Evaluation of the Impact of Concentration and Extraction Methods on the Targeted Sequencing of Human Viruses from Wastewater

Jiang, Minxi; Wang, Audrey W.; Be, Nicholas A.; Mulakken, Nisha; Nelson, Kara L.; Kantor, Rose S.

doi:10.1021/acs.est.4c00580

Title: Evaluation of the Impact of Concentration and Extraction Methods on the Targeted Sequencing of Human Viruses from Wastewater

Journal Article · 30 April 2024 · Environmental Science and Technology

DOI: https://doi.org/10.1021/acs.est.4c00580 · OSTI ID:2438165

^[1]; Wang, Audrey W. ^[1]; Be, Nicholas A. ^[2]; Mulakken, Nisha ^[2];

^[1];

^[1]

University of California, Berkeley, CA (United States)
Lawrence Livermore National Laboratory (LLNL), Livermore, CA (United States)

Sequencing human viruses in wastewater is challenging due to their low abundance compared to the total microbial background. This study compared the impact of four virus concentration/extraction methods (Innovaprep, Nanotrap, Promega, and Solids extraction) on probe-capture enrichment for human viruses followed by sequencing. Different concentration/extraction methods yielded distinct virus profiles. Innovaprep ultrafiltration (following solids removal) had the highest sequencing sensitivity and richness, resulting in the successful assembly of several near-complete human virus genomes. However, it was less sensitive in detecting SARS-CoV-2 by digital polymerase chain reaction (dPCR) compared to Promega and Nanotrap. Across all preparation methods, astroviruses and polyomaviruses were the most highly abundant human viruses, and SARS-CoV-2 was rare. These findings suggest that sequencing success can be increased using methods that reduce nontarget nucleic acids in the extract, though the absolute concentration of total extracted nucleic acid, as indicated by Qubit, and targeted viruses, as indicated by dPCR, may not be directly related to targeted sequencing performance. Further, using broadly targeted sequencing panels may capture viral diversity but risks losing signals for specific low-abundance viruses. Overall, this study highlights the importance of aligning wet lab and bioinformatic methods with specific goals when employing probe-capture enrichment for human virus sequencing from wastewater.

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Research Organization:: Lawrence Livermore National Laboratory (LLNL), Livermore, CA (United States)

Sponsoring Organization:: USDOE National Nuclear Security Administration (NNSA)

Grant/Contract Number:: AC52-07NA27344

OSTI ID:: 2438165

Report Number(s):: LLNL-JRNL-867766; 1103258

Journal Information:: Environmental Science and Technology, Journal Name: Environmental Science and Technology Journal Issue: 19 Vol. 58; ISSN 0013-936X

Publisher:: American Chemical Society (ACS)Copyright Statement

Country of Publication:: United States

Language:: English

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59 BASIC BIOLOGICAL SCIENCES
human virus
nucleic acid extraction
probe-capture enrichment
targeted sequencing
virus concentration
wastewater-based epidemiology
wastewater-based surveillance

Title: Evaluation of the Impact of Concentration and Extraction Methods on the Targeted Sequencing of Human Viruses from Wastewater

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References (54)

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