Does It Makes Sense To Assemble All My Rna-Seq Samples As Though They Were One?

Question

[Ryan Thompson]

I am doing reference-guided transcript assembly and quantitifaction using Tophat and Cufflinks. Currently, I am running Cufflinks on each sample individually and then merging the assemblies with the cuffmerge tool. Would I get a better assembly by combining all the BAM files from all my samples into one and running Cufflinks on that? Or should it theoretically produce an identical assembly?

(Obviously the second strategy will yield nonsense FPKM values, but I can always requantify against each BAM file individually, so that's not a problem.)

Answer 1

[Bioinfosm]

I guess using all samples to make a topHat assembly would not make sense. I have seen a lot of groups combine the assembly from different samples, and then use that as the guide for tophat to get a better handle on transcripts (similar to what you are suggesting). Using data from all samples could actually confuse the assembly and yield inefficient transcript models to use with topHat!

[Nicolas Rosewick]

Even if the samples are from the same biological group ?

[Bioinfosm]

Unless it is the same sample, you might be better off to run independent tophat analysis to find all the transcripts separately. Then there is always the option of combining all those to form the inclusive 'transcriptome'

Answer 2

[Nicolas Rosewick]

Hi,

Little additional question : Is it a good idea if the samples are from the same biological group ?

Thanks

N.