A gene-specific RNA enrichment protocol for nanopore direct-RNA sequencing

Area of Science:

• Molecular Biology
• Genomics
• Bioinformatics

Background:

• Third-generation sequencing technologies like Oxford Nanopore direct-RNA sequencing enable native RNA analysis.
• Cellular RNA is dominated by ribosomal RNA and housekeeping transcripts, hindering the study of lowly expressed genes.
• Targeted sequencing offers a solution by enriching specific transcripts of interest.

Purpose of the Study:

• To present a detailed protocol for gene-specific RNA enrichment prior to Oxford Nanopore direct-RNA sequencing.
• To demonstrate the effectiveness of this enrichment protocol using the MYCN oncogene as a target.
• To improve the efficiency and coverage of sequencing for low-abundance RNA transcripts.

Main Methods:

• Development of a protocol utilizing biotinylated DNA capture probes for gene-specific RNA enrichment.
• In-solution hybridization of probes to total RNA, followed by stringent washes and RNA elution.
• Application of Oxford Nanopore direct-RNA sequencing to enriched and unenriched RNA samples.
• Bioinformatic analysis of sequencing data to assess enrichment efficiency and read mapping.

Main Results:

• The enrichment protocol successfully isolated MYCN gene-specific RNAs with a purification factor of 4.8 × 10^3.
• Following enrichment, 65% of primary mapped reads from direct-RNA sequencing aligned to MYCN transcripts.
• The protocol effectively removed unwanted RNA species, demonstrating robust target gene enrichment.

Conclusions:

• The developed gene-specific RNA enrichment protocol significantly enhances Oxford Nanopore direct-RNA sequencing efficiency.
• This method is highly effective for increasing read coverage of low-abundance or specific target genes.
• The protocol provides a valuable tool for detailed analysis of native RNA molecules, particularly for specific transcripts of interest.