Problems aligning to a very small "genome"

[id0]

I was sequencing a transgenic gene product. Since it does not exist in any genome, I created a custom genome by using the expected sequence. I then used BWA to align to that genome.

The problem is that the results of the alignment look extremely messy. Here is a screenshot:


I've never seen anything like this for genomes like hg19 or mm9. I think the problem may be that this genome is just over 1kb in length. Is that too small? I know that there may be memory problem with large genomes and that different read lengths are not compatible with all aligners, but I didn't think there would be a problem with a genome too small. Are there some settings that I have to adjust or use a different aligner altogether?

[swbarnes2]

I think you messed up your command lines somewhere. Reads with so many mismatches shouldn't have aligned at all.

I've gotten IGV plots like that when I scrambled file names in the bwa samse or sampe step. So double-check that.

A small genome shouldn't be too big a problem, though you will get a few reads being forced to align there, when they really belong in a highly similar spot in the host genome. I don't think thsi will be a significant problem, though.

The most accurate way to do what you are doing is to align to a reference genome of whole host organism + your transgene. That would prevent the problem I described above, but it would take a lot longer. Your way should work, I'd just double-check to see if there was a silly error in your command line.

[id0]

I think you messed up your command lines somewhere. Reads with so many mismatches shouldn't have aligned at all.

You were right! That was my mistake. It turns out the F and R SAI files I was giving BWA were actually the same one. I was using the same command as always and assumed that could not be a problem. I forgot that the file names were slightly different this time. I thought the reference genome size was the only variable I introduced.

Apparently specifying the same SAI file twice does not generate any error and the alignment is still generated.