ORF Capture-Seq as a versatile method for targeted identification of full-length isoforms.
Gloria M SheynkmanKatharine S TuttleFlorent LavalElizabeth TsengJason G UnderwoodLiang YuDa DongMelissa L SmithRobert SebraLuc WillemsTong HaoMichael A CalderwoodDavid E HillMarc VidalPublished in: Nature communications (2020)
Most human protein-coding genes are expressed as multiple isoforms, which greatly expands the functional repertoire of the encoded proteome. While at least one reliable open reading frame (ORF) model has been assigned for every coding gene, the majority of alternative isoforms remains uncharacterized due to (i) vast differences of overall levels between different isoforms expressed from common genes, and (ii) the difficulty of obtaining full-length transcript sequences. Here, we present ORF Capture-Seq (OCS), a flexible method that addresses both challenges for targeted full-length isoform sequencing applications using collections of cloned ORFs as probes. As a proof-of-concept, we show that an OCS pipeline focused on genes coding for transcription factors increases isoform detection by an order of magnitude when compared to unenriched samples. In short, OCS enables rapid discovery of isoforms from custom-selected genes and will accelerate mapping of the human transcriptome.
Keyphrases
- genome wide
- genome wide identification
- bioinformatics analysis
- dna methylation
- single cell
- rna seq
- endothelial cells
- transcription factor
- copy number
- small molecule
- genome wide analysis
- cancer therapy
- high resolution
- loop mediated isothermal amplification
- dna binding
- fluorescence imaging
- single molecule
- high density
- genetic diversity
- binding protein