GFF3 annotation file

[svl]

tophat --solexa1.3-quals Mus_musculus.NCBIM37.56 bic.txt -G mus_musculus.NCBIM37.56.gff3

Maybe you have to put all options before the index-base and reads. The manual says:

Usage: tophat [options]* <index_base> <reads1_1[,...,readsN_1]> [reads1_2,...readsN_2]

[Wei-HD]

Thanks Shurjo and svl!

I just want to provide my own junctions. Therefore I should write (I put data file: bic.txt, and index file as well as GFF3 file in the same folder):

tophat --solexa1.3-quals Mus_musculus.NCBIM37.56 bic.txt -G mus_musculus.NCBIM37.56.gff3

But I got en error: Error: you must set the mean inner distance between mates with -r
And my data is not pair-end data.

Thanks in advance!

[shurjo]

Neither before nor after but during the TopHat run :-). Use it with the -G option to Tophat

Like so:

tophat --mate-inner-dist 240 --mate-std-dev 25 ~/bin/bowtie/bowtie-0.12.1/indexes/hg18_inclusive 108971.read1.fa 108971.read2.fa -m 2 -p 4 -G /home/sensh/pipeline_test/GFF3/UCSC_knowngenes_hg18_tweaked.gff3

[svl]

I am not sure what exactly you want, but if you:

1) want to use a GFF file to find out about gene-expression, then tophat since version 1.0.12 says: "TopHat no longer calculates gene expression. Users interested in expression calculations should consider using Cufflinks for gene- and isoform-level expression calculations."

or

2) want to provide your own junctions, then search the manual for "Supplying your own junctions" and you'll see the "-G/--GFF <GFF3 file>" flag explained

svl

[Wei-HD]

Hi shurjo,

Thanks your reply. I already have the GFF3 file of mouse Mus_musculus.NCBIM37.56.gff3. But still have no clue when I should use this GFF file, before or after tophat running? sorry I am a bit confused.

Many thanks!

[shurjo]

This depends on how you want to treat your data. Giving TopHat the annotation file will force it look for the junctions contained therein even if it would not have considered them otherwise. There is a gtf2gff3 script available online (google the term) that you can use to make a GFF3 file for hg18 from the hg18 knownGenes table (which is downloadable in GTF format).

HTH,

Shurjo

[Wei-HD]

Hi All,

I want to consult everyone how to use this GFF3 annotation file. Since I use bowtie index in which the name of chromosome has been changed as "1","2","3"..., instead of "chr1","chr2","chr3"..., therefore I could not upload the junction to UCSC since the name is case sensitive.

I just read the tophat manual providing TopHat with an annotation file. But I don't know how to use this annotation file. I just simply run "--solexa1.3-quals", then got the result. Should I use this file before running this command?
Can some experienced SEQers give me some hints?

Really appreciate your help