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Related Concept Videos

Ribosome Profiling02:24

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Ribosome profiling or ribo-sequencing is a deep sequencing technique that produces a snapshot of active translation in a cell. It selectively sequences the mRNAs protected by ribosomes to get an insight into a cell’s translation landscape at any given point in time.
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Splicing is the process by which eukaryotic RNA is edited before its translation into protein. The RNA strand transcribed from eukaryotic DNA is called the primary transcript. The primary transcripts that become mRNAs are called precursor messenger RNAs (pre-mRNAs). Eukaryotic pre-mRNA contains alternating sequences of exons and introns. Exons are nucleotide sequences that code for proteins, whereas introns are the non-coding regions. In RNA splicing, introns are removed and exons are bonded...
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Identification of Alternative Splicing and Polyadenylation in RNA-seq Data
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Identification of Protein Isoforms Using Reference Databases Built from Long and Short Read RNA-Sequencing.

Aidan P Tay1,2,3, Joshua J Hamey1, Gabriella E Martyn1

  • 1School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, New South Wales 2052, Australia.

Journal of Proteome Research
|May 25, 2022
PubMed
Summary

Long read, direct RNA sequencing significantly improves the discovery of protein isoforms by revealing transcripts missed by short-read methods. This enhances proteomic identification and database generation for novel protein variants.

Keywords:
IlluminaMS/MSOxford Nanopore TechnologyRNA-seqalternative splicingdirect RNA-sequencinglong read RNA sequencingprotein isoformproteogenomics

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Area of Science:

  • Genomics
  • Proteomics
  • Molecular Biology

Background:

  • Alternative splicing generates diverse protein isoforms with varied functions.
  • Accurate identification of these isoforms is crucial for understanding cellular processes and development.
  • Existing short-read sequencing methods may miss a significant portion of transcriptomic and proteomic diversity.

Purpose of the Study:

  • To evaluate the utility of long read, direct RNA sequencing for identifying protein isoforms in human K562 cells.
  • To compare the performance of long-read RNA sequencing with traditional short-read RNA sequencing for transcript and isoform discovery.
  • To assess the impact of long-read RNA sequencing-based databases on proteomic identification of protein isoforms.

Main Methods:

  • Utilized long read, nanopore-based, direct RNA sequencing (RNA-seq) on human K562 cells.
  • Compared identified transcripts and alternatively spliced genes with data from Illumina-based short read RNA-seq.
  • Co-analyzed proteomic and transcriptomic data using custom databases derived from long-read and short-read data.
  • Generated peptide evidence to validate identified protein isoforms.

Main Results:

  • Long direct reads identified substantially more Ensembl transcripts and alternatively spliced genes than short paired-end reads.
  • A significant number of peptides, proteins, and protein isoforms were identified using long-read-based databases but missed with Illumina-derived databases.
  • Unequivocal peptide evidence confirmed novel protein isoforms not present in reference or short-read-derived databases.

Conclusions:

  • Long read, direct RNA sequencing enhances the discovery of protein isoforms compared to short-read methods.
  • This approach improves the comprehensiveness of reference databases for proteomic analysis.
  • Long-read RNA sequencing is valuable for identifying novel protein isoforms and expanding our understanding of the proteome.