Problem with Tophat and Cufflinks

[gongchang]

It is my first time working with RNA-Seq data, and I am having problems with Tophat and Cufflinks. Hope someone out there can give me some suggestions for the problems I encountered.

Thanks a lot,

Jie

1) Problems with tophat:

I downloaded the mouse GTF file and converted it to gff3 file ,using the script mentioned in a post from this forum, and also put the fasta file generated from the bowtie index to the folder where the bowtie indexes are.

Here are the shell script I used:
./tophat -G mouse.gff3 /path/to/indexes/ m_musculus input.fasta

And the the following output was generated:
[Wed Apr 28 18:59:09 2010] Beginning TopHat run (v1.0.11)

-----------------------------------------------

[Wed Apr 28 18:59:09 2010] Preparing output location ./tophat_out/

[Wed Apr 28 18:59:09 2010] Checking for Bowtie index files

[Wed Apr 28 18:59:09 2010] Checking for reference FASTA file

[Wed Apr 28 18:59:09 2010] Checking for Bowtie

Bowtie version: 0.12.2.0

[Wed Apr 28 18:59:09 2010] Checking reads

seed length: 37bp

format: fasta

[Wed Apr 28 18:59:15 2010] Reading known junctions from GFF file


And then the cursor went back to the system console, and the program just stopped. Although there was a folder called “tophat_out” generated, but no real output data.

Any parameters I need to adjust?

2)Problem with Cufflinks:
The system is “Ubuntu linux”.

First of all, the binary cufflinks(X86 linux version) cannot be executed. I assume it is due to the “boost” library being incorrectly installed.

Second, I tried to compile cufflinks and received error some messages when “sudo make” was executed:

Anybody has experience with the "libboost" compilation/installation? Thanks~

[john_mu]

I can possibly help you with your second problem...

The "boost" library is only needed if you want to build cufflinks from scratch. If you just want to run the binary, you don't need to have boost installed.

Please check you have downloaded the correct version. I can run it fine on Linux x86.

[sjm]

I think I had similar issues with TopHat when I was first using it a year or so ago. Check the chromosome descriptors in your gff3 file and make sure they match those in your bowtie index (you can look in the fasta file you've made, or run the bowtie-inspect command). If these don't match, I think you run into this problem.

Also, are you aware that the tophat -G function has been deprecated in recent versions of the program, and that the authors now recommend the use of an additional module (Cufflinks) which performs similar functions, but uses gtf files (which are easier to find)? This stuff has been recently published (Trapnell et al, Nat. Biotechnol. a month or so ago).