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

Area of Science:

• Genomics
• Molecular Biology
• Bioinformatics

Background:

• RNA sequencing (RNA-seq) is crucial for transcriptomic profiling.
• Systematic comparisons of long-read and short-read RNA-seq platforms using identical samples are limited.
• Understanding platform-specific performance is essential for accurate transcriptomic analysis.

Purpose of the Study:

• To conduct a multi-faceted, side-by-side comparison of long-read (PacBio) and short-read (Illumina) RNA-seq.
• To evaluate platform performance across gene expression, transcript variants, fusion genes, pri-miRNAs, and CDR3 regions.
• To provide a practical framework for comparing transcriptomic features using standardized pipelines and identical input material.

Main Methods:

• Utilized identical peripheral blood cell RNA from four healthy donors for both long-read and short-read RNA-seq.
• Employed widely available software for data analysis across platforms.
• Assessed performance in gene expression, alternative splicing, novel isoforms, fusion genes, pri-miRNAs, and immune receptor CDR3 sequences.

Main Results:

• Long-read RNA-seq demonstrated superior detection of complex alternative splicing, novel transcript isoforms, and full-length immune receptor sequences (e.g., immunoglobulin heavy chains), improving clonotype resolution.
• Both platforms captured overlapping pri-miRNAs and CDR3 sequences, with each identifying unique elements.
• Short-read RNA-seq showed higher quantification accuracy for highly expressed genes and greater concordance with microarray data.

Conclusions:

• Long-read and short-read RNA-seq technologies possess complementary strengths for transcriptomic profiling.
• Total RNA can be used as a proxy for pri-miRNA and CDR3 analysis when specialized kits are unavailable.
• This study provides a reproducible framework for comparing transcriptomic features, emphasizing the use of identical samples and standard analysis pipelines.