Concetta Dipace is a Technical Services Manager with Oxford Nanopore Technologies and presented at the London Calling 2022 a masterclass introduction entitled “Getting started with nanopore sequencing and planning your experiment.” This session was similar in title to one from the Nanopore Community Meeting 2022, though there is always something new to learn in these video masterclasses! They stated the Oxford Nanopore Technologies goal: “to enable the analysis of anything, by anyone, anywhere.” I love that! Dipace also mentioned that nanopore sequencing offers a range of investigations with long and ultralong sequencing. Oxford Nanopore, Dipace noted, offers direct sequencing of a molecule. This ability to read molecules directly without, for example, bisulfite conversion, allows a researcher to investigate methylation signatures in real-time. Dipace also noted that adaptive sampling allows a user to select for certain sequences. Dipace noted that there is a range of Nanopore devices: the PromethION, GridION, and the MinION family. Dipace explained that the Mk1b was released for early access use in 2014. The device has been used in extreme environments, to investigate outbreaks, and even onboard the International Space Station. Dipace explained how Nanopore sequencing works when a DNA or RNA strand passes through a nanopore producing a unique electrical signal that is interpreted by the base caller into sequence data. The longest molecule that has been sequenced was over four million bases! The motor protein modulates the speed of the nucleic acid traveling through the pore.
Dipace recommended planning an experiment with the end first. This approach sounds like backward design! Choosing an appropriate extraction protocol will also depend on the application and system. The choice of kit will depend on parameters such as preparation time, input of nucleic acid available, the type of throughput needed to address the biological question, and the need to multiplex, if appropriate. The choice of sequencing system will depend on the experiment and project too. Dipace also listed several tools for data analysis, including the web-based EPI2ME suite, EPI2ME Labs Workflows, and research softwares and third-party options. I’ve been using EPI2ME Labs based on Jupyter notebooks and enjoy the simplicity of the workflows. Right now, I am using the bacterial genome assembly and annotation pipeline.
Each experimental protocol available from Oxford Nanopore Technologies has an experiment checklist. This information is available as a PDF or website with the necessary reagents and equipment for the protocol. Dipace also mentioned that the Nanopore website has on-demand learning experiences. I have done several of these short online courses and enjoyed them: there are videos and information as well as links to useful tips. The Oxford Nanopore Technologies website has a Getting Started Guide and interactive tool. The tool helps a user select an approach through a series of questions. I want to continue learning from these masterclasses because they usually include suggestions that are sometimes buried in the documentation.
