RNA-Seq Analysis

RNA-Seq Analysis

RNA-Seq, gene product abundance.

Understanding what transcriptome is present in a tissue or cell type is crucial to any functional genomic analysis, and many reviews on the topic of measuring RNA with next-generation sequencing technology abound [1].

Here is one protocol we have used at the Neuro: there are many others, and we encourage you to discuss alternatives with us.

Short description of the workflow and its purpose:

This workflow takes as input RNA sequence reads in FASTQ format and uses Salmon to perform a “pseudo-alignment.” Pseudo-alignment consists of mapping reads against a reference transcriptome index and quantifying the expression of transcripts.  Transcript quantification and variance estimation are generated by bootstraps sampling and modelling sample-specific parameters and biases using a Variational Bayesian EM algorithm [2].

  • Original Link if a similar workflow exists in GenPipes (using kalisto) [3]
  • Workshop notes from the Griffith lab at Wash U (St-Louis): Intro to RNAseq Analysis [4]

Bibliography:

  1. RNA sequencing: the teenage years. Stark R, Grzelak M, Hadfield J. Nat Rev Genet. 2019 Nov;20(11):631-656. DOI 10.1038/s41576-019-0150-2 PMID: 31341269
  2. Alignment and mapping methodology influence transcript abundance estimation. Srivastava A, Malik L, Sarkar H, Zakeri M, Almodaresi F, Soneson C, Love MI, Kingsford C, Patro R. Genome Biol. 2020 Sep 7;21(1):239. DOI 10.1186/s13059-020-02151-8 PMID: 32894187 
  3. GenPipes: an open-source framework for distributed and scalable genomic analyses. Bourgey M, Dali R, Eveleigh R, Chen KC, Letourneau L, Fillon J, Michaud M, Caron M, Sandoval J, Lefebvre F, Leveque G, Mercier E, Bujold D, Marquis P, Van PT, Anderson de Lima Morais D, Tremblay J, Shao X, Henrion E, Gonzalez E, Quirion PO, Caron B, Bourque G. Gigascience. 2019 Jun 1;8(6):giz037. DOI:10.1093/gigascience/giz037. PMID: 31185495  
  4. Obi L. Griffith, Malachi Griffith, Zachary L. Skidmore, Huiming Xia, Erica Barnell, Sharon Freshour, Jeff Szymanski, and Katie Campbell. Wahington University, St-Louis, MO, USA. https://griffithlab.org/ - Intro to RNAseq Analysis.