Question: The right pipline with cufflinks, cuffmerge and cuffdiff
1
gravatar for seta
3.1 years ago by
seta1.2k
Sweden
seta1.2k wrote:

Hi all,

I'm trying to use cufflinks and related programs (cuffmerge and cuffdiff) to find differentially expressed genes and some splicing events between two conditions (3 replicate for each sample) for a plant. This is my first experience and just based on my reading, I decided to use cufflinks for transcript assembly along with GTF file as a guide (-g option in cufflinks) for each bam file, then merge all assembly files together and with reference annotation (GTF file) to make a single annotation as the input for cuffdiff. Could you please help me on the following issue:

1) is right the above pipeline? 2) The default value of "--overlap-radius" in cufflinks is 50, please kindly tell me to go ahead with default or reduce it (what number suggest)? 3) The experiment was done at 4 time points, so for cuffdiff analysis, I should just "-T" option in the command, is there anything to care about it?

Thanks so much

cuffdiff cufflinks annotation • 2.0k views
ADD COMMENTlink modified 3.1 years ago by WouterDeCoster43k • written 3.1 years ago by seta1.2k
0
gravatar for geek_y
3.1 years ago by
geek_y10k
Barcelona
geek_y10k wrote:

The two following resources helps you to understand more about the pipeline

http://www.nature.com/nprot/journal/v7/n3/full/nprot.2012.016.html

Informatics of RNA-Seq.

ADD COMMENTlink modified 3.1 years ago • written 3.1 years ago by geek_y10k
0
gravatar for WouterDeCoster
3.1 years ago by
Belgium
WouterDeCoster43k wrote:

Cufflinks and related programs (the "old" tuxedo tools) are superseded by HISAT2, Ballgown and stringtie, see http://www.nature.com/nprot/journal/v11/n9/full/nprot.2016.095.html (if you can't access that publication, sci-hub or let me know and I'll share)

ADD COMMENTlink written 3.1 years ago by WouterDeCoster43k

Thank you. I overviewed both papers, very helpful. I used STAR for mapping instead of tophat, and I thought the cufflinks package is enough appropriate for the rest of work. Could you please let me know the main superiority of StringTie over cufflinks package and if this software is suitable for surveying splicing events, actually, does it a similar work to cufflinks?

Thanks

ADD REPLYlink modified 3.1 years ago • written 3.1 years ago by seta1.2k
1

StringTie does almost exactly the same thing as cufflinks. We have found it a) to assemble few false positive transcripts b) Be an order of magnitude quicker and have a lower memory footprint than cufflinks.

ADD REPLYlink written 3.1 years ago by i.sudbery7.0k

Thank you friend. As I mentioned I used STAR for mapping so that cufflink can use it, using the below command:

STAR --runThreadN 8 --genomeDir /home/seta/wrkdir/software/star-2.5.2b/genome --sjdbGTFfile    /home/seta/wrkdir/software/star-2.5.2b/genome/anno.gtf --outSAMstrandField intronMotif --outFilterIntronMotifs RemoveNoncanonical --outSAMtype BAM SortedByCoordinate --outFileNamePrefix map1 --readFilesIn /home/seta/wrkdir/Sequensing_files/file_1.fastq.gz --readFilesCommand zcat

Could you please let me know if the bam file from the above command is appropriate for StringTie?

ADD REPLYlink written 3.1 years ago by seta1.2k

Yeah, I'm pretty sure the input requirements are the same as cufflinks.

ADD REPLYlink written 3.1 years ago by i.sudbery7.0k
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