The right pipline with cufflinks, cuffmerge and cuffdiff

Question

[seta]

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

Answer 1

[GouthamAtla]

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.

Answer 2

[WouterDeCoster]

Cufflinks and related programs (the "old" tuxedo tools) are superseded by HISAT2, Ballgown and stringtie, see http://www.nature.com/nprot/journal/v11/n9/full/nprot.2016.095.html (if you can't access that publication, sci-hub or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

[seta]

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

[i.sudbery]

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.

[seta]

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?

[i.sudbery]

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