[Siva]

By default, the accepted_hits.bam file is co-ordinate sorted and does not need any further sorting to run cufflinks on it. Have you tried the accepted_hits.bam 'as is' generated by Tophat?

Also, would suggest you use the GTF from igenomes, if you are mapping to one of the 10 genomes available here:

http://tophat.cbcb.umd.edu/igenomes.html.

Hi Parthav
I work on maize genome sequence available from http://ftp.maizesequence.org/current/assembly. You will find the file <ZmB73_RefGen_v2.tar.gz>. On untaring we get separate files for each chromosome. So before making the bowtie index, I concatenated all the chr.fasta files. (10 chromosomes, 1 Mt, 1 Pt and 1 UNKNOWN...a total of 13). I also built another index with only the ten chromosomes (hoping that by getting rid of Mt and Pt, I might circumvent the issue)

Whatever I do, I still end up with

Error: sort order of reads in BAMs must be the same

when trying to run cufflinks.

I think it may be to do with all the pre-processing I am having to do with the genome files. I will have to talk to some one from maizesequence.org.

[syintel87]

By default, the accepted_hits.bam file is co-ordinate sorted and does not need any further sorting to run cufflinks on it. Have you tried the accepted_hits.bam 'as is' generated by Tophat?

Also, would suggest you use the GTF from igenomes, if you are mapping to one of the 10 genomes available here:

http://tophat.cbcb.umd.edu/igenomes.html.

Converting GFF to GTF is not necessary as you can use the 'genes.gtf' available within the igenomes directory structure of your genome of interest.

Also, highly advised to run tophat with a bowtie index that is built on the same genome, that you are providing the GTF file for, in the cufflinks run.

Thanks
Parthav

I have tried
- bowtie-build,
- both accepted_hits.bam and the sorted one,
- accepted_hits.sam (converted from accepted_hits.bam)
- GFF (same one was used in running tophat and cufflinks)
- GTF (converted from the GFF)

However, all of these combinations did not work, if gff/gtf is provided when running cufflinks.
Also, there is no genome that I need on http://tophat.cbcb.umd.edu/igenomes.html.
Thank you for your tips though.

[eieneg]

You must remove colons in the FASTA file and GFF also. Cufflinks and downstream programs of Tophat that use a SAM file get confused with the colons in the sequence (genome reference) names as it interferes in the SAM header.