A Systematic Benchmark of High-Accuracy PacBio Long-Read RNA Sequencing for Transcript-Level Quantification

Abstract

PacBio long-read RNA sequencing resolves transcripts with greater clarity than short-read technologies, yet its quantitative performance remains under-evaluated at scale. Here, we benchmark the high-throughput PacBio Kinnex platform against Illumina short-read RNA-seq using matched, deeply sequenced datasets across a time course of endothelial cell differentiation. Compared to Illumina, Kin-nex achieved comparable gene-level quantification and more accurate transcript discovery and transcript quantification. While Illumina detected more transcripts overall, many reflected potentially unstable or ambiguous estimates in complex genes. Kinnex largely avoids these issues, producing more reliable differential transcript expression (DTE) calls, despite a mild bias against short transcripts (shorter than 1.25 kb). When correcting Illumina for inferential variability, Kinnex and Illumina quantifications were highly concordant, demonstrating equivalent performance. We also benchmarked long-read tools, nominating Oarfish as the most efficient for our Kinnex data. Together, our results establish Kinnex as a reliable platform for full-length transcript quantification.