Question: (Closed) cufflinks splicing analysis drosophila - 'notest' in output
0
gravatar for fing16
2.1 years ago by
fing160
United Kingdom
fing160 wrote:

Hi everyone,

I'm attempting to use cufflinks to examine alternative splicing in my RNAseq data from drosophila. I'm new to the analysis and running into some problems, I'd really appreciate any help offered.

I aligned my data to the dm6 reference genome from UCSC, using the R/BioConductor platform, with qAlign function in QuasR. I had no problems using these bam files to count reads and analyse differential expression in other related BioC packages.

When I try to use cufflinks to analyse alternative splicing in these bam files, I read that the output files can be blank if the reference genome lacks some features, so I updated my reference genome to prepare a .gff file with the correct features using cuffcompare:

cuffcompare -s . -CG -r dmel-all-r6.05.gff dmel-all-r6.05.gff 

This produces the file cuffcmp.combined.gtf, which I use with cuffdiff:

mkdir spliceresults

cuffdiff -o ./spliceresults cuffcmp.combined.gtf \

sample1.bam,sample2.bam,...,sampleN.bam \

sample1B.bam,sample2B.bam,...,sampleNB.bam

This produces an output file with thousands of genes, all of them have the status 'notest' in the splicing.diff output file. I am aware that problems can be caused by mismatch of the reference and data, but am I right in thinking that I have got over that initial problem with the cuffcompare command? If so, and my files match correctly, why am I getting this results? I read something about not having enough reads in the analysis produces the 'notest' output, but I cannot see that this would be the case here. I have used these bam files for other parts of the RNA analysis and these have worked fine, and I also have 30 biological replicates within each treatment.

I'm sure I am missing something obvious, but my lack of experience with splicing analysis and cufflinks is letting me down. Thanks very much in advance for any help or advice anyone can give me.

Fiona

cufflinks drosophila splicing • 567 views
ADD COMMENTlink modified 2.1 years ago • written 2.1 years ago by fing160

Ps: One simple guess: check for the "XS" tag in the BAM data. If missing, NOTEST can result. http://cole-trapnell-lab.github.io/cufflinks/cufflinks/index.html#cufflinks-input-files

For more help, please see a general question forum. Best! Jen, Galaxy team

ADD REPLYlink written 2.1 years ago by Jennifer Hillman Jackson23k
0
gravatar for Jennifer Hillman Jackson
2.1 years ago by
United States
Jennifer Hillman Jackson23k wrote:

Hello fing16!

We believe that this post is does not fit the main topic of this site.

For general bioinformatics questions, including tool line-command usage, perhaps try https://www.biostars.org? This Biostars forum is focused on Galaxy usage (http://galaxyproject.org). Thanks!

For this reason we have closed your question. This allows us to keep the site focused on the topics that the community can help with.

If you disagree please tell us why in a reply below, we'll be happy to talk about it.

Cheers!

ADD COMMENTlink written 2.1 years ago by Jennifer Hillman Jackson23k
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