samtools pileup after bowtie

[a11msp]

Hi everyone,

I'm new here - and new to short-read sequencing (although I can't say I'm new to bioinformatics...) and would appreciate your help with the following problem:

I'm using Illumina GA _sequence files to do bowtie alignments with a reference genome as follows:

$ bowtie -t --chunkmbs 1024 -p 14 -k 1 --best -y -S --solexa1.3-quals -S <ref-index> <illumina _sequence file> > s3_bowtie.sam

# reports 87.5% reads with at least one alignment

Convert SAM to BAM and generate a pileup:

$ samtools view -uS s3_bowtie.sam | ../../samtools/samtools sort - s3_bowtie
$ ./samtools pileup -vcf <REF.fa> s3_bowtie.bam > s3_bowtie.raw.pileup

When I open this file, I see virtually no positions matching the reference. Since the bowtie alignment generally worked, I'm suspecting that something went wrong at the samtools stage.

In fact, when I use eland alignments instead (the _export file that comes from the GA pipeline converted to SAM using samtools' export2sam.pl), I'm having the same problem.

Would greatly appreciate any ideas as to why this may be happening.

Many thanks!

[seq_GA]

May be try converting sam to bam using reference genome and then sort.

$/samtools import /Genome/hg18/hg18.fa.fai $file.sam $file.bam

[a11msp]

Ok, this has now been resolved. Turns out I was explicitly specifying the -v option with samtools pileup that excluded from the output all positions matching to the reference....