A low-cost library construction protocol and data analysis pipeline for Illumina-based strand-specific multiplex...

Area of Science:

• Genomics and Bioinformatics
• Molecular Biology

Background:

• Next-generation sequencing (NGS) technologies have revolutionized transcriptome exploration.
• Illumina sequencing platforms are widely used for gene expression analysis, but cost and throughput can be limiting.
• Existing RNA-sequencing (RNA-seq) protocols may not be optimal for high-throughput, low-cost applications.

Purpose of the Study:

• To develop a low-cost, robust protocol for preparing Illumina-compatible RNA-seq libraries.
• To establish a streamlined bioinformatics pipeline for RNA-seq data analysis, including differential gene expression and intron retention.
• To characterize the rice leaf transcriptome using the developed protocol and pipeline.

Main Methods:

• Designed balanced adapter sequences for multiplexing and incorporated dUTP for strand-specific library construction.
• Simplified RNA purification, fragmentation, and size-selection steps to increase throughput.
• Developed a customizable bioinformatics pipeline with significance tests for differential gene expression and intron retention analysis.

Main Results:

• Successfully generated Illumina-compatible RNA-seq libraries with improved efficiency and reduced cost.
• The developed pipeline enabled robust analysis of differential gene expression and intron retention events.
• Characterized the rice leaf transcriptome, identifying novel gene models and improving genome annotation.

Conclusions:

• The developed low-cost RNA-seq library preparation protocol and data analysis pipeline offer a robust and efficient solution for transcriptome studies.
• Strand-specific RNA-seq is advantageous for detecting anti-sense transcripts and intron retention events.
• This method has significant potential for improving genome annotation and facilitating gene expression research across various organisms.