BWA sam -> bam in pipeline

[Virome]

I am trying to use BWA mem to align pair-end reads to a single genome and pipe the sam file output to a bam and then a bedfile. The pipeline doesn't seem to be working.

It creates the output sam, bam and bed files but the bam and bed files are empty. It completes the alignment but an error message keeps appearing: Failed to open BAM file stdin.

Here is the code I am using:

bwa mem NC_005148.fa ./seqtk_1/subsample_1/sub_NC_005148_1.fq ./seqtk_1/subsample_1/sub_NC_005148_2.fq > NC_005148_BWA.sam | \
samtools view -S -h -u - | \
samtools sort - | \
samtools rmdup -s - | \
tee NC_005148_BWA_sorted.bam | \
bamToBed > NC_005148_BWA_sorted.bed

I am new to coding so any help would be appreciated.

[GenoMax]

This is probably based on this Biostars thread. Compare your command with the one there and you will notice that you are missing a couple of inputs (-).

[Virome]

That the thread that I used for the basis of the pipeline. I tried with the '- -' like it said in the thread the first time but it didn't work also.

Also, do I need to index the resulting bam files?

[GenoMax]

Since new version of samtools has slightly different options try this variation

bwa mem index R1.fq.gz R2.fq.gz | samtools view -Shu - | samtools sort - | samtools rmdup -s - - | tee NC_005148_BWA_sorted.bam | bamToBed > NC_005148_BWA_sorted.bed

When using pipes you should keep adding one additional operation to see where things are going wrong to debug starting at left side of the command.

[Virome]

Thank you. I've tried the code that was posted. It provided the sam file and a sorted bam file. However, it appears to be empty.

I used samtools flagstat on the sorted bam file to just to check the alignment. This was the output:
0 + 0 in total (QC-passed reads + QC-failed reads)
0 + 0 secondary
0 + 0 supplementary
0 + 0 duplicates
0 + 0 mapped (N/A : N/A)
0 + 0 paired in sequencing
0 + 0 read1
0 + 0 read2
0 + 0 properly paired (N/A : N/A)
0 + 0 with itself and mate mapped
0 + 0 singletons (N/A : N/A)
0 + 0 with mate mapped to a different chr
0 + 0 with mate mapped to a different chr (mapQ>=5)

If I use samtools on flagstat on the sam file. This is the output:
2000000 + 0 in total (QC-passed reads + QC-failed reads)
0 + 0 secondary
0 + 0 supplementary
0 + 0 duplicates
2000000 + 0 mapped (100.00% : N/A)
2000000 + 0 paired in sequencing
1000000 + 0 read1
1000000 + 0 read2
1999996 + 0 properly paired (100.00% : N/A)
2000000 + 0 with itself and mate mapped
0 + 0 singletons (0.00% : N/A)
0 + 0 with mate mapped to a different chr
0 + 0 with mate mapped to a different chr (mapQ>=5)


I am struggling to think what is happening to the bam file.

[GenoMax]

The command is working for me with Samtools v.1.4 and BedTools v.2.25.0. If you are using older version of these packages then try to upgrade.

As for debugging why the command is not working for you, start at the left and keep adding one pipe at a time to figure out which component fails. I assume you are using real index/file names as applicable in your case.