March 2023

Differential Gene Expression Analysis Workflow


QC of aligned reads

  • Alignment Rate
  • Duplication Rate
  • Insert Size
  • Transcript coverage

QC of aligned reads - Alignment Rate

  • Depends on:
    • Quality of Reference Genome
    • Quality of library prep and sequencing
    • For human and mouse > 95%

QC of aligned reads - Duplication Rate

  • Human exome is ~30 Mb therefore there are < 30 million possible reads
  • Duplication rates in RNAseq can be > 40%

QC of aligned reads - Insert Size

  • Insert size is the length of the fragment of mRNA from which the reads are derived

QC of aligned reads - Transcript coverage


QC Goals

  • Ensure the experiment generated the expected data
  • Check is the sequencing depth and alignment rates are similar across samples
  • Identify poor alignment parameters (sample quality, library prep ?)
  • Discover contamination from another organism or from DNA
  • Identify biases present in the data