Rapid and Cost-Effective Digital Quantification of RNA Editing and Maturation in Organelle Transcripts by Oxford Nanopore Target-Indexed-PCR (TIP) Sequencing.

Abstract

RNA editing and maturation are critical regulatory mechanisms in plant organelles, yet their quantification remains technically challenging. Traditional Sanger sequencing lacks sensitivity and reproducibility, whereas advanced next-generation sequencing (NGS) approaches, such as rRNA-depleted long non-coding (lnc) RNA-seq or targeted amplicon-seq, involve high costs, complex workflows, and limited accessibility. To address these limitations, I developed a rapid and cost-effective long-read sequencing approach, termed Target-Indexed-PCR (TIP) sequencing, for digital quantification of RNA editing and intron retention events in targeted chloroplast transcripts. This method combines multiplexed high-fidelity PCR amplification with Oxford Nanopore sequencing and custom in-house Perl and Python scripts for streamlined data processing, including barcode-based demultiplexing, strand reorientation, alignment to a pseudo-genome, manual editing-site inspection, and splicing variant identification. As a proof of concept, TIP sequencing was applied to ndhB and ndhD transcripts, two chloroplast NAD(P)H dehydrogenase genes with the highest number of known editing sites in Arabidopsis thaliana. These transcripts were analyzed both in an inducible CRISPR interference (iCRISPRi) system targeting MORF2, a key RNA-editing factor, and in MORF2-YFP transgenic lines with either overexpression or co-suppression silencing. My findings revealed dose- and development-dependent impacts of MORF2 on C-to-U editing efficiency. Moreover, I identified an accumulation of intron-retaining ndhB transcripts, specifically in Dex-treated iCRISPRi lines and in both MORF2-YFP overexpression and silencing rosette leaves, indicating impaired chloroplast splicing functions when MORF2 expression is perturbed beyond an as-yet-undefined threshold. The platform achieves single-molecule resolution, robust reproducibility, and high read coverage across biological replicates at a fraction of the cost of lncRNA-seq. Collectively, this study establishes TIP sequencing as a versatile, scalable, and affordable tool for targeted post-transcriptional analysis in plant organelles and expands our understanding of MORF2's role in chloroplast RNA maturation. By overcoming key limitations of existing approaches, TIP sequencing enables routine, site-specific quantification of post-transcriptional regulation in organelles, including RNA editing and splicing, making it broadly accessible to researchers studying plastid biology, stress responses, and organelle-nucleus communication.