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High-Throughput Next Generation Sequencing

Volume 733 of the series Methods in Molecular Biology pp 93-103

Date:

Ribosomal RNA Depletion for Massively Parallel Bacterial RNA-Sequencing Applications

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Abstract

RNA-sequencing (RNA-Seq) is a digital display of a transcriptome using next-generation sequencing technologies and provides detailed, high-throughput view of the transcriptome. The first step in RNA-Seq is to isolate whole transcriptome from total RNA. Since large ribosomal RNA (rRNA) constitutes approximately 90% RNA species in total RNA, whole transcriptome analysis without any contamination from rRNA is very difficult using existing RNA isolation methods. RiboMinus purification method provides a novel and efficient method to isolate RNA molecules of the transcriptome devoid of large rRNA from total RNA for transcriptome analysis. It allows for whole transcriptome isolation through selective depletion of abundant rRNA molecules from total RNA. The rRNA depleted RNA fraction is termed as RiboMinus RNA fraction, which is enriched in polyadenylated RNA, nonpolyadenylated RNA, preprocessed RNA, tRNA, numerous regulatory RNA molecules, and other RNA transcripts of yet unknown function. Using RiboMinus method to isolate RiboMinus RNA results in up to 99.0% removal of 16S and 23S rRNA molecules from 0.5 to 10 μg total bacterial RNA based on Bioanalyzer analysis. It enables efficient whole transcriptome sequencing analysis without major contamination from highly abundant rRNA. Residual rRNA accounts for less than 10% of entire transcriptome based on both SOLiD and Genome Analyzer RNA-Seq data.

Key words

Transcriptome RNA-Seq Ribosomal RNA Bacteria 16S 23S Next-generation sequencing Ribominus Polyadenylation