Interview: OnRamp – a tool for rapid, multiplexed validation of plasmids using nanopore sequencing

Mel Englund is a Postdoctoral Researcher in the Boyle Lab at the University of Michigan, USA. Her research uses a nanopore-based sequencing workflow – OnRamp (Oxford Nanopore-based Rapid Analysis of Multiplexed Plasmids) – to validate plasmids used in plasmid-based reporter assays for the characterisation of cis-regulatory elements in the human genome. We caught up with Mel to discuss her research into cis-regulatory elements, how nanopore sequencing is changing our understanding of cis-regulatory elements and the importance of plasmid validation in these studies.

You can hear more about Mel's work in the recent webinar 'OnRamp rapid multiplexed validation of plasmids using nanopore sequencing'.

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What are your current research interests?
I am currently working on development of a novel high-throughput assay for characterisation of cis-regulatory elements in the human genome. This project has given me the opportunity to branch out to investigate a number of fascinating interconnected topics including cis-regulatory element mechanisms of action, transcription and how mRNA untranslated regions impact transcript stability and translation, insulator function, CRISPR biology, and the complexities of rational plasmid engineering and reporter assay design.

What first ignited your interest in molecular biology and what led you to focus on non-coding sequences in the human genome?
My interest was sparked initially by two excellent molecular biology courses I took at the University of California, Berkeley. Biophysical chemistry revealed the organised chaos that underlies molecular and cellular function. In genetics, I found an unending source of interest in unravelling the code that determines why humans are the way they are. My focus on non-coding sequences fit naturally with my desire to work on engineering tools and methods for molecular biologists to use in mapping the as-of-yet undeciphered portions of the human genome.

How is nanopore sequencing changing the way we understand cis-regulatory elements? How has it benefited your work?
Nanopore sequencing has the potential to impact the study of cis-regulatory elements in a number of ways. First, in the rapid expansion of our ability to fully map out genomes across species. Recent work in our lab has helped support how frequently mobile elements can become co-opted by the genome as potentially functional regulatory elements. However, our inability to properly map these elements using short-read sequencing, due to the presence of many highly similar copies of these elements in the human genome, has limited our ability to characterise these regulatory functions. Nanopore sequencing’s long reads solve this problem, allowing much more effective and accurate mapping of mobile element sequences in the human genome.

What is the impact for researchers of having a scalable and accessible method to perform plasmid sequence validation?
The impact of scalable plasmid validation is also the second answer to how nanopore sequencing helps us to better understand cis-regulatory elements. One of the fundamental tools for studying cis-regulatory element activity is the plasmid-based reporter assay. Obtaining accurate and reproducible data from these assays requires that plasmid sequences are precise and do not contain cryptic errors. However, current tools for validation of plasmid sequences have been limited in scale, often meaning only partial sequences are validated. Using nanopore for plasmid sequencing allows for rapid, scalable validation of the entire plasmid, as the long reads easily span the entirety of large plasmids. This reduces the potential for introduction of undetected errors which impact function and therefore lead to inaccurate interpretation of assay results.

What have been the main challenges in your work and how have you approached them?
The main challenge related to my specific projects has been the frustrating way in which plasmid-based reporter assays are truly such a good model for regulatory activity in many ways. Frustrating, because it means that while we tend to visualise vectors as a simple, precisely manipulable, engineered arrangement of parts, they often are not. Many of the complexities of cis-regulatory function that apply in a genomic context (element interactions, context-dependence, antagonistic and synergistic relationships) are also present in plasmids. Through my work developing novel, complex reporter assays I have developed a growing respect for all that we still have to learn about them.

What’s next for your research?
I am looking to take the lessons I have learned in assay engineering and regulatory biology characterisation and translate these skills into supporting novel tool and treatment development in the biotechnology industry. I am excited to break into new topics and develop new skills. I also keep coming back to an interest in better understanding what happens after cis-regulatory elements act during transcription. I am interested in deciphering the rules guiding how RNAs are encoded, spliced, stabilised, how mechanisms of transcription and cis-regulatory element activity might be intertwined, and how this impacts assay design and RNA-based treatment development.