Week 3: Overview of RNA Seq - bcb420-2025/Izumi_Ando GitHub Wiki

⏰ - expected 1.5 hours : actual 3 hours

RNA Sequencing: the teenage years

Citation

Stark, R., Grzelak, M., & Hadfield, J. (2019). RNA sequencing: the teenage years. Nature reviews. Genetics, 20(11), 631–656. https://doi.org/10.1038/s41576-019-0150-2

Below is a short summary of the biggest takeaways from the paper. Smaller notes and highlights are on my iPad.
image
Screenshot of Figure 1 from the paper. Super intuitive.

overview of rna-seq evolution

  • outlines standard workflow: rna extraction, mRNA enrichment, cDNA synthesis, adaptor ligation, sequencing, and computational processing
  • tracks rna-seq’s growth from early bulk dge assays to current single-cell and spatial transcriptomics

technical challenges and solutions

  • discusses reproducibility issues due to variable library preps, software versions, and mapping ambiguities
  • short-read limitations: difficulty in resolving isoforms, problems with multi-mapped reads, transcript length biases
  • emerging long-read technologies (pacbio, nanopore, direct rna-seq) promise full-length transcript capture and detection of rna modifications (eg, m6a)

future directions

  • mentions how improved computational methods (isoform reconstruction, novel statistical models) are addressing limitations
  • emphasizes the continuous evolution and broadening of rna-seq applications beyond simple differential expression