Accurate expression quantification from nanopore direct RNA sequencing with NanoCount

Area of Science:

• Genomics
• Molecular Biology
• Bioinformatics

Background:

• Accurate gene and isoform expression quantification is vital for understanding cellular functions, differentiation, and disease mechanisms.
• Direct RNA sequencing (DRS) offers potential advantages over traditional methods by avoiding RNA fragmentation, cDNA synthesis, and PCR.

Purpose of the Study:

• To develop and validate a tool (NanoCount) for fast and accurate transcript isoform quantification in DRS data.
• To characterize the capability of DRS in identifying differential gene and isoform expression in complex organisms.

Main Methods:

• Development of NanoCount, a novel tool for DRS data analysis.
• Application of NanoCount to synthetic RNA controls and human SH-SY5Y cell differentiation models.
• Comparative analysis of NanoCount performance against existing methods.

Main Results:

• NanoCount demonstrated superior performance in transcript isoform quantification compared to similar methods.
• DRS successfully quantified RNA expression and identified differential expression of genes and isoforms in differentiating SH-SY5Y cells.
• Differential expression analysis revealed 231 genes, 333 isoforms, and 27 isoform switches between undifferentiated and neuron-like cells, with upregulated genes linked to neurogenesis.

Conclusions:

• DRS, enhanced by NanoCount, accurately quantifies RNA expression and isoform changes.
• DRS is capable of identifying biologically relevant differential gene and isoform expression, including novel isoforms.
• The findings establish DRS as a powerful tool for studying complex biological processes and diseases at the transcriptomic level.