How to convert GTF to RefFlat?

[feralBiologist]

My use case is as follows:

I have aligned RNA-Seq data using tophat2 and a GTF file I got from Ensembl. Now I would like to produce alignment stats (number of reads mapping to exons, introns, intergenic regions) and, it seems, the CollectRnaSeqMetrics script from the Piccard toolset would be the most straight-forward to use. However, it requires annotation in RefFlat format (why on Earth there have to be so many different annotation formats??). So now I would like to convert GTF to RefFlat format. There is, apparently, a tool called gtfToGenePred which is supposed to do this but I could not find a manual or documentation for it. Can anyone provide info on converting GTF files to RefFlat format? Alternatively, can you suggest a way I can get the alignment stats using a GTF annotation file?

[dpryan]

If you just run gtfToGenePred without any arguments then it prints its usage instructions. This is by far the simplest method to generate the file you want.

[feralBiologist]

If you just run gtfToGenePred without any arguments then it prints its usage instructions. This is by far the simplest method to generate the file you want.

Thanks. I tried it but it does not seem to work:

localhost:Downloads nnikolo$ ls -lt gtf*
-rwxr-xr-x@ 1 nnikolo  staff  12944032 19 Feb 12:18 gtfToGenePred
localhost:Downloads nnikolo$ ./gtfToGenePred
-bash: ./gtfToGenePred: cannot execute binary file
localhost:Downloads nnikolo$ gtfToGenePred
-bash: /Users/nnikolo/Downloads/gtfToGenePred: cannot execute binary file

[dpryan]

chmod a+x gtfToGenePred

[GenoMax]

@feralBiologist: Looks like you may not have download the correct binary (or did you compile this yourself)? What OS are you using?

[sarvidsson]

chmod a+x gtfToGenePred

[B]-rw[B]x[/B]r-[B]x[/B]r-[B]x[/B][/B]@ 1 nnikolo  staff  12944032 19 Feb 12:18 gtfToGenePred

Doesn't seem to be the problem

[dpryan]

Yeah, I expect that GenoMax is correct and the wrong executable was downloaded. I should note that I just downloaded and tested that program on a GTF file on my workstation (running Ubuntu) and it worked fine.

[feralBiologist]

@feralBiologist: Looks like you may not have download the correct binary (or did you compile this yourself)? What OS are you using?

I am on MacOS X. Actually, I could not find a proper download page - just opening one of the Google search results ended up automatically downloading the binary.

[sarvidsson]

What about this one? http://hgdownload.cse.ucsc.edu/admin.../gtfToGenePred

[feralBiologist]

What about this one? http://hgdownload.cse.ucsc.edu/admin.../gtfToGenePred

Thanks - I think it's robots.txt that UCSC put which prevented Google from displaying that site.

[feralBiologist]

I ran into troubles again. I was able to run gtfToGenePred and it produced a refFlat file from the Ensembl GTF file. However, when I tried using it with Picard's CollectRnaSeqMetrics I got

Exception in thread "main" picard.annotation.AnnotationException: Wrong number of fields in refFlat file mm75.genePred at line 1
        at picard.annotation.RefFlatReader.load(RefFlatReader.java:80)
        at picard.annotation.RefFlatReader.load(RefFlatReader.java:66)
        at picard.annotation.GeneAnnotationReader.loadRefFlat(GeneAnnotationReader.java:37)
        at picard.analysis.CollectRnaSeqMetrics.setup(CollectRnaSeqMetrics.java:105)
        at picard.analysis.SinglePassSamProgram.makeItSo(SinglePassSamProgram.java:98)
        at picard.analysis.SinglePassSamProgram.doWork(SinglePassSamProgram.java:53)
        at picard.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:187)
        at picard.cmdline.PicardCommandLine.instanceMain(PicardCommandLine.java:95)
        at picard.cmdline.PicardCommandLine.main(PicardCommandLine.java:105)

So, gtfToGenePred has, apparently, produced an invalid file. Has anyone, actually, used gtfToGenePred successfully?

[GenoMax]

While you wait for someone to sort out the refFlat issue you could try this tool: http://rseqc.sourceforge.net/

Hopefully will get you stats similar to what you are looking for.

[feralBiologist]

Has anyone, actually, used gtfToGenePred successfully?

If anyone is interested - here is the GTF I used, the refFlat produced, and here are the command line arguments I used for CollectRnaSeqMetrics:

java -jar picard.jar CollectRnaSeqMetrics INPUT=sample.sam REF_FLAT=mm75.genePred STRAND=NONE MINIMUM_LENGTH=100 CHART=chart.out OUTPUT=accepted_hits_sam.stat

I'd be interested if anyone can make CollectRnaSeqMetrics work using these input files.

[GenoMax]

Based on the answer Pierre provided: https://www.biostars.org/p/107779/

Try your luck with this refFlat.txt file: http://hgdownload.cse.ucsc.edu/golde...refFlat.txt.gz

Note: If you need Ensembl ID's then this would not help. At least this file does not produce an error with CollectRnaSeqMetrics.

[feralBiologist]

Based on the answer Pierre provided: https://www.biostars.org/p/107779/

Try your luck with this refFlat.txt file: http://hgdownload.cse.ucsc.edu/golde...refFlat.txt.gz

Note: If you need Ensembl ID's then this would not help. At least this file does not produce an error with CollectRnaSeqMetrics.

Unfortunately, I did quite a bit of work already using the Ensembl GTF - I would have to start afresh and discard all the work done so far as I can't be certain that the Ensembl version I am using (75) has an exact UCSC equivalent. Seems to me to be too much of a hassle if the only bit missing is the stats on intergenic regions and introns.