Wastewater surveillance has emerged as a pivotal tool in public health epidemiology. Particularly catalyzed by the Covid-19 pandemic, modern culture-independent sequencing methods have become indispensable due to their ability to offer a comprehensive perspective. This study presents an innovative, integrated approach for simultaneous pathogen detection, antimicrobial resistance (AMR) monitoring, and functional analysis within wastewater systems. By harnessing advanced sample preparation technologies provided by Zymo Research and leveraging PacBio Onso™ short-read sequencing, our objective is to elevate our comprehension of microbial dynamics within wastewater environments.
Methods
To evaluate the efficacy of Zymo Research’s nucleic acid extraction technology, a study was conducted using real-world wastewater samples. Samples were processed using Zymo Research’s Quick-DNA/RNA™ Water kit, then analyzed the samples using the latest sequencing methodologies tailored for wastewater surveillance.
Sample Information
Wastewater samples pose significant challenges for nucleic acid extraction, primarily due to their high turbidity and the abundance of PCR inhibitors. These samples have a diverse array of microbes, including viruses, bacteria, and fungi, all crucial targets for comprehensive wastewater surveillance. Notably, for effective pathogen detection, processing large volumes (>30 mL) is imperative, a task often daunting for conventional extraction kits. Here, 30 mL of wastewater (Samples 1 & 2) and 250 mg of primary sludge (Sample 3) were processed with the Quick-DNA/RNA™ Water Kit.
DNA/RNA Extraction
DNA/RNA extraction serves as the foundational step for all downstream molecular analysis applications. The quality of the extracted nucleic acid is critical to ensuring accurate analysis results. Zymo Research’s Quick-DNA/RNA™ Water Kit offers a solution that delivers high-quality DNA/RNA eluates free of inhibitors, ready for next-generation sequencing (NGS) and polymerase chain reaction (PCR). Notably, the kit features an innovative concentration buffer and inhibitor removal technology that enables the concentration of microbes from large volume water samples and removes PCR inhibitors yielding ultra-pure DNA.
| Sample ID | Sampling Site | Description |
|---|---|---|
| Sample 1 | Influent from wastewater treatment plant 1 | The raw wastewater without treatment |
| Sample 2 | Influent from wastewater treatment plant 2 | The raw wastewater without treatment |
| Sample 3 | Primary Sludge | The sediment from primary treatment process |
Next Generation Sequencing (NGS) Platforms
- Illumina NextSeq 2000™: The Illumina NextSeq 2000™ platform is a widely utilized system for wastewater and environmental surveillance, particularly for pathogen detection and the identification of antimicrobial resistance (AMR) and mobile genetic elements (MGEs). Illumina sequencing employs a robust technology that utilizes reversible terminator chemistry, allowing for accurate and high-throughput sequencing of DNA fragments. Its versatility and scalability make it suitable for a wide range of applications, including metagenomic analysis and microbial community profiling.
- PacBio Onso™ Short-Read Sequencing System: The PacBio Onso™ short-read sequencing system utilizes sequencing by binding (SBB) technology. PacBio sequencing achieves high accuracy (Q40+) by measuring light signals emitted from fluorescently labeled nucleotides when they are bound, but not incorporated, by a polymerase on a DNA strand. This technology offers advantages in analyzing complex genomic regions, such as repetitive sequences and structural variations, making it valuable for comprehensive genomic characterization.
Onso data was downsampled from 130 M to 8 M to generate the same data size with NextSeq, then utilized the advanced bioinformatics pipeline provided by Zymo Research Microbiome Sequencing Services to generate a comprehensive wastewater report. These reports highlighted key functional bacterial groups vital for water monitoring plants. Specifically, the analysis was focused on nitrogen removal bacteria, essential for effective wastewater treatment, and methylotrophs, pivotal in the anaerobic digestion of sludge.
Results
The data generated below was provided in a customized report from Zymo Research Sequencing Services.
Figure 1. Reads number assigned to AMR class in 2 sequencing systems
Figure 2. Read number assigned to AMR class in 2 sequencing systems.
Figure 3. Read number assigned to methylotrophs in 2 sequencing systems.
Figure 4. Read number assigned to nitrogen removal bacteria in 2 sequencing systems.
Zymo Research Microbiome Sequencing Services also provided a customized data report for the read number assigned to specific bacterial groups that are significant for wastewater monitoring. (Figure 3 & 4) Read number for data generated using PacBio Onso™ and Illumina NextSeq 2000™ sequencing methods are compared.
Conclusion
This study underscores the effectiveness of the Quick-DNA/RNA™ Water Kit in conjunction with next-generation sequencing (NGS) for wastewater surveillance, offering crucial insights into microbial dynamics and antibiotic resistance patterns. By employing two sequencing technologies and analyzing microbial taxa, functional groups, and antibiotic resistance genes (ARGs) in real-world wastewater samples, notable insights have emerged.
The PacBio Onso™ system emerges as a standout technology boasting high-throughput short-read sequencing capabilities that provide detailed and precise microbial community structures, antibiotics resistance profiles, and functional bacteria detection. This precision equips researchers with the tools necessary for predicting disease outbreaks and optimizing both public health management strategies and water treatment processes, thereby bolstering environmental and public health outcomes.
Despite lower accuracy and read counts compared to the PacBio Onso™ system, the Illumina NextSeq 2000™ system offers unparalleled speed, enabling real-time transmission tracking—a vital asset in scenarios where rapid response is paramount.
In essence, the integration of the Quick-DNA/RNA™ Water Kit with NGS technologies holds immense promise for advancing wastewater surveillance practices.