Different reflections from the techincal replicates.

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6.7 years ago

Shaurya Jauhari ▴ 50

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 • 1.2k views

ADD COMMENT • link 6.7 years ago by Shaurya Jauhari ▴ 50

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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 REPLY • link 6.7 years ago by GenoMax 141k

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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 REPLY • link 6.7 years ago by WouterDeCoster 47k

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