Targeted RNA-sequencing aims to focus coverage on areas of interest that are inadequately sampled in standard RNA-sequencing experiments. In a recent study, researchers from Cornell University present a novel approach for targeted RNA-sequencing that uses OligoMix pools of reverse transcription primers to enable sequencing enrichment at user-selected locations across the genome. Multiplexed Primer Extension sequencing, or MPE-seq. They demonstrate this approach by targeting pre-mRNA splice junctions in S. cerevisiae, revealing high-precision detection of splice isoforms, including rare pre-mRNA splicing intermediates.

Array-based oligonucleotide synthesis can be used to generate primer pools for use in MPE-seq

LC Sciences

Obtaining adequate amounts of primer pools for MPE-seq from cost-effective array based oligonucleotide synthesis can be achieved in four steps: (1) PCR amplification of the oligonucleotide synthesis pool using a 5’ blocked sense primer and a biotinylated antisense primer. (2) Restriction digestion to cleave off the PCR primer handle. (3) Lambda exonuclease digestion of free 5’ ends. (4) Streptavidin purification of biotinylated PCR handle. The unbound fraction is the desired primer pool product.

 

Reference
H Xu, BJ Fair, Z Dwyer, M Gildea, JA Pleiss (2018) Multiplexed Primer Extension Sequencing Enables High Precision Detection of Rare Splice Isoforms bioRxiv. doi: 10.1101/331629 [abstract]

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