Problems creating custom genome for short oligo

[Kory Plakos]

I'm having trouble creating a custom genome for a short oligo. I have a construct, under 80 nt in length, which I've sequenced and successfully aligned to a predicted sequence using Bowtie2. With Bowtie, I was able to create an index using a custom fasta file that looks like this:

>Custom_oligo

[<80 nt SEQUENCE]

The alignment proceeded quickly and without trouble. I've converted the files from sam to bam, sorted and indexed, and now I'm trying to visualize the data using IGV. I've attempted to create a custom genome following the instructions under "Creating a .genome file" on the Loading a Genome page, and it seems like it works, but when I load my .bam file, I get the following error message:

File: [filepath]/Custom_oligo_align.sorted.bam

does not contain any sequence names which match the current genome.

File:      Custom_oligo,

Genome:     

It looks like IGV doesn't see any sequence names in my fasta file.

Any suggestions to help with this problem?

Thanks!

Kory

[Jim Robinson]

Hi Kory,

This is curious.  First I would try just loading the fasta file directly using "Genome > Load from file" and select the fasta.    Then load your bam.   Report here the results and we'll take the next step.

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[Kory Plakos]

Thanks for the quick response! I got this error when loading from file:

An error occurred while accessing: [filepath]/igv/genomes/Custom_oligo.fasta.txt
Blank line at line number 7, followed by data line at 8, in contig null Blank lines are only allowed at the end of a contig     

 

I created this fasta using textedit on my mac, saved it as a .txt, then changed the file extension to .fasta. I originally didn't think this would be the problem since the Bowtie alignments worked well with this file, but now I'm not so sure.

Kory

[Jim Robinson]

Is the file small enough to attach?   The fasta indexer is fussy about format.

To view this discussion on the web visit https://groups.google.com/d/msgid/igv-help/aac79cbf-5a0d-495b-89b8-7f1baea6c194%40googlegroups.com.

[Kory Plakos]

I solved it! The problem was using TextEdit on my mac to work with the fasta file. When I tried copying and pasting the text into TextWrangler then saving as a .fasta extension, that file loaded into IGV with no problems and I was able to load my bam file and view my alignments.

Thanks for all the help!

Kory

[Jim Robinson]

OK, great. We've also had problem using TextEdit, it saves non-ascii
characters even when saving as "text".

Jim