Hello.

I'm encountering the problem with viral RNA ngs data alignement. I wrote two kinds of scripts; first:

INPATH=/home/dir/usr
OUTPATH=/home/dir/usr
reference=/dir/ref.fasta
cd /mnt/home/usr/tools/bwa-0.7.17
./bwa index $reference
for file1 in $INPATH/*.fastq.gz;
do
        bname=$(basename $file1 )
        sample=$( cut -d'_' -f 1,2,3<<<"$bname")
        echo $bname $sample
        input1=$INPATH/$sample"_R1_001.unpaired.fastq.gz"
        output1=$OUTPATH/$sample"_R1_001.unpaired.sai"
        input2=$INPATH/$sample"_R2_001.unpaired.fastq.gz"
        output2=$OUTPATH/$sample"_R2_001.unpaired.sai"
        outfile=$OUTPATH/$sample"_pe.sam"
        echo $input1 $output1 $input2 $output2
        ./bwa mem -t 4 $reference $input1 | gzip -3 > $output1
        ./bwa mem -t 4 $reference $input2 | gzip -3 > $output2
        ./bwa sampe -a $reference  $output1 $output2 $input1 $input2 > $outfile
        echo $outfile
done

gives output like:

[M::bwa_idx_load_from_disk] read 0 ALT contigs
[M::process] read 1034 sequences (71414 bp)...
[M::mem_process_seqs] Processed 1034 reads in 0.058 CPU sec, 0.017 real sec
[gzclose] buffer error
[M::bwa_idx_load_from_disk] read 0 ALT contigs
[gzclose] buffer error

Usage:   bwa sampe [options] <prefix> <in1.sai> <in2.sai> <in1.fq> <in2.fq>

Options: -a INT   maximum insert size [0]
         -o INT   maximum occurrences for one end [100000]
         -n INT   maximum hits to output for paired reads [3]
         -N INT   maximum hits to output for discordant pairs [10]
         -c FLOAT prior of chimeric rate (lower bound) [1.0e-05]
         -f FILE  sam file to output results to [stdout]
         -r STR   read group header line such as `@RG\tID:foo\tSM:bar' [null]
         -P       preload index into memory (for base-space reads only)
         -s       disable Smith-Waterman for the unmapped mate
         -A       disable insert size estimate (force -s)

Notes: 1. For SOLiD reads, <in1.fq> corresponds R3 reads and <in2.fq> to F3.
       2. For reads shorter than 30bp, applying a smaller -o is recommended to
          to get a sensible speed at the cost of pairing accuracy.

But second:

INPATH=/home/dir/usr
    OUTPATH=/home/dir/usr
    reference=/dir/ref.fasta
cd /mnt/home/usr/tools/bwa-0.7.17
./bwa index $reference
for file1 in $INPATH/*.fastq.gz;
do
        bname=$(basename $file1 )
        sample=$( cut -d'_' -f 1,2,3<<<"$bname")
        echo $bname $sample
        input1=$INPATH/$sample"_R1_001.unpaired.fastq.gz"
        output1=$OUTPATH/$sample"_R1_001.unpaired.sai"
        input2=$INPATH/$sample"_R2_001.unpaired.fastq.gz"
        output2=$OUTPATH/$sample"_R2_001.unpaired.sai"
        outfile=$OUTPATH/$sample"_pe.sam"
        echo $input1 $output1 $input2 $output2
        ./bwa mem $reference $input1 $input2 | gzip -3 > $outfile
        echo $outfile
done

has output like

[M::bwa_idx_load_from_disk] read 0 ALT contigs
[W::bseq_read] the 2nd file has fewer sequences. 
[M::process] read 4726 sequences (334488 bp)... 
[W::bseq_read] the 2nd file has fewer sequences.
[M::mem_pestat] # candidate unique pairs for (FF, FR, RF, RR): (0, 654, 734, 0)
[M::mem_pestat] skip orientation FF as there are not enough pairs
[M::mem_pestat] analyzing insert size distribution for orientation FR... 
[M::mem_pestat] (25, 50, 75) percentile: (2337,4712, 7183) 
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 16875)
[M::mem_pestat] mean and std.dev: (4752.09,2805.37)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 21721)
[M::mem_pestat] analyzing insert size distribution for orientation RF... 
[M::mem_pestat] (25, 50, 75) percentile: (2388,4788, 7274) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 17046) [M::mem_pestat] mean and std.dev: (4832.84, 2813.06)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 21932) [M::mem_pestat] skip orientation RR as there are not enough pairs [mem_sam_pe] paired reads have different names:
"M06108:35:000000000-J52R8:1:1101:9702:1150",
"M06108:35:000000000-J52R8:1:1101:19124:1560"

Is that somehow related with low quality of reads/wrong trimmommatic tool, or I just have to recheck the spelling of bname and sample variables?