by Shreya Palakurthi
Introduction and Goals:
This summer, I worked in the Thio Lab as an independent researcher. My objective was to
leverage single cell sequencing to investigate the partial dynamics of viral diversity within the
precore and basal core promoter regions in the hepatitis B virus genome to determine if spatial
clustering exists and whether there is evidence for cell-to-cell spread or clonal proliferation. I
adapted a methodology that has been used with success to perform single cell sequencing
targeting the Pol region of the HBV genome, which includes single cell laser capture
microdissection, DNA/RNA extraction from single cells, RT-Polymerase Chain Reaction, PCR
amplification, agarose gel loading and electrophoresis, and Sanger sequencing. My goals for
this project were to: (a) attempt to determine if spatial clustering of HBV exists, and if so create
new hypothesis concerning cell-to-cell spread of infection or clonal proliferation of infected
hepatocytes; (b) gain proficiency in laser capture microdissection and potentially find relevant
applications of techniques such as digital droplet PCR; and (c) learn how to collaborate,
network, and gain independence in driving a project forward and troubleshooting experimental design.
Key Experiences:
Although I intended to process and sequence all~288 clinical hepatocyte single-cell samples by the end of the summer, that did not go quite as planned! I ran into a slew of contamination issues throughout the course of the summer. I had to address these issues in a multitude of ways, from modifying my sterile technique to altering my protocol to diminish contamination risk. In terms of key experiences, many of them center around revelations I made while troubleshooting my protocol while processing samples. Initially, I ran into multiple issues related to switching my positive and negative controls. I had to switch from HB6
(clinical) samples to NDRI57 (clinical sample with a known result) to test where I was running into issues with my protocol. Despite being familiar with the DNA/RNA extraction protocol due to previous experience with extractions, I had to reacquaint myself with the process to determine whether I was switching tubes or if I was running into some other kind of unanticipated error. Moreover, I was supervised by one of the postdoctoral fellows in my lab who was familiar with the protocol to see if she would be able to determine where I was running into issues. Despite being supervised twice, she and I were unable to determine the issue at hand, which was a frustrating experience to say at the very least. I had to resort to adding an extra layer of separation between my tubes when executing my protocol to dry to diminish any carry-over contamination. and tube switching, which would show up in the agarose gel I ran at the end of the protocol. The
nature of the protocol in itself posed to be a serious hurdle, as I would only be able to determine
contamination at the very end of the protocol while running an agarose gel, which would
visualize my positive and negative controls. Moreover, as I was working with known positive and
negative controls, there was no real way for me to determine whether or not I was receiving a
false positive or negative with my HB6 samples, as the positivity rate is variable between clinical
sample sets and even spatially within a hepatocyte slide sample itself. Despite these setbacks,
an achievement I made was being able to address the tube switching issue, as in my most
recent set of samples I had no issues with tube switching issues and now just need to address
carry-over contamination.
Skills and Knowledge Gained:
Despite gaining a variety of technical skills, including DNA/RNA extraction, PCR, gel agarose
preparation and electrophoresis, and Sanger sequencing, I began to shadow other skills such
as laser capture microdissection, which is a skill I intend on further developing after I am able to process my first set of 96 clinical samples from HB6. This is an interesting skill to possess, and is heavily relevant to both my project and further investigations as it enables me to prepare my own set of single cell samples from a slide of tissue, which I can later use in other types of experiments. Throughout the summer, I also gained a variety of soft skills related to literature review, presentation, and general networking with other people. I used to be nervous when presenting slides to share my results, but through regular practice in weekly lab meetings and troubleshooting sessions with other members of my lab, I grew to appreciate this skill and plan to further develop it in both academic and recreational settings. Moreover, I gained a new understanding of the troubleshooting process, specifically modular troubleshooting. Before, I used to just repeat my entire protocol and hope I did not receive the same results. However, after talking to senior members of my lab, they told me to rerun certain segments of my protocol using results/samples from previous segments, as it would enable me to better determine at which step something may have gone wrong. This skill has saved me a great deal of time, and also has helped me become more efficient by exposing technical deficits faster.
Impact of OKRs:
My OKRs have greatly helped guide me throughout the summer, and have helped me stay on
track in terms of developing the skills I wished to develop both in my project. My OKRs were to
gain proficiency in the literature review and experimental design processes, to determine the
differences between pure science research and translational medicine with respect to virology,
and to produce a model of cell-to-cell transmission of HBV infection within hepatocytes. While
working towards my OKRs, I had to present my results and perform literature reviews. My key
result of reading at least 10 papers about translational virology research and comparing them
with 10 papers about typical pure science virology research was particularly impactful, as it
greatly informed my future career aspirations, as I realized I enjoy experimentation that is less
hypothesis driven, as is typical of NIH grant-driven research, and is more “exploratory” in nature, which is more acceptable by NSF. I found that I also enjoy the troubleshooting process, which is typical of more exploratory research avenues, which has led me to believe that I would like to have more of an emphasis on engineering new biotechnological methods and techniques in my future career.
Lessons Learned:
I learned a great deal during my experience over the summer. I realized that more than anything
else, patience is key in science. A range of issues emerged at the most inopportune times
during the course of my project, and having the patience to troubleshoot and address these
issues was a necessary part of my experience. Moreover, as aforementioned, the process of
modular troubleshooting is vital to any type of research, and was a skill I greatly developed
during my experience. In terms of workplace dynamics, not being afraid to ask questions and
advocate for yourself is very valuable, as towards the beginning of my experience my fear to
ask questions added additional days of delay to my project. Not being afraid to take up space in the workplace and ask questions of people is necessary, as becoming acquainted with a lab
environment and learning new skills is a process that everyone has to go through and is
necessary for any future development.
Future Applications:
The molecular biology skills I learned will definitely be useful in my future career, which will likely be within the realm of virology and academia. Moreover, the soft skills I gained in terms of presentation skills and literature review are greatly relevant to any future pursuit, as they are
necessary in conveying information and even advocating for yourself. The most major career
change I experienced was realizing my love of the engineering process, as I found myself very
invested in troubleshooting my protocol and finding ways to improve the process for myself and others. I hope to further explore avenues within this exploratory pathway, whether they are industry-based or entrepreneurial in nature, and I highly recommend anyone who is interested in something similar to do the same
