**2.2. Transcriptome assembly**

#### *2.2.1. Reference-based transcriptome assembly and profiling*

Typically, in a reference-based transcriptome profiling study, the computational workflow starts with aligning the quality-checked reads to the reference genome or transcriptome using a suitable read aligner. The aligned reads are then used to quantitate the genomic features (genes/isoforms). The quantity of the features needs to be normalized before comparison between different experimental conditions. The normalized feature counts are then used for drawing statistical inference on their difference in expression between samples under study. Finally, the differentially expressed set of genes is processed to derive biological insights relevant to the experimental setup. The success of this analysis depends very much on decisions that the user takes while choosing reference genome, annotation, tools, and associ‐ ated parameter values at every step of the analysis. Steps involved in reference-based tran‐ scriptome assembly and analysis are described below.
