use custom perl script for SmartSeq-3 filtering (remove BBMap and fastq_pair dependencies)

 && gcc -std=gnu99   ../main.c ../robstr.c ../fastq_pair.c ../is_gzipped.c  -o fastq_pair \

 && cp fastq_pair /bin/fastq_pair

RUN wget https://sourceforge.net/projects/bbmap/files/latest/download ; mv download BBMap_38.87.tar.gz \

 && tar -xvzf BBMap_38.87.tar.gz

# RUN wget https://sourceforge.net/projects/bbmap/files/latest/download ; mv download BBMap_38.87.tar.gz \

#  && tar -xvzf BBMap_38.87.tar.gz

ENV PATH bbmap:$PATH

# ENV PATH bbmap:$PATH

    #Smart-Seq3

    if [[ "$technology" == "smartseq"* ]];then

        echo "Note: SmartSeq-3 requires additional dependencies, install fastq_pair and bbduk.sh and add these to the PATH"

        echo "  ...processsing for ${technology}"

        if [[ $verbose ]]; them

            echo "Note: SmartSeq-3 requires additional filtering for UMIs"

        fi

        for convFile in "${convFiles[@]}"; do

            read=$convFile

            convR1=$read

            convR2=$(echo $read | perl -pne 's/(.*)_R1/$1_R2/' )

            convI1=$(echo $read | perl -pne 's/(.*)_R1/$1_I1/' )

            convI2=$(echo $read | perl -pne 's/(.*)_R1/$1_I2/' )

start=$(date +%s.%N)

            # reads matching adapter sequence for R1

            grep -A 2 -B 1 ATTGCGCAATG $convR1  | sed '/--/d' > ${convR1}.umi.fastq

            #match R2 to R1 containing UMI

            fastq_pair  ${convR1}.umi.fastq $convR2

end=$(date +%s.%N)

runtime=$(python -c "print(${end} - ${start})")

echo "Runtime for filtering with grep was $runtime"

            # uses bbduk (decontamination using k=mers) from BBMap(BBTools) vesion 38.87

            # step 1: process as paired ends to match k-mers (adapters matched are filtered) (clean = internal; fail = 5' UMI reads)

start1=$(date +%s.%N)

            bbduk.sh in1=${convR1}.umi.fastq.paired.fq in2=${convR2}.paired.fq outu1=$crIN/clean_R1.fq outu2=$crIN/clean_R2.fq outm1=$crIN/fail_R2.fq outm2=$crIN/fail_R1.fq outs=$crIN/pass_singletons.fq literal=ATTGCGCAATG k=10

end1=$(date +%s.%N)

runtime=$(python -c "print(${end1} - ${start1})")

echo "Runtime for filtering with bbduk.sh was $runtime"

            # step 2: take matched (adapter filtered) reads and remove adapter (and all bases to the left)

start2=$(date +%s.%N)

            bbduk.sh in1=$crIN/fail_R1.fq in2=$crIN/fail_R2.fq out1=$crIN/trimmed_R1.fq out2=$crIN/trimmed_R2.fq literal=ATTGCGCAATG ktrim=l k=11

            # match indexes to R1

            fastq_pair  ${convR1}.umi.fastq ${convI1}

            fastq_pair  ${convR1}.umi.fastq ${convI2}

             mv ${convI1}.fastq.paired.fq ${convI1}

             mv ${convI2}.fastq.paired.fq ${convI2}

             mv $crIN/trimmed_R1.fq ${convR1}

             mv $crIN/trimmed_R2.fq ${convR2}

             rm $crIN/*fastq.paired.fq $crIN/*fastq.single.fq $crIN/clean_R1.fq $crIN/clean_R2.fq $crIN/fail_R1.fq $crIN/fail_R2.fq $crIN/pass_singletons.fq ${convR1}.umi.fastq

end2=$(date +%s.%N)

runtime=$(python -c "print(${end2} - ${start2})")

echo "Runtime for trimming with bbduk.sh was $runtime"

runtime=$(python -c "print(${end2} - ${start1})")

echo "Runtime for trimming and filtering with bbduk.sh was $runtime"

start=$(date +%s.%N)

            perl sub/FilterSmartSeqReadUMI.pl --r1=${convR1}--r2=${convR2} --i1=${convI1} --i2=${convI2} --out .

end=$(date +%s.%N)

runtime=$(python -c "print(${end} - ${start})")

echo "Runtime for trimming and filtering with custom perl was $runtime"

            # filter UMI reads by matching tag sequence ATTGCGCAATG (bases 1-11 of R1) and remove as an adapters 

            perl sub/FilterSmartSeqReadUMI.pl --r1=${convR1} --r2=${convR2} --i1=${convI1} --i2=${convI2} --out $crIN

            # returns R1 with tag sequence removed (left trim) starting with 8pbp UMI and corresponding reads for I1, I2, and R2

            mv $crIN/SmartSeq3_parsed_R1.fastq ${convR1}

            mv $crIN/SmartSeq3_parsed_R2.fastq ${convR2}

            mv $crIN/SmartSeq3_parsed_I1.fastq ${convI1}

            mv $crIN/SmartSeq3_parsed_I2.fastq ${convI2}

        done

    fi

exit 0

    #converting barcodes

    echo " adjusting barcodes of R1 files"

    if [[ $barcodeadjust != 0 ]]; then