TopHat output and USCS genome browser question

sdarko · 07-27-2009, 08:45 AM

Hi folk! This is my first post and I'm quite new to next-gen sequencing analysis, so please be kind

Here is some background on my data:
mRNA from rat heart tissue was isolated and sequenced using a Heliscope from Helicos. I then converted the sms file generated by Helicos to a fasta file using a script in the Helisphere software. From there I used TopHat to look for splice junctions.

From what I can see, everything went fine. The TopHat output includes all the files that I would expect it to include. Now, when I tried to view the .BED and .WIG files using the UCSC genome browser I have problems.

Using the input found here --> http://genome.ucsc.edu/cgi-bin/hgGateway I tried to view my files after selecting genome->rat and the most recent assembly. I try to upload my .BED file and received the following error:
"Error File 'junctions.bed' - Unrecognized format line 2 of custom track: gi|34868215|ref|NW_047355.1|Rn11_WGA1875_4 1124643 1138858 JUNC00000001 2 - 1124643 1138858 255,0,0 2 11,10 0,14205 (note: chrom names are case sensitive)"

I am wondering if I'm even attempting to use the right tool to view the file or if there is some upstream problem?

Any help would be appreciated.

Thanks,
Sam

glh · 08-03-2009, 03:50 PM

The pre-built index for rat is built from NCBI contigs. In the first column of the bed file where it should be giving you a chromosome name you're getting the contig instead. You can build your own index from the UCSC fasta files with bowtie-build and it won't have this problem.

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07-27-2009, 08:45 AM	#1
sdarko Member Location: Bethesda, MD Join Date: Apr 2009 Posts: 51	TopHat output and USCS genome browser question Hi folk! This is my first post and I'm quite new to next-gen sequencing analysis, so please be kind Here is some background on my data: mRNA from rat heart tissue was isolated and sequenced using a Heliscope from Helicos. I then converted the sms file generated by Helicos to a fasta file using a script in the Helisphere software. From there I used TopHat to look for splice junctions. From what I can see, everything went fine. The TopHat output includes all the files that I would expect it to include. Now, when I tried to view the .BED and .WIG files using the UCSC genome browser I have problems. Using the input found here --> http://genome.ucsc.edu/cgi-bin/hgGateway I tried to view my files after selecting genome->rat and the most recent assembly. I try to upload my .BED file and received the following error: "Error File 'junctions.bed' - Unrecognized format line 2 of custom track: gi\|34868215\|ref\|NW_047355.1\|Rn11_WGA1875_4 1124643 1138858 JUNC00000001 2 - 1124643 1138858 255,0,0 2 11,10 0,14205 (note: chrom names are case sensitive)" I am wondering if I'm even attempting to use the right tool to view the file or if there is some upstream problem? Any help would be appreciated. Thanks, Sam

08-03-2009, 03:50 PM	#2
glh Junior Member Location: Kansas City, MO Join Date: Aug 2009 Posts: 2	The pre-built index for rat is built from NCBI contigs. In the first column of the bed file where it should be giving you a chromosome name you're getting the contig instead. You can build your own index from the UCSC fasta files with bowtie-build and it won't have this problem.