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!
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!
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