GEOPATHS - Ani Chaudhary

Data points measuring the duration and magnitude of various signals generated by particles in varying solutions of pond water being dragged through a solid-state nanopore. The various solutions can be seen to the upper right-hand side of the figure, with the large yellow, purple, and red signals being from solutions treated with nucleic enzymes.

Ani Chaudhary

Intern with the Deamer Molecular Biology Lab

Goals for Summer 2020

During this summer internship, I hope to develop comfortability with biology lab techniques as well as using a nanopore processor, the instrument we will be using to conduct our study this summer. I want to become more familiar with nanopores in general as a way to analyze organic macromolecules such as DNA. I hope our study this summer results in a published paper as I would like to know what that whole process is like, from hypothesis formulation to finally publishing. This internship would give me experience working in a biology lab as well as published research under my belt, allowing me to pursue more advanced projects after this summer is over, and making my resume/CV more competitive for future internships.

My ultimate career goals are to pursue paleontology, so while this project directly has little to do with my interests for graduate studies, I believe the molecular techniques I will learn will be useful, especially as genetics is becoming increasingly important in understanding phylogenies.

How Covid-19 changed my summer plans

Before covid-19, I had an internship lined up and planned to visit friends all summer. And to some extent that actually hasn’t changed. I had been accepted for an internship at the Cleveland Museum of Natural History studying placoderm fossils which was unfortunately cancelled because of the virus. This was going to be my first hands on experience with fossils and now I only have the summer after my junior year to be able to develop those skills. I had also planned to visit a few friends around California but obviously that too was no longer possible. Thankfully the Geopaths internship came in like a saving grace so I’m no longer stressed about getting research experience this summer. For the weeks where I would’ve been in Ohio scrubbing rock matrix off dunkleosteus fossils, I’m now sifting through seawater for bacteriophages and analyzing their genomes.

Since social distancing hasn’t allowed me to visit the friends I had planned on spending time with over the summer, we instead opted to keep in touch virtually via a Minecraft server; where we dealt with our collective boredoms exploring and building together. While in any other scenario I wouldn’t be sitting at home all day, I’ve adjusted to these stationary modifications to my original summer plans. At the end of the day, this too shall pass.

GeoScience Career Panel Reflection

My post-graduation goals are to go into a graduate program and eventually into academia, so a lot of the career advice didn’t personally resonate with me. However one thing that I did appreciate learning about was how valuable it was to have a diverse array of experiences. Something I have been paranoid about is getting experience working with fossils before applying to graduate school. Even if I get an internship next summer doing work with fossils, I was scared I wasn’t going to be as competitive for a graduate program as an applicant who has had years of experience. After listening to Friday’s career panel, I’m more confident that having a diverse set of experiences, with geoscience and biological lab skills, is going to help me down the line.

Final Reflection

Before Covid-19, I had an internship lined up and had planned to visit friends all summer. To some extent that actually hasn’t changed. I had been accepted for an internship at the Cleveland Museum of Natural History studying placoderm fossils which was unfortunately cancelled because of the pandemic. This was going to be my first hands-on experience working with fossils and now I will have to wait for the summer after my junior year to develop those skills. I had also planned to visit a few friends around California but obviously that too was no longer possible. Thankfully the Geopaths internship came in like a saving grace so I wasn’t stressed about getting research experience this summer. For the weeks where I would’ve been in Ohio scrubbing rock matrix off dunkleosteus fossils I sifted through seawater for bacteriophages.

My internship involves, and still continues to involve, working with Dr. Deamer and at Ontera Inc. where we analyzed seawater samples for marine bacteriophages using a NanoCounter. The focus is on marine viruses called bacteriophages that specialize in parasitizing prokaryotic hosts. Marine phages make up the most abundant biomass in the ocean, with up to 250 million phages per milliliter of seawater, outnumbering their bacterial hosts nearly ten to one. As many of these phages attack photosynthetic cyanobacteria, they represent a large yet uncounted part of our global carbon cycling.

Literature on marine bacteriophages is extremely limited, as it is difficult to isolate and quantify them from solution. On top of this, many of their hosts are difficult to culture in lab conditions, which renders the phages themselves difficult to culture and study. The purpose of our study is to test a new solid-state nanopore device called a NanoCounter to see if it can detect and count different bacteriophages in seawater. The NanoCounter, designed and manufactured by biotech company Ontera Inc., counts macromolecules like DNA, RNA, and other polymers and characterizes them by size. It does this by using a voltage to drag molecules in a solution inserted into a test strip through a nanopore and records the change in ionic current as the various particles move across the pore. The accompanying software then characterizes the detected particles by their overall disturbance in current and the duration of the signal abnormality.

Seawater for this study was collected at various locations along the northern California coastline. Preliminary samples were collected from Davenport landing; approximately 100mL from tide pools and 50mL from knee-deep seawater. These were then diluted with a 1:1 ratio of sample and lithium chloride buffer. The resulting solutions were then injected into their respective test strips and tested with the NanoCounter for particles. To our knowledge, this is the first time that a nanopore device has been used to monitor the phage content of water samples.

As a preliminary test, the above mentioned test strip of diluted tide pool sea water was inserted into the NanoCounter to see if the instrument’s detection capabilities were sufficient for our study. The test ran smoothly for approximately the first ten minutes of running the device, however, the 45nm pore eventually became incurably clogged and results after the ten minute mark were of ambiguous quality.

From numerous samples of varying concentrations and solutions, tests similar to the one described above were run, and data was collected in terms of the magnitude and duration of signals passing through the pore. We learned that detergents generally do not work with solid-state pores because they tend to clog the pore as well as make passing polymers appear ‘stickier’. The most interesting observation made, however, were the signals present after the addition of RNAse and DNAse to a diluted sample of pond water.

The addition of these two enzymes produces many new data points (yellow, red, and purple) with very high signals, such as the one in the image below. That means these enzymes are reacting with something much larger than the pore or any typical strand of nucleic acid and allowing them to pass through the pore.

I learned so much from this internship. First of all, Dr. Deamer walked me through the scientific process, from coming up with a hypothesis and reading primary literature to writing a report. Dr. Deamer still finally has to run a control sample of known phages and send me the data, but after that I will learn how to synthesize a cohesive paper from that. I also learned how nanopores work, and in particular nanopore sequencing, a third generation genetic sequencing technique that I can apply to many other future projects.

The signature of a sizable particle quickly passing through the pore. The particle only briefly blocks the current passing through the pore, creating the downward disturbance seen in the graph.