RNA-Seq: Differential Expression Analysis

Be careful about experimental design : avoid putting all the
replicates in the same lane, using the same barcode for the
replicates, putting different number of samples in lanes etc...
Non- uniformity of the per base read distribution (Illumina Random
Hexamer Priming bias visible on the 13 first bases)
Bias hierarchy : biological condition concentration run/flowcell lane
At equivalent expression level, a long gene will have more reads than a short one.
Non random coverage along the transcript.
Multiple hit for some reads alignments.

Keywords: Differential Expression, RNA-seq, transcriptomics

RNA-Seq: Differential Expression Analysis https://tess.elixir-europe.org/materials/rna-seq-differential-expression-analysis Be careful about experimental design : avoid putting all the replicates in the same lane, using the same barcode for the replicates, putting different number of samples in lanes etc... Non- uniformity of the per base read distribution (Illumina Random Hexamer Priming bias visible on the 13 first bases) Bias hierarchy : biological condition concentration run/flowcell lane At equivalent expression level, a long gene will have more reads than a short one. Non random coverage along the transcript. Multiple hit for some reads alignments. Differential Expression, RNA-seq, transcriptomics