Pileup loosing consensus sequence?

[MatthewVC]

Hello,

I am doing a project involving re-sequencing mitochondrial genomes and am having some trouble with the SAM Tools pileup function in Galaxy.

Using Galaxy, I am mapping my Illumina reads onto a FASTA copy of the mitochondrial genome using BWA. This is to avoid my reads inappropriately being mapped onto the numerous places in the nuclear genome where fragments of the mitochondrial genome have integrated. This improves my coverage considerably (from roughly 50X when mapping reads to entire genome to 4000+ when mapping only to the mtDNA).

However, when I generate a pileup from the .bam file, the third column, which should have the consensus sequence, consists entirely of “N”s. This is preventing me from continuing with the bioinformatic analysis.

I wanted to see if anyone could offer any suggestions as to why my consensus sequence is lost.

Thanks,
Matt

[MatthewVC]

I figured it out a few minutes after posting this. I had used the "build in" reference genome when generating the pileup file instead of using the reference from my history that had also been used to map the reads. I guess I was too quick to post.

Matt