London Calling 2023 opens with a full day of masterclasses delivered by in-house nanopore sequencing experts
This year’s London Calling conference got off to a busy start, with seven masterclasses walking the virtual audience through everything they need to know to perform their nanopore sequencing experiments. Oxford Nanopore’s own in-house sequencing experts delivered sample-to-answer training, with topics spanning from sample preparation, to data analysis, and a new topic this year, how to perform tumour-normal sequencing. The online attendees also had the option to learn from a breadth of content on the virtual platform, from posters to pre-recorded Mini Theatre presentations and product information in the Live Lounge.
Here's a quick overview of the masterclass sessions, all of which are available to watch on-demand in the conference platform until 26th May and following that, on our website.
How to get started with nanopore sequencing and plan your experiment
Akelia kicked off the masterclass series with an introduction to how nanopore sequencing technology works and its benefits across multiple areas of research. Akelia talked through the considerations for planning your nanopore sequencing experiment, before introducing each step of an experiment, from sample to answer. She first provided an introduction to DNA and RNA extraction and an overview of the library preparation kits that can be used — pointing out that precious libraries can be re-used by loading on a new flow cell in order to ‘max out the data from a single library prep’. Akelia then introduced the range of nanopore sequencing devices available before finally discussing data analysis solutions for every level of experience. Akelia summed up her masterclass with an example workflow — characterisation of variants across the human genome at high throughput — and provided links for resources for every stage of the nanopore sequencing process.
How to extract high-quality DNA and RNA
Vânia began her masterclass by explaining how to determine the right extraction method for your sample type and what to consider before performing an extraction. Vânia then provided recommendations on how to optimise extractions for different sample types, with a walk-through of different extraction methods for different matrices, followed by a description of best practices for storing DNA and RNA and how to perform and interpret quality checks in order to maximise sequencing output. Vânia finished up the masterclass by sharing an example — extracting DNA from human blood clinical research samples to optimise for characterising and phasing methylation — to demonstrate the decision-making process behind selecting the right extraction method.
How to select the right library prep workflow for your experiment
Covering the next stage of preparing samples for sequencing, Nikita delivered her masterclass on choosing a library preparation method to best suit your experimental goals. Nikita began by highlighting the Kit 14 chemistry from Oxford Nanopore, which has been optimised for both >99% accuracy and high sequencing output for all read lengths. Nikita then explained the difference between ligation-based and rapid library preparation methods, before introducing the different options available for nanopore sequencing of DNA, RNA, and cDNA. The applications and benefits of different sequencing techniques were discussed, including targeted approaches with PCR-free and PCR-based options, plus multiplexing solutions. Finally, Nikita highlighted the support available online for choosing a library prep method.
How to load a PromethION Flow Cell
In this interactive demo, Thomas began by introducing the range of PromethION sequencing devices, from the compact PromethION 2 Solo to the ultra-high-throughput PromethION 24 and 48 devices. He then talked viewers through the anatomy of a high-output PromethION Flow Cell, before demonstrating how to load a flow cell on to a PromethION 2 Solo, how to prepare and flush the flow cell, and how to load a sequencing library. Thomas also introduced how to load Flongle and MinION Flow Cells. Last but not least, Thomas invited viewers – who had received PromethION Flow Cell loading demo packs in advance of the masterclass – to follow along with him, as they learned step-by-step how to prepare and load their own demonstration PromethION Flow Cell.
How to get started with data analysis
Beginning the masterclasses on data analysis, Bryant introduced the basics of how nanopore sequencing data analysis works, and introduced the file formats involved. He described how to basecall nanopore sequencing data, and how to choose the right basecalling model to suit the specifics of an experiment. Bryant went on to describe the options available for analysis of nanopore sequencing data and introduced ideal software for those who are newer to data analysis: MinKNOW — the software onboard nanopore sequencing devices — and EPI2ME solutions, offering local and cloud-based data analysis. Finally, Bryant outlined how to directly call methylation — including both 5mC and 5hmC — in PCR-free nanopore sequencing datasets, highlighting example data from an imprinted gene.
How to take your data analysis further
Building on Bryant’s introduction to data analysis, Dilrini introduced how to assemble nanopore sequencing data, with guidance on how the assembly process works and options for high-quality genome assemblies for small genomes, large genomes, and from mixed microbial samples. Dilrini shared how, using nanopore sequencing data alone, human genomes can be assembled to near-chromosome-level contiguity, for reference-quality assemblies. Next, Dilrini introduced how to call variants in nanopore sequencing data, including SNVs, structural variants (SVs), and methylation. Finally, Dilrini described the options available for nanopore transcriptomic data analysis, up to the single-cell level.
How to perform tumour-normal sequencing
Finally, collating all the expertise learned throughout the masterclass series, Philipp explained how to perform an end-to-end sequencing workflow — from DNA extraction to data analysis — using tumour-normal research samples. Philipp began by highlighting the benefits of nanopore sequencing for cancer research and introducing how tumour-normal sequencing can be used to identify somatic and germline variants in cancer research samples. He then explained how to extract DNA and prepare libraries for sequencing, with best-practice guidance throughout. Philipp described the range of nanopore sequencing devices available, and their potential uses and benefits within the workflow. Next, he reminded us how to call variants within nanopore sequencing data, using the latest available data analysis tools. Finally, Philipp shared a worked example using tumour-normal clinical research samples across multiple tissue types, highlighting how nanopore sequencing enabled haplotype-resolved characterisation of genomic and epigenomic variants.
This year’s conference is a hybrid event and you can still register for online attendance for the live presentations over the next two days and watch all of these masterclasses on demand on the virtual platform too.