London Calling 2021: Day 1

For the second year running London Calling finds itself online, but with over 5600 people registered to attend the first day, the virtual format is still demonstrating its ability to reach wider audiences than previously possible. Delegates tuning in today could view and take part in our Masterclass and Showcase Stage content, covering experiment planning and design, live demos, data analysis sessions and networking. Keep reading below for more details.

Masterclass: How to plan your experiment with Tim Walker

'A good scientific experiment always starts with a hypothesis' were the wise words from Oxford Nanopore's Tim Walker to get the day started. When considering experiment design, whilst people may commonly start with their sample and work through to their answer, Tim took care to highlight that with nanopore sequencing we encourage users to start with the end of their workflow - what is it you're looking for in your data? Once you know this you can work backwards from there to ensure you're using the correct device, flow cell, kit and extraction to achieve your required coverage depth, read length etc. He then went on with specific examples for whole genome, targeted, RNA or cDNA and metagenomic sequencing.

Masterclass: Extracting high-quality nucleic acid with Vania Costa

If you haven't encountered Vania before, you should have done. As the Godmother of sample extraction there is no-one better placed to guide you through the extraction of high-quality DNA and RNA. In this masterclass she first introduced her 'lab hacks 101' for guidelines on what to consider when planning your experiment and choosing your extraction method, before giving more specific practical examples on ultra-long DNA, structural variants and methylation. Touching on fragment length, tissue types, extraction kits, contaminants and QC methods, her mantra pervades through this talk 'You cannot sequence what you do not have'.

Masterclass: Choosing the right nanopore sequencing kit for you with Akelia Wauchope-Odumbo

To continue the theme established with Tim at the start of the day, with Oxford Nanopore's different kit offerings it is good to have guidance on which one you should select to permit arrival at your desired biological answer. Here, Akelia made an important point regarding methylation data and nanopore sequencing - there is no additional lab prep required - before going on to elaborate on Oxford Nanopore's native DNA, amplified DNA, RNA and targeted sequencing kit offerings. There was emphasis placed upon the difference between ligation and transposase-based sample preparation approaches, and for both of these an explanation of the multiplexing options available to ensure you can make best use of the sequencing yield available to you. With a suite of options available Akelia's summary was simple; 'Whatever your application we have a kit to suit your sequencing needs'.

Demo: Flow cell loading with Andrada Tomoni

To take a break from presentations and allow users to begin to get hands-on with the technology, Andrada gave an interactive demo for users in over 50 countries who had pre-registered to receive the Demo Flongle flow cell loading kit. In the first half of the session she covered exactly what you would do in the lab to load your Flongle flow cell; prepare it, prime it, load it, seal it. Care should always be taken to avoid bubbles. After the theory, viewers were then invited to don their goggles and gloves so they could follow along with their demo kits at home.

Showcase stage: Rare disease - Part 1

Continuing the demo theme, a team composed of clinical applications scientists, wet lab scientists and bioinformaticians then used our Live Lounge Showcase Stage to introduce us to an applied example of using Oxford Nanopore's technology in a real-world scenario. Prader-Willi syndrome is a rare human genomic disorder focused around problems arising in the 5 Mbase Prader-Willi region on Chromosome 15, which normally is a result of spontaneous deletion in early embryogenesis or the inheritance of two maternal alleles at the Prader-Willi region. However there is a third mechanism which suggests a hereditary basis for the disease. In this session the team covered the mechanisms for this, before displaying how samples to permit research into this topic could be prepared and QCd on the Flongle and MinION Mk1C.

Masterclass: Getting started with data analysis and basecalling with Bryant Catano

Back to our Masterclass topics and our delegates now moved into into analysis of sequence data. Bryant Catano opened with an explanation of file types and formats (raw data, alignment formats, reference genome files, annotated files and result files), before delving into how to interact with the command line - not as scary as it sounds - to carry out alignment of your basecalled sequence. 'The command line is used because it's very fast, it's very flexible, it gives you much greater control', but Bryant does recommend keeping a cheat sheet of common commands to hand when you first start out as this will help significantly as you find your feet. To finish up he introduced EPI2ME Labs tutorials, a didactic approach enabling users to get to grips with command line analysis concepts. Those who wanted to take it to the next level needed to stay tuned for the following session.

Masterclass: Taking data analysis further with Anthony Doran

The end of the day saw Anthony Doran taking a deep breath and plunging into the world of secondary analysis with your sequence data. Bringing it right back to the start of the day with Tim and designing your experiment, Anthony mirrored this by stating how the final answer from your analysis needed to be considered in scope of the steps which would come before it. If you didn't start with the correct data and file formats, your final answer wouldn't be possible to achieve (to paraphrase Vania, you cannot analyse what you do not have!). Genome assembly, structural variant detection and methylation were all covered, with the analysis consistently being tied back to your earlier experimental decisions; did you PCR, have you used the Ultra-long sequencing kit, and do you have enough coverage? As well as the tools to use, Anthony elaborated on the publicly available datasets with which you could try the nanopore data before you buy, allowing the end user to demonstrate themselves that nanopore sequencing is capable of answering their specific biological question.

Tune in tomorrow for the start of our talks and presentations among much much more!