Dataset Information

Robust Performance of SARS-CoV-2 Whole-Genome Sequencing from Wastewater with a Nonselective Virus Concentration Method.

ABSTRACT: The sequencing of human virus genomes from wastewater samples is an efficient method for tracking viral transmission and evolution at the community level. However, this requires the recovery of viral nucleic acids of high quality. We developed a reusable tangential-flow filtration system to concentrate and purify viruses from wastewater for genome sequencing. A pilot study was conducted with 94 wastewater samples from four local sewersheds, from which viral nucleic acids were extracted, and the whole genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was sequenced using the ARTIC V4.0 primers. Our method yielded a high probability (0.9) of recovering complete or near-complete SARS-CoV-2 genomes (>90% coverage at 10× depth) from wastewater when the COVID-19 incidence rate exceeded 33 cases per 100 000 people. The relative abundances of sequenced SARS-CoV-2 variants followed the trends observed from patient-derived samples. We also identified SARS-CoV-2 lineages in wastewater that were underrepresented or not present in the clinical whole-genome sequencing data. The developed tangential-flow filtration system can be easily adopted for the sequencing of other viruses in wastewater, particularly those at low concentrations.

SUBMITTER: Segelhurst E

PROVIDER: S-EPMC10005814 | biostudies-literature | 2023 Apr

REPOSITORIES: biostudies-literature

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Publications

Robust Performance of SARS-CoV-2 Whole-Genome Sequencing from Wastewater with a Nonselective Virus Concentration Method.

Segelhurst Emily E Bard Jonathan E JE Pillsbury Annemarie N AN Alam Md Mahbubul MM Lamb Natalie A NA Zhu Chonglin C Pohlman Alyssa A Boccolucci Amanda A Emerson Jamaal J Marzullo Brandon J BJ Yergeau Donald A DA Nowak Norma J NJ Bradley Ian M IM Surtees Jennifer A JA Ye Yinyin Y

ACS ES&T water 20230302 4

The sequencing of human virus genomes from wastewater samples is an efficient method for tracking viral transmission and evolution at the community level. However, this requires the recovery of viral nucleic acids of high quality. We developed a reusable tangential-flow filtration system to concentrate and purify viruses from wastewater for genome sequencing. A pilot study was conducted with 94 wastewater samples from four local sewersheds, from which viral nucleic acids were extracted, and the ...[more]

PMID: 37406038

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Project description:Wastewater SARS-CoV-2 surveillance has been deployed since the beginning of the COVID-19 pandemic to monitor the dynamics in virus burden in local communities. Genomic surveillance of SARS-CoV-2 in wastewater, particularly efforts aimed at whole genome sequencing for variant tracking and identification, are still challenging due to low target concentration, complex microbial and chemical background, and lack of robust nucleic acid recovery experimental procedures. The intrinsic sample limitations are inherent to wastewater and are thus unavoidable. Here, we use a statistical approach that couples correlation analyses to a random forest-based machine learning algorithm to evaluate potentially important factors associated with wastewater SARS-CoV-2 whole genome amplicon sequencing outcomes, with a specific focus on the breadth of genome coverage. We collected 182 composite and grab wastewater samples from the Chicago area between November 2020 to October 2021. Samples were processed using a mixture of processing methods reflecting different homogenization intensities (HA + Zymo beads, HA + glass beads, and Nanotrap), and were sequenced using one of the two library preparation kits (the Illumina COVIDseq kit and the QIAseq DIRECT kit). Technical factors evaluated using statistical and machine learning approaches include sample types, certain sample intrinsic features, and processing and sequencing methods. The results suggested that sample processing methods could be a predominant factor affecting sequencing outcomes, and library preparation kits was considered a minor factor. A synthetic SARS-CoV-2 RNA spike-in experiment was performed to validate the impact from processing methods and suggested that the intensity of the processing methods could lead to different RNA fragmentation patterns, which could also explain the observed inconsistency between qPCR quantification and sequencing outcomes. Overall, extra attention should be paid to wastewater sample processing (i.e., concentration and homogenization) for sufficient and good quality SARS-CoV-2 RNA for downstream sequencing.

Dataset Information

Robust Performance of SARS-CoV-2 Whole-Genome Sequencing from Wastewater with a Nonselective Virus Concentration Method.

Publications

Robust Performance of SARS-CoV-2 Whole-Genome Sequencing from Wastewater with a Nonselective Virus Concentration Method.

Similar Datasets

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