How To Interpret Cufflinks Results?

Question

[Lisanne]

Dear All,

I am using Tophat and Cufflinks to analyse RNA-seq data. Tophat, Cufflinks, cuffcompare and cuffdiff produce multiple results. I want to know if there is a way to interpret all these results. I am very interested in differential splicing and the visualization of this kind of information.

I am running the programs with all the defaults.

Thank you all!

Lisanne

Answer 1

[Malachi Griffith]

TopHat will produce a BAM file and junctions.bed, both of which can be visualized in Next-Gen Genome Browsers such as IGV and the Savant Genome Browser. The GTF file produced by Cufflinks can also be loaded in these browsers along with known gene annotations for comparison. Savant has a plugin specifically designed for Cufflinks.

For visualizing Cuffdiff output, you should also check out CummeRbund. This is an R package that is designed to aid and simplify the task of analyzing Cufflinks RNA-Seq output.

CummeRbund takes the various output files from a cuffdiff run and creates a SQLite database of the results describing appropriate relationships betweeen genes, transcripts, transcription start sites, and CDS regions. Once stored and indexed, data for these features, even across multiple samples or conditions, can be retrieved very efficiently and allows the user to explore subfeatures of individual genes, or genesets as the analysis requires. We have implemented numerous plotting functions as well for commonly used visualizations.

[bharata1803]

Hello, I am beginner to tophat - cufflinks combo for RNA-seq ddata analysis. My goal is basically to get the expression level of several genes that I have interest in. I checked the fpkm file from cufflinks with excel and tried to compare for a gene id from several experiment data and I find some result, around 500-700 value even though 1 data has almost 0 value. So, can I safely conclude that the gene expression level is around that or is there any other step to calculate some statistic testing to get the differential expression level? I am really confused with the manual from the cufflinks website and there is no real step-by-step from start to end about how to get the value. Now, I will try to visualize it in Savant Genome Browser but I still don't know what to do. Thank you.

[charbel.feghaly]

hello Mr Griffith,

I'am Facing a similar problem, the illumina rna seq results i had contain in the file g option novel transcripts for each of my patients but i dont have the .diff files to use them in IPA software and i have CUFF.ID for the genes and transcripts and also files such as genes.fpkm_tracking, and GTF files for transcripts. So what can i do with these files in order to view alternative splicing and their expression

thank you charbel

Answer 2

[Rm]

Cufflinks generates : transcripts.gtf : Its a GTF file you can visualise it in a genome browser (gbrowser ucsc etc) isoforms.fpkm_tracking : Expression values for the transcripts expressed genes.fpkm_tracking: Expression values for the genes expressed

Cuffdiff: uses all the bam files from tophat output and can compare (differential) across samples or group of samples and will generate similar files with suffix .diff ; you can use reported fold change information for comparing samples.