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Organism-specific depletion of highly abundant RNA species from bacterial total RNA

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Engelhardt, Florian ; Tomasch, Jürgen ; Häussler, Susanne. / Organism-specific depletion of highly abundant RNA species from bacterial total RNA. In: Access Microbiology. 2020 ; Vol. 2, No. 10. pp. acmi000159.

Bibtex

@article{95d51b2e24d94dc9a52cd62a9c5554de,
title = "Organism-specific depletion of highly abundant RNA species from bacterial total RNA",
abstract = "High-throughput sequencing has become a standard tool for transcriptome analysis. The depletion of overrepresented RNA species from sequencing libraries plays a key role in establishing potent and cost-efficient RNA-seq routines. Commercially available kits are known to obtain good results for the reduction of ribosomal RNA (rRNA). However, we found that the transfer-messenger RNA (tmRNA) was frequently highly abundant in rRNA-depleted samples of Pseudomonas aeruginosa , consuming up to 25 % of the obtained reads. The tmRNA fraction was particularly high in samples taken from stationary cultures. This suggests that overrepresentation of this RNA species reduces the mRNA fraction when cells are grown under challenging conditions. Here, we present an RNase-H-based depletion protocol that targets the tmRNA in addition to ribosomal RNAs. We were able to increase the mRNA fraction to 93-99% and therefore outperform not only the commercially Ribo-off kit (Vazyme) operating by the same principle but also the formerly widely used Ribo-Zero kit (Illumina). Maximizing the read share of scientifically interesting RNA species enhances the discriminatory potential of next-generation RNA-seq experiments and, therefore, can contribute to a better understanding of the transcriptomic landscape of bacterial pathogens and their used mechanisms in host infection.",
author = "Florian Engelhardt and J{\"u}rgen Tomasch and Susanne H{\"a}ussler",
note = "{\textcopyright} 2020 The Authors.",
year = "2020",
doi = "10.1099/acmi.0.000159",
language = "English",
volume = "2",
pages = "acmi000159",
journal = "Access Microbiology",
issn = "2516-8290",
publisher = "Microbiology Society",
number = "10",

}

RIS

TY - JOUR

T1 - Organism-specific depletion of highly abundant RNA species from bacterial total RNA

AU - Engelhardt, Florian

AU - Tomasch, Jürgen

AU - Häussler, Susanne

N1 - © 2020 The Authors.

PY - 2020

Y1 - 2020

N2 - High-throughput sequencing has become a standard tool for transcriptome analysis. The depletion of overrepresented RNA species from sequencing libraries plays a key role in establishing potent and cost-efficient RNA-seq routines. Commercially available kits are known to obtain good results for the reduction of ribosomal RNA (rRNA). However, we found that the transfer-messenger RNA (tmRNA) was frequently highly abundant in rRNA-depleted samples of Pseudomonas aeruginosa , consuming up to 25 % of the obtained reads. The tmRNA fraction was particularly high in samples taken from stationary cultures. This suggests that overrepresentation of this RNA species reduces the mRNA fraction when cells are grown under challenging conditions. Here, we present an RNase-H-based depletion protocol that targets the tmRNA in addition to ribosomal RNAs. We were able to increase the mRNA fraction to 93-99% and therefore outperform not only the commercially Ribo-off kit (Vazyme) operating by the same principle but also the formerly widely used Ribo-Zero kit (Illumina). Maximizing the read share of scientifically interesting RNA species enhances the discriminatory potential of next-generation RNA-seq experiments and, therefore, can contribute to a better understanding of the transcriptomic landscape of bacterial pathogens and their used mechanisms in host infection.

AB - High-throughput sequencing has become a standard tool for transcriptome analysis. The depletion of overrepresented RNA species from sequencing libraries plays a key role in establishing potent and cost-efficient RNA-seq routines. Commercially available kits are known to obtain good results for the reduction of ribosomal RNA (rRNA). However, we found that the transfer-messenger RNA (tmRNA) was frequently highly abundant in rRNA-depleted samples of Pseudomonas aeruginosa , consuming up to 25 % of the obtained reads. The tmRNA fraction was particularly high in samples taken from stationary cultures. This suggests that overrepresentation of this RNA species reduces the mRNA fraction when cells are grown under challenging conditions. Here, we present an RNase-H-based depletion protocol that targets the tmRNA in addition to ribosomal RNAs. We were able to increase the mRNA fraction to 93-99% and therefore outperform not only the commercially Ribo-off kit (Vazyme) operating by the same principle but also the formerly widely used Ribo-Zero kit (Illumina). Maximizing the read share of scientifically interesting RNA species enhances the discriminatory potential of next-generation RNA-seq experiments and, therefore, can contribute to a better understanding of the transcriptomic landscape of bacterial pathogens and their used mechanisms in host infection.

U2 - 10.1099/acmi.0.000159

DO - 10.1099/acmi.0.000159

M3 - Journal article

C2 - 33195973

VL - 2

SP - acmi000159

JO - Access Microbiology

JF - Access Microbiology

SN - 2516-8290

IS - 10

ER -

ID: 62343136