RNA-seq Analisys Course

Appendix on Ribo-seq

Resource for Ribo-seq

Pipeline

Introduction: Ribo-Seq or ARTSeq

Ribosome profiling (Ribo-Seq), also called active mRNA translation sequencing (ARTseq), isolates RNA that is being processed by the ribosome in order to monitor the translation process (Ingolia et al., 2009). In this method, ribosome-bound RNA first undergoes digestion. The RNA is extracted, and the rRNA is depleted. The extracted RNA is reverse-transcribed to cDNA. Deep sequencing of the cDNA provides the sequences of RNAs bound by ribosomes during translation. This method has been refined to improve both the qualitative and quantitative nature of the results. Careful attention should be paid to:

  1. generation of cell extracts in which ribosomes have been faithfully halted along the mRNA they are translating in vivo;
  2. nuclease digestion of RNAs that are not protected by the ribosome, followed by recovery of the ribosome-protected mRNA fragments;
  3. quantitative conversion of the protected RNA fragments into a DNA library that can be analyzed by deep sequencing. The addition of harringtonine (an alkaloid that inhibits protein biosynthesis) causes ribosomes to accumulate precisely at initiation codons and assists in their detection.

  • Advantages:

    Reveals a snapshot with the precise location of ribosomes on the RNA More closely reflects the rate of protein synthesis than mRNA levels No prior knowledge of the RNA or open-reading frames (ORFs) is required Surveys the whole genome Can identify protein-coding regions

  • Disadvantages:

    Initiation from multiple sites within a single transcript makes it challenging to define all ORFs Does not provide the kinetics of translational elongation

Pipelines

You can see many information on ths site:

REF : https://www.bioconductor.org/packages/release/bioc/html/RiboProfiling.html

Pipeline 1