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Isolation and Sequencing of Bacterial Metagenomic DNA From Eighteen Mile Creek

at a location in Eden N.Y.

Elizabeth Agle, Madilyn Brodzinski, Alexandria DePan, Peter Mroz, Patrick Schmitz,

Meeah Wegrzynowski, Emma Willet, and Keith Kwas

from Eden Central Schools

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Introduction

This research project exposed us to the world of data collection, technical analysis and scientific research, all of which aim to build skills that can be used in future scientific endeavors. The object of this project was to gain insight into the local, microscopic world around us and the proper procedures to understand such a world. It was a privilege to be given the opportunity to work with updated technological software, such as the Oxford Nanopore Sequencers, in a true collegiate lab, alongside the guidance of faculty and teachers. The project served as a connection for us students; working and networking with the local UB Jacobs School of Medicine and Biomedical Sciences and the Buffalo Niagara Waterkeeper, placing our science class in a real world work environment. The project helped create a strong base for our futures and gain real life experience.

Methods

• 2 liters of water was collected from 18 Mile Creek on October 8th, 2022.
• Metadata was collected and included air and water temperature, GPS coordinates and photos of the collection site and some basic biochemical characteristics of the water sample using Tetra EasyStrips 6-in-1 Freshwater and Saltwater Aquarium Test Strips.
• Water was filtered sequentially through 1.2 µm GFC and 0.22 µM Durapore filters.
• DNA was extracted from the 0.22 µM filter using a modification of a Qiagen PowerWater DNA Isolation Kit¹.
• Metagenomic DNA was amplified using a Rapid PCR Barcoding Kit from Oxford Nanopore Technologies.
• Amplified DNA was size selected using SPRI beads (Beckman Coulter) and sequenced on a Flongle flow cell in a MK1C sequencer (Oxford Nanopore).
• Sequencing reads were uploaded to EPI2ME for a WIMP (What’s In My Pot) workflow to identify bacteria in the sample.

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Figure 2. Water collection filters. Left, 1.2 µM GFC showing debris collected. Right, 0.22 µM filter enriched with bacteria passing through the 1.2 µM filter

Results – Water Filtration

References

1. GigaScience, Volume 9, Issue 6, June 2020 (https://tinyurl.com/5bb484cv)
2. Methods can be accessed at: https://tinyurl.com/ye28433f
3. https://river-runner.samlearner.com/
4. https://www.google.com/maps/

Department of Biotechnical and Clinical Laboratory Sciences�Jacobs School of Medicine and Biomedical Sciences

buffalo.edu

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Figure 3. Summary of sequencing run data.

Summary and Conclusions

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We collected our water samples from the local 18 Mile Creek, in Eden, New York. The top 30 genera from our sample are pictured above. After our samples were cultivated on a petri dish, we chose one sample to focus on. This specific bacteria, named Proteus Vulgaris was chosen due to its fuzzy visual appearance, which in turn was attributed to the swarming feature of this bacteria. Our sequenced sample was full of other bacteria, all of the same Proteus genus, and all also had the swarming feature. Thus, giving reason to believe that is how our sequenced sample included many other reads. The general water sample contained many typical freshwater or “normal” bacteria, such as Hydrogenophaga and Limnohabitans. However, some bacteria found in the general water sample, like the Pseudomonas, was found to be a common, yet, malicious pathogen with antibiotic resistant properties. Additionally, the Flavobacterium, are pathogens that cause columnaris in fish. It affects the species of catfish, tilapia, and trout. Due to the location of the creek in comparison to local farms, this could be the reason for the types of bacteria found. The creek we collected from is also a fishing hotspot, the frequent human visitation and the animals that habituate there, could also add to the variety of bacteria found.

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Figure 5. Phylogeny of the top 30 genera present in the metagenomic sample with a minimum of 0.1% abundance in the sample. Full WIMP data can be viewed at https://tinyurl.com/4nbnz3tz

Acknowledgements

1. Funded by the National Institutes of Health: R25GM137364
2. Special thanks to Buffalo Niagara Waterkeeper, Erie Niagara and Rural AHECs, Dr. Stephen Koury, Sandra Small and Sunha Kim for their assistance with project activities.

Results - Water Collection

Figure 1. 18 Mile Creek Water Collection Site in Eden, N.Y..

A. Overview of area and flow of water from sampling site from River Runner.

B. Aerial view of site from Google Maps. Arrow indicates collection site.

C. Photo of students collecting water samples from site.

Results – DNA Extraction and Sequencing

Figure 4. Identification of microbial genera detected after submitting sequencing data to the EPI2ME site for a WIMP analysis. Homo genus is likely the result of contamination of human DNA in the PCR amplification of isolated metagenomic DNA.