Native RNA or cDNA Sequencing for Transcriptomic Analysis: A Case Study on Saccharomyces cerevisiae.

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• Nanopore sequencing enables direct, amplification-free analysis of native RNA and complementary DNA (cDNA).
• Both native RNA sequencing and cDNA sequencing are cost-effective transcriptome profiling methods.
• Understanding the differences between these approaches is crucial for selecting appropriate long-read sequencing technologies.

Purpose of the Study:

• To compare native RNA sequencing and cDNA sequencing using matched samples.
• To evaluate the performance and characteristics of both sequencing methods.
• To guide researchers in choosing the best long-read sequencing method for their studies.

Main Methods:

• Matched native RNA and cDNA sequencing were performed on *Saccharomyces cerevisiae* under glucose and ethanol growth conditions.
• Comparative analysis of sequence yields, read quality, read length distribution, and mapping ability.
• Evaluation of differential gene expression and RNA modification detection.

Main Results:

• Library preparation for direct RNA sequencing was shorter than for direct cDNA sequencing.
• Sequence characteristics like yield, quality, and read length differed between the two methods.
• Differential gene expression analysis results were comparable between native RNA and cDNA sequencing.
• Direct RNA sequencing underestimated RNA modifications at the 5' end due to 3' bias.

Conclusions:

• Native RNA and cDNA sequencing provide comparable differential gene expression data.
• Direct RNA sequencing offers insights into RNA modifications but exhibits 5' end underestimation.
• This comprehensive evaluation aids researchers in selecting optimal long-read sequencing methods for transcriptome analysis.