Question: CuffDiff error, "Uncaught exception in exposed API method," job runs, but no results
0
igboin.2 • 0 wrote:
Hello,
I’ve run into a problem while analyzing some RNAseq data on usegalaxy.com. I am looking at bacterial and host gene expression during infection. The host is Drosophila and the bacterium is F. nucleatum ATCC 10953 (Fn). I made and sequenced 3 RNA libraries: Fn alone (RNA from in vitro grown bacteria), Drosophila alone (RNA from uninfected flies), Fn/Drosophila mix (RNA from Fn infected flies).
I aligned the Drosophila reads with Tophat and performed differential analysis (Drosophila alone vs. Drosophila/Fn mix) using Cuffdiff, with no problem.
Problem: I aligned the Fn reads with Bowtie, but when I try to perform differential analysis using Cuffdiff (Fn alone vs. Drosophila/Fn mix) I keep getting errors as follows:
1st time: I ran CuffDiff on BAM files generated by Bowtie (I didn't filter out unmapped reads).
I got the following error in red in the History: “An error occurred while getting updates from the server. Please contact Galaxy administrator if the problem persists.” I clicked on “details,” and saw the following (this is not the original, just the most recent one, dated Sept 3rd. I’ve run the analysis multiple times since August when it first happened and gotten the same error):
user username christinaigboin quota_percent 55 total_disk_usage 147709821229 nice_total_disk_usage 137.6 GB email igboin.22osu.edu
is_admin false
tags_used
model_class User
id a175da8e4b898694
source HistoryCOntents(7f397613db43f024,270)
xhr readyState 4
responseText {"err_msg": "Uncaught exception in exposed API method:", "err_code": 0}
responseJSON err_msg Uncaught exception in exposed API method:
err_code 0
status 500
statusText Internal Server Error
responseHeaders Server nginx/1.4.7
Date Tue, 03 Sep 2014 00:08:34 GMT
Content-Type application/json
Transfer-Encoding chunked
Connection keep-alive
Cache-Control max-age=0,no-cache,no-store
options data
parse true
The job still ran but every gene got a “NO TEST” under status and “0” under value_1 and value_2 (FPKM for each of the two samples/conditions being compared).
2nd time: Thinking it was a server issue, I repeated the exact same analysis. Again I got the error in red, the job ran, but every gene got a “NO TEST” under status and “0” under value_1 and value_2.
3rd time: I filtered out the unmapped reads from the Bowtie output, and repeated the analysis on BAM files containing only mapped reads. Again I got the error in red, the job ran, but every gene got a “NO TEST” under status and “0” under value_1 and value_2.
4th time: I changed the header of the fasta file, which was originally: “>gi|254302039|ref|NZ_CM000440.1| Fusobacterium nucleatum subsp. polymorphum ATCC 10953 chromosome, whole genome shotgun sequence”, to: “>NZ_CM000440.1”, in order to match what is written in the .gff file, under the Seqid column for each gene (“NZ_CM000440.1”). I performed the analysis using this updated fasta file and the BAM files containing only mapped reads. Again I got the error in red, the job ran, but every gene got a “NO TEST” under status and “0” under value_1 and value_2.
5th time (ran overnight Sept 2-3): I reran the analysis using the updated fasta file and SAM files containing only the mapped reads (did a BAM to SAM conversion). Again I got the error in red, the job ran, but every gene got a “NO TEST” under status and “0” under value_1 and value_2.
The Cufflinks page says that Cuffdiff can be used with RNA-seq data from bacteria (http://cufflinks.cbcb.umd.edu/faq.html#bact), so I am not sure what I am doing wrong.
I appreciate any help you can give me.
Thanks,
Christina
ADD COMMENT
• link
•
modified 4.2 years ago
by
Jennifer Hillman Jackson ♦ 25k
•
written
4.2 years ago by
igboin.2 • 0
