Comparison of rRNA depletion methods for efficient bacterial mRNA sequencing

Area of Science:

• Molecular Biology
• Genomics
• Bioinformatics

Background:

• High-throughput RNA sequencing in prokaryotes requires deep sequencing for detecting weakly expressed genes.
• High ribosomal RNA (rRNA) and transfer RNA (tRNA) content in total RNA reduces the proportion of coding RNA reads.
• Efficient rRNA depletion is crucial for increasing sequencing depth and reducing costs, but the widely used RiboZero kit has been discontinued.

Purpose of the Study:

• To compare the efficiency of commercially available rRNA depletion kits (riboPOOLs, RiboMinus, MICROBExpress) against the discontinued RiboZero kit.
• To evaluate a self-developed rRNA depletion method using biotinylated probes and magnetic beads.
• To assess the impact of different rRNA depletion methods on sequencing depth and the detection of weakly expressed genes.

Main Methods:

• Comparison of three commercial rRNA depletion kits (riboPOOLs, RiboMinus, MICROBExpress) and a self-developed biotinylated probe method.
• Application of these methods to prokaryotic total RNA (E. coli) followed by RNA sequencing.
• Analysis of rRNA depletion efficiency, mRNA read counts, and detection of weakly expressed genetic elements.

Main Results:

• The self-made biotinylated probes and riboPOOLs demonstrated comparable rRNA depletion efficiency to the former RiboZero kit.
• RiboMinus and MICROBExpress showed lower rRNA depletion efficiencies compared to RiboZero, riboPOOLs, and the self-made method.
• Both the self-made probes and riboPOOLs significantly increased mRNA sequencing depth and improved the identification of weakly expressed genes.

Conclusions:

• Self-made biotinylated probes and riboPOOLs serve as effective replacements for the discontinued RiboZero kit for prokaryotic RNA sequencing.
• These methods enhance sequencing efficiency by reducing rRNA content and increasing the yield of mRNA reads.
• The developed in-house protocol allows for customized, species-specific depletion of rRNA, tRNA, or other RNA targets.