Hi,

I am using viGEN pipeline to identify viruses in RNA-Seq data. I am getting following error when I run the script.

stat: No such file or directory Warning: Could not open read file "/home/venura/viGEN-master/unmapped_fastq/RRV4_un/RRV4_un__1.fq" for reading; skipping... Error: No input read files were valid (ERR): bowtie2-align exited with value 1

As one other post suggested I looked whether it has to something to do with the path using head -4 /home/venura/viGEN-master/unmapped_fastq/RRV4_un/RRV4_un_1.fq It reads the first four lines of the .fq file without any problem.

@V300020467L4C001R0020000146 GAAGAAGTTAACATCTTTAAGGATGATGTTGTTATTCAGTTCATCAACCCTAAGGTTCAAGCTTCAATTGCTGCAAACACATGGGTTGTTAGCGAAGTCG + eefeeeeeeeeeeeeeeefeeeeeeeeeeeeeeeeeeeeeeedeeeee_fedefeeefeeeefeefeffdfefffefffffffeeeefcfeeeeeecfef

Can some one help me to troubleshoot the error? Thank you!

Complete script is given below( I removed the functionFlagstatBam given in the original script since authors forgot to include the function in the script uploaded there at github. I opened an issue there requesting author to include the missing function.)

#!/bin/bash

# Alignment using Bowtie2
functionBowtie2() {
    echo "Start - Align using Bowtie2 local algin" >> $LOG
    $BOWTIE2_SW"bowtie2" -x $BOWTIE2_REF --local -1 $I_FOLDER$ID"_1.fq" -2 $I_FOLDER$ID"_2.fq" --threads $N_THREAD --al $O_FOLDER$ID/$ID".bowtie2.align" --un $O_FOLDER$ID/$ID".Bowtie2.unaligned" -S $O_FOLDER$ID/$ID".bowtie2.sam" --sensitive > $O_FOLDER$ID/$ERR2 2> $O_FOLDER$ID/$ERR3
    echo "End - Align using Bowtie2 local algin" >> $LOG
}

#Convert SAM file to BAM file
functionConvertSamtoBam() {
    echo "Start - convert sam to bam file using Samtools" >> $LOG
    $SAMTOOLS"samtools" view -bS $O_FOLDER$ID/$ID".bowtie2.sam" > $O_FOLDER$ID/$ID".bowtie2.bam"
    echo "End - convert sam to bam file using Samtools" >> $LOG
}

#Sort BAM coordinate wise
functionSortBam() {
    echo "Start - Samtools sort bam file" >> $LOG
    $SAMTOOLS"samtools" sort $O_FOLDER$ID/$ID".bowtie2.bam" -o $O_FOLDER$ID/$ID".bowtie2.sorted.bam" -@ $N_THREAD
    echo "End - Samtools sort bam file" >> $LOG
} 

#Index BAM file
functionIndexBam() {
    echo "Start - Samtools index bam file" >> $LOG
    $SAMTOOLS"samtools" index $O_FOLDER$ID/$ID".bowtie2.sorted.bam"
    echo "End - Samtools index bam file" >> $LOG
}

# Samtools idx -  genome level counts
functionIdxStatsBam() {
    echo "Start - Samtools Idx" >> $LOG
    $SAMTOOLS"samtools" idxstats $O_FOLDER$ID/$ID".bowtie2.sorted.bam" > $O_FOLDER$ID/$ID".bowtie2.idxstats.txt"
    echo "End - Samtools Idx" >> $LOG
 }

########################################
# Values to set by the user
N_THREAD=8
I_FOLDER="/home/venura/viGEN-master/unmapped_fastq/RRV4_un/"
O_FOLDER="/home/venura/viGEN-master/output_bowtie2/"

LOG=$O_FOLDER"/pipeline_log.txt"
ERR2="system.err.bowtie2.txt"
ERR3="log.bowtie2.txt"
SAMTOOLS="/usr/bin/"
BOWTIE2_SW="/usr/bin/"
BOWTIE2_REF="/home/venura/viGEN-master/vir_ref/virus.bowtie2.refB"

for x in $(cat samplenames.txt)
do
    echo $x
    ID=${x%*}

    echo "-----------------------------" >> $LOG
    echo $ID >> $LOG

    START=$(date +%s) 

    mkdir $O_FOLDER$ID

    functionBowtie2

    functionConvertSamtoBam

    functionSortBam

    functionIndexBam

    functionIdxStatsBam

    END=$(date +%s)
    DIFF=$(( ($END - $START)/60 )) 
    echo "It took $DIFF minutes" >> $LOG
done