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Dear colleagues,
Thanks for this source of information that is Seqanswers, and thanks to many experts that share their knowledge and time with us, the unexperienced.
I adress you to ask for help about a topic, splicing variant detection, that has been addressed before. Although I feel not quite clearly.
I am working with a splicing mutant in Arabidopsis, and I have the same common problem that other users have reported with cuffdiff
My issue is that I cannot get cuffdiff v2.1.1 to perform the splicing tests, even afters etting the --min-reps-for-js-test option
I have RNAseq samples for 2 controls and 3 mutants, but either if I run 2controls VS 3mutants or 2controls VS 2mutants cuffdiff give and empty splicing.diff file.
I do get the isoform_exp.diff file. Although is very probable that such file does not contain all the information since it uses the isoforms reported in wt and my mutant would have abnormal ones
My pipeline is as follows:
so far I have managed to perform
Map with Tophat -> count with HTseq -> DEseq (gene expression differences detected)
Map with Tophat -> cufflinks -> cuffmerge -> ASTALAVISTA (abnormal splicing detected)
Now I want to know wich genes are the ones affected in the alternative splicing.
In addittion to that, it could be great if i can get some quantification too
The plan is to do:
Tophat -> cuffdiff -> cummeRbund ->SpliceR
The choice of SpliceR is because it recognizes Intron retention as one of the splicing forms and IR is the main splicing event in Arabidopsis
Other recommended tools are exon centered and perhaps not suitable for my needs
DEXseq: exon centred
DSGseq: exon centred
flipflop: isoform prediction, gives genomic ranges output
The commnad I have run so far is
cuffdiff -o ./cuffdiff_output -p 8 --FDR 0.05 --min-reps-for-js-test 2 -L wt,mutant ~/path_to_genome/TAIR10.gff wt1.bam,wt2.bam mutant1.bam,mutant2.bam,mutant3.bam
Any kind of help, hint or advice is greatly appreciated
Thanks for this source of information that is Seqanswers, and thanks to many experts that share their knowledge and time with us, the unexperienced.
I adress you to ask for help about a topic, splicing variant detection, that has been addressed before. Although I feel not quite clearly.
I am working with a splicing mutant in Arabidopsis, and I have the same common problem that other users have reported with cuffdiff
My issue is that I cannot get cuffdiff v2.1.1 to perform the splicing tests, even afters etting the --min-reps-for-js-test option
I have RNAseq samples for 2 controls and 3 mutants, but either if I run 2controls VS 3mutants or 2controls VS 2mutants cuffdiff give and empty splicing.diff file.
I do get the isoform_exp.diff file. Although is very probable that such file does not contain all the information since it uses the isoforms reported in wt and my mutant would have abnormal ones
My pipeline is as follows:
so far I have managed to perform
Map with Tophat -> count with HTseq -> DEseq (gene expression differences detected)
Map with Tophat -> cufflinks -> cuffmerge -> ASTALAVISTA (abnormal splicing detected)
Now I want to know wich genes are the ones affected in the alternative splicing.
In addittion to that, it could be great if i can get some quantification too
The plan is to do:
Tophat -> cuffdiff -> cummeRbund ->SpliceR
The choice of SpliceR is because it recognizes Intron retention as one of the splicing forms and IR is the main splicing event in Arabidopsis
Other recommended tools are exon centered and perhaps not suitable for my needs
DEXseq: exon centred
DSGseq: exon centred
flipflop: isoform prediction, gives genomic ranges output
The commnad I have run so far is
cuffdiff -o ./cuffdiff_output -p 8 --FDR 0.05 --min-reps-for-js-test 2 -L wt,mutant ~/path_to_genome/TAIR10.gff wt1.bam,wt2.bam mutant1.bam,mutant2.bam,mutant3.bam
Any kind of help, hint or advice is greatly appreciated