The number of studies using third-generation sequencing utilizing Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) is rapidly increasing in many different research areas. Among them, plant full-length single-molecule transcriptome studies have mostly used PacBio sequencing, whereas ONT is rarely used. Therefore, in this study, Yangzhou University researchers examined ONT RNA sequencing methods in plants. They performed a detailed evaluation of reads from PacBio, Nanopore direct cDNA (ONT Dc), and Nanopore PCR cDNA (ONT Pc) sequencing including characteristics of raw data and identification of transcripts. In addition, matched Illumina data were generated for comparison.
ONT Pc showed overall better raw data quality, whereas PacBio generated longer read lengths. In the transcriptome analysis, PacBio and ONT Pc performed similarly in transcript identification, simple sequence repeat analysis, and long non-coding RNA prediction. PacBio was superior in identifying alternative splicing events, whereas ONT Pc could estimate transcript expression levels.
Context-specific errors of a PacBio subreads, b ONT Dc 1D reads, and c ONT Pc 1D reads. The error types shown are insertions, deletions, and mismatches. For insertions (deletions), the large base above the plot indicates the inserted (deleted) base. For mismatch errors, the large base to the left (above) indicates the expected (observed) base. Blocks of coloured tiles show the error frequency in specific contexts for each error; the small base to the left (above) indicates the base preceding (following) the error. Error frequency is plotted on separate scales for insertions, deletions, and mismatches