Question: Different reflections from the techincal replicates.
gravatar for Shaurya Jauhari
7 months ago by
Shaurya Jauhari40 wrote:

An experimental instance (mock-treated) was analyzed to have 24 upregulated genes (significantly differentially expressed). The technical replicate had none, which is apparently unusual. Is it all good here computationally or something went wrong? Could the biological provenance be questioned? I am using the tophat-cufflinks pipeline and have cross-checked my commands.

rna-seq • 339 views
ADD COMMENTlink written 7 months ago by Shaurya Jauhari40

Are you referring to pure technical replicates for sequencing (same library run in multiple lanes)? Then that result is not unusual. Technical replication is not needed for sequencing.

ADD REPLYlink written 7 months ago by genomax43k

You should know that the old 'Tuxedo' pipeline of Tophat and Cufflinks is no longer the "advisable" tool for RNA-seq analysis. The software is deprecated/ in low maintenance and should be replaced by HISAT2, StringTie and ballgown. See this paper: Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown. (If you can't get access to that publication, let me know and I'll -cough- help you.) There are also other alternatives, including alignment with STAR and bbmap, or pseudo-alignment using salmon.

ADD REPLYlink written 7 months ago by WouterDeCoster26k
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