RNA-seq Tophat2 errors

[LiamT]

Hi everyone

I'm rather new to this field, so am trying my best and have spent the better part of a day trying to get Tophat2 to work with RNA-seq data and the hg19 reference. I can smell that I'm close, but I just don't or can't see what is wrong with the way I'm running the program. I have installed everything as per instructions, the programs are all in the path, the indexes installed, and so I start with the following:

tophat2 -p 4 -o hivA --library-type fr-firststrand --keep-tmp --b2-very-sensitive $HOME/programs/bowtie2/hg19 $HOME/lincrna/fastq/A_1.fastq, $HOME/lincrna/fastq/A_2.fastq

which gives


[2012-05-09 09:20:23] Beginning TopHat run (v2.0.0)
-----------------------------------------------
[2012-05-09 09:20:23] Checking for Bowtie
Bowtie version: 2.0.0.5
[2012-05-09 09:20:23] Checking for Samtools
Samtools version: 0.1.18.0
[2012-05-09 09:20:23] Checking for Bowtie index files
[2012-05-09 09:20:23] Checking for reference FASTA file
[2012-05-09 09:20:23] Generating SAM header for /home/thompsonl/programs/bowtie2/hg19
Traceback (most recent call last):
File "/home/thompsonl/programs/tophat2/tophat", line 3778, in ?
sys.exit(main())
File "/home/thompsonl/programs/tophat2/tophat", line 3645, in main
params.read_params = check_reads_format(params, reads_list)
File "/home/thompsonl/programs/tophat2/tophat", line 1676, in check_reads_format
zf = ZReader(f_name, params.system_params)
File "/home/thompsonl/programs/tophat2/tophat", line 1629, in __init__
self.file=open(filename)
IOError: [Errno 2] No such file or directory: ''

Now obviously, it can't find something, but what exactly can't it find ? I've checked the logs in the output directory but that doesn't yield any additional info. I've also tried a precompiled and source version of tophat2, but it doesn't seem to make a difference.

Any suggestions for my myopia ?

Thanks
Liam

[rnaseek]

The error is complaining that it could not open the sequence file. I think you don't need that comma after the first fastq file.
" $HOME/lincrna/fastq/A_1.fastq,"

[LiamT]

Sigh..this seems to work now, thanks for clearing that up . Just a further question though, as the A_1.fastq and A_2.fastq are sequencing replicates should they instead be named A1_1.fastq A2_1.fastq ? Keeping in mind that this is single end RNA-seq not paired end.

[rnaseek]

I thought it was a Paired End run. Since it is not a paired end you might want to rename the files and try running with the comma.

[LiamT]

The system dislikes any combination of commas that I try, I'll try without a comma, use "--library-type fr-firststrand" in the command line and see if it worked afterwords. Thanks for the suggestions.

[rnaseek]

Since they are replicates (tech. reps?), you can run independently, compare and decide to merge the bam files later if needed.