Targeted Depletion of rRNAs and Tobamoviruses Enhances Viral Discovery in Wastewater Metatranscriptomes
- 1University of Calgary
Abstract
Wastewater contains a complex mixture of nucleic acids derived from human cells, microorganisms, diets, and agricultural or industrial inputs. Metatranscriptomics has the potential to broaden the understanding of wastewater viromes, but its utility is constrained by high-abundance RNA molecules that prevent the detection of lower-abundance signals, including human pathogens. Two major sources of this interfering background signal are ribosomal RNAs (rRNAs) from various organisms, and plant-infecting tobamoviruses from human diets, which are often the most abundant RNA viruses in wastewater. These rRNA molecules and RNA genomes can account for upwards of 95% of total RNA sequences, thereby obscuring the detection of lower-abundance viruses of public health importance. This study evaluated whether combined depletion of ribosomal RNAs and genomic RNA from abundant plant tobamoviruses can improve the recovery of underrepresented viral sequences from wastewater. RNA extracts from a large municipal wastewater treatment plant in Calgary, Canada, were processed with or without probe-based depletion of rRNAs and eight abundant tobamoviruses. Metatranscriptomes revealed that depletion reduced rRNA reads by 90%. Reads assigned to tobamoviruses, which accounted for up to 25% of viral reads in untreated samples, were reduced to <2% of viral reads after depletion. This decrease in undesired RNA signal led to an order of magnitude increase in viral read signals and improved assembled viral genome quality. Furthermore, the number of reads assigned to human and animal pathogens increased 42-fold after depletion, enabling the detection of important pathogens like influenza, RSV, and hepatitis A, which were undecteable in untreated samples. Together, these findings demonstrate that targeted depletion of abundant background RNAs improves the detection of low abundance viruses, and enhances the utility of metatranscriptomic approaches for wastewater viral surveillance.
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BibTeX
@misc{bautista2026targeted,
title = {{Targeted Depletion of rRNAs and Tobamoviruses Enhances Viral Discovery in Wastewater Metatranscriptomes}},
author = {M. A. Bautista and T. Bhardwaj and D. J. Barbera and S. J. Rulli and J. Zorz and J. McCalder and C. Papparis and X. Pang and B. E. Lee and J. Qiu and R. G. Clark and N. Acosta and C. O' Grady and M. D. Parkins and C. R. Hubert},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.08.757481},
url = {https://www.biorxiv.org/content/10.64898/2026.10.08.757481v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Targeted Depletion of rRNAs and Tobamoviruses Enhances Viral Discovery in Wastewater Metatranscriptomes
AU - M. A. Bautista
AU - T. Bhardwaj
AU - D. J. Barbera
AU - S. J. Rulli
AU - J. Zorz
AU - J. McCalder
AU - C. Papparis
AU - X. Pang
AU - B. E. Lee
AU - J. Qiu
AU - R. G. Clark
AU - N. Acosta
AU - C. O' Grady
AU - M. D. Parkins
AU - C. R. Hubert
PY - 2026
DA - 2026/10/08
PB - bioRxiv
DO - 10.64898/2026.10.08.757481
UR - https://www.biorxiv.org/content/10.64898/2026.10.08.757481v1
AB - Wastewater contains a complex mixture of nucleic acids derived from human cells, microorganisms, diets, and agricultural or industrial inputs. Metatranscriptomics has the potential to broaden the understanding of wastewater viromes, but its utility is constrained by high-abundance RNA molecules that prevent the detection of lower-abundance signals, including human pathogens. Two major sources of this interfering background signal are ribosomal RNAs (rRNAs) from various organisms, and plant-infecting tobamoviruses from human diets, which are often the most abundant RNA viruses in wastewater. These rRNA molecules and RNA genomes can account for upwards of 95% of total RNA sequences, thereby obscuring the detection of lower-abundance viruses of public health importance. This study evaluated whether combined depletion of ribosomal RNAs and genomic RNA from abundant plant tobamoviruses can improve the recovery of underrepresented viral sequences from wastewater. RNA extracts from a large municipal wastewater treatment plant in Calgary, Canada, were processed with or without probe-based depletion of rRNAs and eight abundant tobamoviruses. Metatranscriptomes revealed that depletion reduced rRNA reads by 90%. Reads assigned to tobamoviruses, which accounted for up to 25% of viral reads in untreated samples, were reduced to <2% of viral reads after depletion. This decrease in undesired RNA signal led to an order of magnitude increase in viral read signals and improved assembled viral genome quality. Furthermore, the number of reads assigned to human and animal pathogens increased 42-fold after depletion, enabling the detection of important pathogens like influenza, RSV, and hepatitis A, which were undecteable in untreated samples. Together, these findings demonstrate that targeted depletion of abundant background RNAs improves the detection of low abundance viruses, and enhances the utility of metatranscriptomic approaches for wastewater viral surveillance.
ER -