Assessing multiplex tiling PCR sequencing approaches for detecting genomic variants of SARS-CoV-2 in municipal wastewater v1

2021 ◽  
Author(s):  
Xuan Lin ◽  
Melissa not provided Glier ◽  
Kevin Kuchinski ◽  
Tenysha Ross-Van Mierlo ◽  
David McVea ◽  
...  
Keyword(s):  
Municipal Wastewater ◽  
Amplicon Sequencing ◽  
Primary Sludge ◽  
Genomic Variants ◽  
Dna Library ◽  
Oxford Nanopore ◽  
Influent Wastewater

In this work, we aim to access the performance of three different multiplex primer schemes, i.e. Swift amplicon SARS-CoV-2 panel (150bp amplicons), ARTIC V3 panel (400bp amplicons), and SARS-CoV-2 midnight panel (1200bp amplicons), for metatranscriptomic sequencing of SARS-CoV-2 for influent wastewater and primary sludge. This protocol is adapted from the Swift amplicon™ SARS-COV-2 protocol (150bp amplicon), ARTIC V3 protocol (400bp amplicon), and "midnight" protocol (1200bp amplicon). Sequencing libraries are prepared with 1) Oxford Nanopore Ligation Sequencing Kit (SQK-LSK109) with Native Barcoding kit (EXP-NEB104 and EXP-NEB114), or 2) NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® with NEBNext® Multiplex Oligos for Illumina®. Links to the protocols are: Swift amplicon protocol (150bp): https://swiftbiosci.com/swift-amplicon-sars-cov-2-panel/ ARTIC V3 protocol (400bp): https://www.protocols.io/view/ncov-2019-sequencing-protocol-v3-locost-bh42j8ye?version_warning=no "midnight" protocol V4 (1200bp): dx.doi.org/10.17504/protocols.io.bh7hj9j6

scholarly journals Assessing multiplex tiling PCR sequencing approaches for detecting genomic variants of SARS-CoV-2 in municipal wastewater

2021 ◽  
Author(s):  
Xuan Lin ◽  
Melissa Glier ◽  
Kevin Kuchinski ◽  
Tenysha Ross-Van Mierlo ◽  
David McVea ◽  
...  
Keyword(s):  
Genome Sequencing ◽  
Municipal Wastewater ◽  
Rna Degradation ◽  
Geographic Region ◽  
Wastewater Sample ◽  
Municipal Wastewater Treatment ◽  
Primary Sludge ◽  
Genome Coverage ◽  
Genomic Variants ◽  
Influent Wastewater

Wastewater-based genomic surveillance of the SARS-CoV-2 virus shows promise to complement genomic epidemiology efforts. Multiplex tiled PCR is a desirable approach for targeted genome sequencing of SARS-CoV-2 in wastewater due to its low cost and rapid turnaround time. However, it is not clear how different multiplex tiled PCR primer schemes or wastewater sample matrices impact the resulting SARS-CoV-2 genome coverage. The objective of this work was to assess the performance of three different multiplex primer schemes, consisting of 150bp, 400bp, and 1200bp amplicons, as well as two wastewater sample matrices, influent wastewater and primary sludge, for targeted genome sequencing of SARS-CoV-2. Wastewater samples were collected weekly from five municipal wastewater treatment plants (WWTPs) in the Metro Vancouver region of British Columbia, Canada during a period of increased COVID-19 case counts from February to April, 2021. RNA extracted from clarified influent wastewater provided significantly higher genome coverage (breadth and median depth) than primary sludge samples across all primer schemes. Shorter amplicons appeared more resilient to sample RNA degradation, but were hindered by greater primer pool complexity in the 150bp scheme. The identified optimal primer scheme (400bp) and sample matrix (influent) was capable of detecting the emergence of mutations associated with genomic variants of concern, of which the daily wastewater load significantly correlated with clinical case counts. Taken together, these results provide guidance on best practices for implementing wastewater-based genomic surveillance, and demonstrate its ability to inform epidemiology efforts by detecting genomic variants of concern circulating within a geographic region.

scholarly journals Bacteria from the Genus Arcobacter Are Abundant in Effluent from Wastewater Treatment Plants

2020 ◽  
Vol 86 (9) ◽  
Author(s):  
Jannie Munk Kristensen ◽  
Marta Nierychlo ◽  
Mads Albertsen ◽  
Per Halkjær Nielsen
Keyword(s):  
Wastewater Treatment ◽  
Pathogenic Bacteria ◽  
Municipal Wastewater ◽  
Wastewater Treatment Plants ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Content Type ◽  
Effluent Quality ◽  
Culture Independent ◽  
Influent Wastewater

ABSTRACT Pathogenic bacteria in wastewater are generally considered to be efficiently removed in biological wastewater treatment plants. This understanding is almost solely based on culture-based control measures, and here we show, by applying culture-independent methods, that the removal of species in the genus Arcobacter was less effective than for many other abundant genera in the influent wastewater. Arcobacter was one of the most abundant genera in influent wastewater at 14 municipal wastewater treatment plants and was also abundant in the “clean” effluent from all the plants, reaching up to 30% of all bacteria as analyzed by 16S rRNA gene amplicon sequencing. Metagenomic analyses, culturing, genome sequencing of Arcobacter isolates, and visualization by fluorescent in situ hybridization (FISH) confirmed the presence of the human-pathogenic Arcobacter cryaerophilus and A. butzleri in both influent and effluent. The main reason for the high relative abundance in the effluent was probably that Arcobacter cells, compared to those of other abundant genera in the influent, did not flocculate and attach well to the activated sludge flocs, leaving a relatively large fraction dispersed in the water phase. The study shows there is an urgent need for new standardized culture-independent measurements of pathogens in effluent wastewaters, e.g., amplicon sequencing, and an investigation of the problem on a global scale to quantify the risk for humans and livestock. IMPORTANCE The genus Arcobacter was unexpectedly abundant in the effluent from 14 Danish wastewater treatment plants treating municipal wastewater, and the species included the human-pathogenic A. cryaerophilus and A. butzleri. Recent studies have shown that Arcobacter is common in wastewater worldwide, so the study indicates that discharge of members of the genus Arcobacter may be a global problem, and further studies are needed to quantify the risk and potentially minimize the discharge. The study also shows that culture-based analyses are insufficient for proper effluent quality control, and new standardized culture-independent measurements of effluent quality encompassing most pathogens should be considered.

Anaerobic Digestion of Primary Sludge Containing Iron Phosphate

1973 ◽  
Vol 8 (1) ◽  
pp. 91-109 ◽  
Author(s):  
M.E. Jack ◽  
G.J. Farquhar ◽  
G.M. Cornwall
Keyword(s):  
Anaerobic Digestion ◽  
Phosphorus Removal ◽  
Municipal Wastewater ◽  
Iron Phosphate ◽  
Chemical Precipitation ◽  
Task Group ◽  
Iron Compounds ◽  
Primary Sludge ◽  
Group Report ◽  
Aluminum Compounds

Abstract The importance of phosphorus as a nutrient in the eutrophication of lakes and rivers has been well established (Fruh 1967). It has been shown in addition that a significant amount of this phosphorus arises from the discharge of treated and untreated municipal wastewater (Task Group Report 1967). Consequently, measures are being taken, notably in the Province of Ontario, for removal of phosphorus from wastewater by means of chemical precipitation. Chemicals exhibiting satisfactory phosphorus removal include lime, iron compounds and aluminum compounds (Leckie and Stumm 1970; Schmid 1968; Wuhrman 1968).

scholarly journals Microalgae as biological treatment for municipal wastewater – effects on the sludge handling in a treatment plant

2018 ◽  
Vol 78 (3) ◽  
pp. 644-654 ◽  
Author(s):  
J. Olsson ◽  
S. Schwede ◽  
E. Nehrenheim ◽  
E. Thorin
Keyword(s):  
Heavy Metals ◽  
Activated Sludge ◽  
Biological Treatment ◽  
Municipal Wastewater ◽  
Treatment Plant ◽  
Waste Activated Sludge ◽  
Activated Sludge Process ◽  
Primary Sludge ◽  
Thermophilic Conditions ◽  
Mesophilic Conditions

Abstract A mix of microalgae and bacteria was cultivated on pre-sedimented municipal wastewater in a continuous operated microalgae-activated sludge process. The excess material from the process was co-digested with primary sludge in mesophilic and thermophilic conditions in semi-continuous mode (5 L digesters). Two reference digesters (5 L digesters) fed with waste-activated sludge (WAS) and primary sludge were operated in parallel. The methane yield was slightly reduced (≈10%) when the microalgal-bacterial substrate was used in place of the WAS in thermophilic conditions, but remained approximately similar in mesophilic conditions. The uptake of heavy metals was higher with the microalgal-bacterial substrate in comparison to the WAS, which resulted in higher levels of heavy metals in the digestates. The addition of microalgal-bacterial substrate enhanced the dewaterability in thermophilic conditions. Finally, excess heat can be recovered in both mesophilic and thermophilic conditions.

scholarly journals Evaluation of the microbial community structure of potable water samples from occupied and unoccupied buildings using16S rRNA amplicon sequencing

2020 ◽  
Author(s):  
Kimothy L Smith ◽  
Howard A Shuman ◽  
Douglas Findeisen
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
Water Samples ◽  
Ad Hoc ◽  
Microbial Community Composition ◽  
Amplicon Sequencing ◽  
The Other ◽  
Water Usage ◽  
16S Rrna Amplicon Sequencing ◽  
Oxford Nanopore

AbstractWe conducted two studies of water samples from buildings with normal occupancy and water usage compared to water from buildings that were unoccupied with little or no water usage due to the COVID-19 shutdown. Study 1 had 52 water samples obtained ad hoc from buildings in four metropolitan locations in different states in the US and a range of building types. Study 2 had 36 water samples obtained from two buildings in one metropolitan location with matched water sample types. One of the buildings had been continuously occupied, and the other substantially vacant for approximately 3 months. All water samples were analyzed using 16S rRNA amplicon sequencing with a MinION from Oxford Nanopore Technologies. More than 127 genera of bacteria were identified, including genera with members that are known to include more than 50 putative frank and opportunistic pathogens. While specific results varied among sample locations, 16S rRNA amplicon abundance and the diversity of bacteria were higher in water samples from unoccupied buildings than normally occupied buildings as was the abundance of sequenced amplicons of genera known to include pathogenic bacterial members. In both studies Legionella amplicon abundance was relatively small compared to the abundance of the other bacteria in the samples. Indeed, when present, the relative abundance of Legionella amplicons was lower in samples from unoccupied buildings. Legionella did not predominate in any of the water samples and were found, on average, in 9.6% of samples in Study 1 and 8.3% of samples in Study 2.SynopsisComparison of microbial community composition in the plumbing of occupied and unoccupied buildings during the COVID-19 pandemic shutdown.

scholarly journals Nanopore amplicon sequencing reveals molecular convergence and local adaptation of opsin genes

2020 ◽  
Author(s):  
Katherine M. Eaton ◽  
Moisés A. Bernal ◽  
Nathan J.C. Backenstose ◽  
Trevor J. Krabbenhoft
Keyword(s):  
Local Adaptation ◽  
Environmental Gradients ◽  
Parallel Evolution ◽  
Amplicon Sequencing ◽  
Species Flock ◽  
Closely Related Species ◽  
Oxford Nanopore ◽  
Opsin Genes ◽  
Potential Case ◽  
Species Specific

AbstractLocal adaptation can drive diversification of closely related species across environmental gradients and promote convergence of distantly related taxa that experience similar conditions. We examined a potential case of adaptation to novel visual environments in a species flock (Great Lakes salmonids, genus Coregonus) using a new amplicon genotyping protocol on the Oxford Nanopore Flongle. Five visual opsin genes were amplified for individuals of C. artedi, C. hoyi, C. kiyi, and C. zenithicus. Comparisons revealed species-specific differences in the coding sequence of rhodopsin (Tyr261Phe substitution), suggesting local adaptation by C. kiyi to the blue-shifted depths of Lake Superior. Parallel evolution and “toggling” at this amino acid residue has occurred several times across the fish tree of life, resulting in identical changes to the visual systems of distantly related taxa across replicated environmental gradients. Our results suggest that ecological differences and local adaptation to distinct visual environments are strong drivers of both evolutionary parallelism and diversification.

scholarly journals Integrated genomic view of SARS-CoV-2 in India

2020 ◽  
Vol 5 ◽  
pp. 184 ◽  
Author(s):  
Pramod Kumar ◽  
Rajesh Pandey ◽  
Pooja Sharma ◽  
Mahesh S. Dhar ◽  
Vivekanand A. ◽  
...  
Keyword(s):  
Host Response ◽  
Genomic Data ◽  
Amplicon Sequencing ◽  
Climatic Conditions ◽  
Molecular Surveillance ◽  
Multiple Introductions ◽  
Oxford Nanopore ◽  
The World ◽  
The Usa ◽  
Local Transmission

Background: India first detected SARS-CoV-2, causal agent of COVID-19 in late January 2020, imported from Wuhan, China. From March 2020 onwards, the importation of cases from countries in the rest of the world followed by seeding of local transmission triggered further outbreaks in India. Methods: We used ARTIC protocol-based tiling amplicon sequencing of SARS-CoV-2 (n=104) from different states of India using a combination of MinION and MinIT sequencing from Oxford Nanopore Technology to understand how introduction and local transmission occurred. Results: The analyses revealed multiple introductions of SARS-CoV-2 genomes, including the A2a cluster from Europe and the USA, A3 cluster from Middle East and A4 cluster (haplotype redefined) from Southeast Asia (Indonesia, Thailand and Malaysia) and Central Asia (Kyrgyzstan). The local transmission and persistence of genomes A4, A2a and A3 was also observed in the studied locations. The most prevalent genomes with patterns of variance (confined in a cluster) remain unclassified, and are here proposed as A4-clade based on its divergence within the A cluster. Conclusions: The viral haplotypes may link their persistence to geo-climatic conditions and host response. Multipronged strategies including molecular surveillance based on real-time viral genomic data is of paramount importance for a timely management of the pandemic.

scholarly journals Tracking the temporal variation of COVID-19 surges through wastewater-based epidemiology during the peak of the pandemic: a six-month long study in Charlotte, North Carolina

2021 ◽  
Author(s):  
Visva Bharati Barua ◽  
Md Ariful Islam Juel ◽  
A. Denene Blackwood ◽  
Thomas Clerkin ◽  
Mark Ciesielski ◽  
...  
Keyword(s):  
North Carolina ◽  
Municipal Wastewater ◽  
Wastewater Treatment Plants ◽  
Strong Positive Correlation ◽  
Municipal Wastewater Treatment ◽  
Fecal Shedding ◽  
Global Pandemic ◽  
High Concentrations ◽  
Municipal Wastewater Treatment Plants ◽  
Influent Wastewater

The global spread of SARS-CoV-2 has continued to be a serious concern after WHO declared the virus the causative agent of the coronavirus disease 2019 (COVID-19) a global pandemic. Monitoring of wastewater is a useful tool for assessing community prevalence given that fecal shedding of SARS-CoV-2 occurs in high concentrations by infected individuals, regardless of whether they are asymptomatic or symptomatic. Using tools that are part of the wastewater-based epidemiology (WBE) approach, combined with molecular analyses, wastewater monitoring becomes a key piece of information used to assess trends and quantify the scale and dynamics of COVID-19 infection in a specific community, municipality, or area of service. This study investigates a six-month long SARS-CoV-2 RNA quantification in influent wastewater from four municipal wastewater treatment plants (WWTP) serving the Charlotte region of North Carolina (NC) using both RT-qPCR and RT-ddPCR platforms. Influent wastewater was analyzed for the nucleocapsid (N) genes N1 and N2. Both RT-qPCR and RT-ddPCR performed well for detection and quantification of SARS-CoV-2 using the N1 target, while for the N2 target RT-ddPCR was more sensitive. SARS-CoV-2 concentration ranged from 103 to105 copies/L for all four plants. Both RT-qPCR and RT-ddPCR showed a significant moderate to a strong positive correlation between SARS-CoV-2 concentrations and the 7-day rolling average of clinically reported COVID-19 cases using a lag that ranged from 7 to 12 days. A major finding of this study is that despite small differences, both RT-qPCR and RT-ddPCR performed well for tracking the SARS-CoV-2 virus across WWTP of a range of sizes and metropolitan service functions.

scholarly journals Recovery of small plasmid sequences via Oxford Nanopore sequencing

2021 ◽  
Vol 7 (8) ◽  
Author(s):  
Ryan R. Wick ◽  
Louise M. Judd ◽  
Kelly L. Wyres ◽  
Kathryn E. Holt
Keyword(s):  
Whole Genome ◽  
Bacterial Isolates ◽  
Small Plasmid ◽  
Dna Library ◽  
Oxford Nanopore ◽  
Sequencing Platforms ◽  
Plasmid Size ◽  
Plasmid Recovery ◽  
Oxford Nanopore Technologies ◽  
Do So

Oxford Nanopore Technologies (ONT) sequencing platforms currently offer two approaches to whole-genome native-DNA library preparation: ligation and rapid. In this study, we compared these two approaches for bacterial whole-genome sequencing, with a specific aim of assessing their ability to recover small plasmid sequences. To do so, we sequenced DNA from seven plasmid-rich bacterial isolates in three different ways: ONT ligation, ONT rapid and Illumina. Using the Illumina read depths to approximate true plasmid abundance, we found that small plasmids (<20 kbp) were underrepresented in ONT ligation read sets (by a mean factor of ~4) but were not underrepresented in ONT rapid read sets. This effect correlated with plasmid size, with the smallest plasmids being the most underrepresented in ONT ligation read sets. We also found lower rates of chimaeric reads in the rapid read sets relative to ligation read sets. These results show that when small plasmid recovery is important, ONT rapid library preparations are preferable to ligation-based protocols.

Polycyclic and Nitro Musks in Canadian Municipal Wastewater: Occurrence and Removal in Wastewater Treatment

2007 ◽  
Vol 42 (3) ◽  
pp. 138-152 ◽  
Author(s):  
Shirley Anne Smyth ◽  
Lori A. Lishman ◽  
Edward A. McBean ◽  
Sonya Kleywegt ◽  
Jian-Jun Yang ◽  
...  
Keyword(s):  
Wastewater Treatment ◽  
Activated Sludge ◽  
Municipal Wastewater ◽  
Treatment Plant ◽  
Waste Activated Sludge ◽  
Secondary Effluent ◽  
Cold Temperatures ◽  
Primary Sludge ◽  
Polycyclic Musks ◽  
Nitro Musks

Abstract The removal and/or partitioning (to sludge) of six polycyclic and five nitro musks through the liquid treatment train of a conventional Canadian secondary activated sludge wastewater treatment plant is characterized. Raw influent, primary effluent, secondary effluent, primary sludge, and waste activated sludge concentrations were correlated to seasonal process temperatures (warm, 22°C; cold, 15°C). Maximum influent concentrations of polycyclic and nitro musks were 7,030 ± 2,120 ng/L for Galaxolide (HHCB) and 158 ± 89 ng/L for musk ketone respectively. Maximum secondary effluent concentrations were 2,000 ± 686 ng/L for HHCB and 51 ± 14 ng/L for musk ketone. Temperature appeared to influence the degree of removal of musks from wastewater during primary clarification (40% median removal at warm temperatures and 9% at cold temperatures) and overall treatment (82% median removal at warm temperatures and 74% at cold temperatures) but not secondary activated sludge treatment (71% median removal at warm temperatures and 70% at cold temperatures). In primary sludge, polycyclic musks were found at concentrations up to 35,000 ng/g for HHCB, and nitro musks were found at concentrations up to 490 ng/g for musk ketone. In waste activated sludge, polycyclic musks were found at concentrations up to 52,000 ng/g (HHCB), and nitro musks were found at concentrations up to 1,100 ng/g (musk ketone). The hydraulic retention time and the suspended solids of the treatment process appeared to influence the degree of partitioning of musks to sludge.

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