
  # awk 'FNR > 1 {print $1"\tTEfinder\tTIP\t"$2 + 1"\t"$3"\t"$5"\t.\t.\tte_name \""$4"\"; tags \""$7"\""}' ${bedo} > ${gtfo}
  # Sorting
  # cp ${workingdir}/${outname}TEinsertions.gtf ${gtfo}
  echo -e "\n\n"
  # cat ${gtfo}
  # bedtools sort -chrThenSizeA -i ${workingdir}/${outname}TEinsertions.gtf > ${gtfo}
  echo -e $(date) " GTF Output: TEfinder output GTF file is now available.\n"
fi

# clean working directory
if [ -z "$intermed" ]
then
 rm ${workingdir}/TEinsertions_putative.bed ${workingdir}/TEinsertions_putative_inrepeat.bed ${workingdir}/reference.fa ${workingdir}/reference.fa.fai \
 ${workingdir}/alignmentInput.sorted.bam ${workingdir}/insertions.txt ${workingdir}/${outname}Alignments.bam ${workingdir}/userTE_noEmptyLines.txt 
 rm -r ${workingdir}/*/
fi

if [ `wc -l <${workingdir}/${outname}TEinsertions.bed` -le "1" ]
then
 echo -e $(date) " Error: TEfinder run unsuccessful."
else
 echo -e $(date) " TE insertion output files have been created. TEfinder completed successfully."
fi