I fear laboratory work in the way my 87-year-old grandmother fears her iPhone: everyone tells me it isn’t that hard, but I constantly worry that if I press the wrong button—or put in an extra microliter of buffer—it’s going to explode.
Like many graduate students in ecology, I spent a few years working as a field technician after completing my undergraduate degree. I fell in love with the theory of genetics in high school, but I switched from a genetics major to an ecology major in my junior year of college after taking a lab-intensive class back-to-back with a field-intensive class. The potential that genetic techniques hold for answering ecological questions spurs an excitement that often keeps me up at night. But standing at a lab bench, pushing around clear liquids, I feel lost. Hiking through the woods, looking for birds and insects, I feel at home.
The opportunity to combine field and laboratory methods fell into my lap unexpectedly when my former supervisor, an avian ecologist in Argentina, connected me with my current Master’s advisor. We brainstormed a project that would link Common Nighthawk migration ecology, which my supervisor and her team were studying in northern Argentina, with diet and prey selection. Before diving into this graduate program, I asked an experienced lab member if she thought I would be in over my head with a DNA metabarcoding project. After an immensely long pause, she answered me: “No, I think you’ll be fine.”
The truth of that statement hangs in the long pause. That pause represents the period of learning that is inherent in every new project, but especially for new graduate students designing their own research for the first time. Three undergraduate genetics classes, including one lab class, had prepared me to understand approximately the first three sentences of the research articles that now made up my lifeline. Eventually, though, I was able to grasp more and more of the content I was reading, and I began to find my feet in the eDNA world. I will now take this opportunity to give a huge shout-out to Alberdi et al. (2019) for the pictures and diagrams—science communicators like you are the real MVPs.
This past summer, I applied for a microgrant through the eDNA Collaborative to help fund my nighthawk diet study. Over the past two years, I captured nighthawks in Florida and northern Argentina, collected fecal samples for diet analysis using DNA metabarcoding, and sampled insect communities to assess nighthawk prey selection. I’d written a dozen grant proposals to fund my field supplies, but this was the first proposal in which I specifically asked for laboratory reagents. After a hot and sticky June night catching nighthawks in Florida, I checked my phone to find a voicemail from members of the eDNA collaborative asking if I was sure I’d requested the right reagent in my proposal. I’m sure I don’t have to describe for you the heart-sinking, stomach-churning self-doubt of a student preparing to do something big for the first time and realizing I might be getting it all wrong.
But I hit the books that night and confirmed that I had, in fact, requested the correct reagent. My extraction protocol uses a beta-mercaptoethanol lysis buffer, silica bead-beating, and SPRI-based beads for DNA purification (Vo and Jedlicka 2014, Snider et al. 2022). The beads that I requested in my eDNA Collaborative proposal were not the SPRI beads typically used, but a DIY alternative that uses Sera-Mag Speedbeads. I relayed this information to the eDNA Collaborative and ultimately received a microgrant that allowed me to purchase the Speedbeads, along with a Denovix dsDNA High-Sensitivity Assay kit for DNA quantification.
I am about two months into my lab work, and the change of pace from the field to the lab never fails to challenge and excite me. In the field, we meticulously executed the tried-and-true methods for capturing nighthawks by setting up L-shaped mist nets with audio lures near territorial males. But, as one of my undergraduate professors liked to say, the critters don’t read the books. So, we also set up V-shaped and W-shaped and octagonal mist nets, and we scrambled through mud and Smilax thorns with our handheld nets to find females on nests. We bleached cloth capture bags and stored them inside out to avoid contamination, but when a nighthawk pooped on our shirtsleeves, we scraped up the feces and put it in ethanol anyway. Once we spilled a sample of hundreds of dead insects on the floor of our field house and spent the wee hours of the morning scooping ethanol and bugs back into a jar. The fieldwork was full of trial and error, permit delays, vehicles stuck in mud, and critters failing to read the books. And in all these ways, it was a typical field research project and a smashing success.
Often a fecal sample could be obtained directly from the cloaca while taking measurements.
In the lab, though, the standards are different. We all know that “good enough” in the field is far from “good enough” at the lab bench. The naked eye can’t differentiate 10mM from 1mM Tris-HCl, or a pH of 7 from a pH of 8, but these are the differences between successful and failed reactions. A tube of blank elution buffer looks exactly the same as a tube of high-quality DNA, and at times, only the lab gods will ever know what went wrong. For these reasons, working in both environments, as is typical for eDNA research, can be immensely challenging but equally rewarding. As a student, I am grateful to have the opportunity to learn and grow in both settings.
I have extracted and quantified nearly all of my nighthawk fecal samples, and despite what I’m sure has been some user error with the pipette, nothing has exploded. If a paper titled “Perils and pitfalls of an improved method for eliminating primer bias in high-throughput sequencing—and how ‘bout those semiqualitative results?” appears in my Web of Science alerts, it will not send me running. So far, the words after the long pause have proven true: with the support of a network of scientists expanding access to eDNA resources, a Master’s student with a limited genetics background is progressing toward completion of a DNA metabarcoding thesis. To all the grandmothers out there: your iPhone is not going to explode. (Long pause filled with learning and reading.) You’ll be fine.
Malaise traps (pictured) and UV lights were used to sample insect abundance.
References
Alberdi, A., O. Aizpurua, K. Bohmann, S. Gopalakrishnan, C. Lynggaard, M. Nielsen, and M. T. P. Gilbert. 2019. Promises and pitfalls of using high‐throughput sequencing for diet analysis. Molecular Ecology Resources 19:327-348.
Snider, A. M., A. Bonisoli-Alquati, A. A. Pérez-Umphrey, P. C. Stouffer, and S. S. Taylor. 2022. Metabarcoding of stomach contents and fecal samples provide similar insights about Seaside Sparrow diet. Ornithological Applications 124.
Vo, A. T. E., and J. A. Jedlicka. 2014. Protocols for metagenomic DNA extraction and Illumina amplicon library preparation for faecal and swab samples. Molecular Ecology Resources 14:1183-1197.