# Systematic evaluation of long- and short-read RNA-seq for human peripheral blood

## TL;DR

This study compares long-read and short-read RNA sequencing technologies using the same blood samples, highlighting their strengths in capturing different aspects of gene expression.

## Contribution

The study provides a systematic comparison of RNA-seq platforms using identical biological samples and standard analysis pipelines.

## Key findings

- Long-read sequencing better detects complex splicing events and full-length immune receptor sequences.
- Short-read sequencing shows superior accuracy for highly expressed genes and microarray concordance.
- Both platforms detect overlapping and unique transcriptomic features when using total RNA.

## Abstract

RNA sequencing (RNA-seq) technologies enable comprehensive transcriptomic profiling, yet systematic comparisons using identical biological samples remain limited. Here, we performed a multi-faceted comparison of long-read (PacBio) and short-read (Illumina) RNA-seq using the same RNA from peripheral blood cells of four healthy donors. Unlike prior studies that aggregate datasets from different sources, this study evaluates platform-dependent performance across gene expression, transcript variants, fusion genes, primary microRNAs (pri-miRNAs), and immune receptor complementarity-determining region 3 (CDR3) regions using widely available software, highlighting both reproducibility and accessibility. Long-read sequencing outperformed short-read sequencing in detecting complex alternative splicing events, novel transcript isoforms, and full-length immune receptor sequences, particularly immunoglobulin heavy chains, enhancing clonotype resolution. Both platforms captured largely overlapping pri-miRNAs and CDR3 sequences, but each also detected unique elements, demonstrating that total RNA can serve as a proxy for these specialized features when dedicated kits are not used. Short-read sequencing retained superior quantification accuracy for highly expressed genes and stronger concordance with microarray data. Collectively, our findings reveal the complementary strengths of long- and short-read RNA-seq and provide a practical framework for systematic, side-by-side comparison of transcriptomic features, emphasizing the benefits of using the same input material and standard analysis pipelines.

Graphical Abstract