11 months ago by
This is a common scenario and you first have to choose what analysis strategy you want to pursue, whether you want to quantify expression levels of just annotated transcripts in a reference database or to quantify expression of all the transcripts in your experiment.
The first strategy is referred to as a reference based transcript evaluation pipeline, where after mapping reads, you quantify expression levels (and differential expression) relative to a reference transcriptome database (for example RefSeq). The second strategy is known as a de novo transcriptome reconstruction pipeline, where after mapping the reads are assembled into transcript structures (in the absence of a reference) to provide a comprehensive view of the transcriptome/sample. These de novo transcriptome structures are then provided to a tool like Cuffmerge or Stringtie-Merge to generate an experiment-specific transcriptome database, which is then used as a reference to generate expression values and differential expression estimates with tools like Cuffdiff and Featurecounts/Deseq2.
Have a look at the two strategies using the provided links. These will take you to step-by-step tutorials that will introduce you to both strategies and commonly used tools (with parameter recommendations) to achieve your goals.
Hope this helps!
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