Yet again the Nanopore Community Meeting found itself online in 2021, but that did not deter hundreds of delegates from tuning in for the Day 1 Masterclass sessions with a number of experts from Oxford Nanopore Technologies. Delegates had the opportunity to learn from presenters on a number of topics including planning their experiment, sample extraction and preparation, and analysis methods including methylation detection and genome assembly. A hands-on, follow-along Flongle Flow Cell loading session brought a tactile dynamic to the day as well.
Nanopore 101: how to get started and plan your first experiment
A familar face to Many, the Director of Segment Marketing James (Jim) Brayer started the day's proceedings with his favourite topic: Nanopore 101. 'Your goal is to answer biological questions, and nanopore sequencing is only a means to that point' he began his talk with, before outlining the range of devices Oxford Nanopore has available to give you access to the nanopore sequencing platform. The core components of the platform and how DNA in solution becomes a basecalled signal were touched upon, before Jim highlighted the unique benefits of nanopore sequencing: Direct sequencing of native molecules, short to ultra-long reads, and real-time analysis.
This lead nicely into Akelia Wauchope-Odumbo, Technical Applications Scientist, covering how new users can go about planning their first experiment. By necessity, this required an overview of our specific sample preparation offerings, but also guidance on which Community resources can help you determine this. Yield and analysis type (One click? Command Line? Guided or self-learning?) were the next options covered, before an applied example demonstrated that you should start at the end (what answer do you need?) before deciding on your analysis, the data volumes, prep and extraction.
Sample prep: how to extract high-quality DNA and RNA
If you haven't encountered Vânia Costa before, you should have done. An Application Scientist steeped in sample extraction expertise, there are few people better placed to give guidance on how to ensure you get a great performing sample for your nanopore sequencing experiment. Detailing her 'roadmap for nucleic acid extractions', Vânia took viewers through several considerations including: planning before you reach the lab; optimal sample storage solutions; choosing the best extraction method for your experimental goals (you cannot sequence what you do not have!); and sample QC. She finished up with an applied example, choosing plant DNA extraction as the topic and showing how all previously-mentioned considerations could be tuned even further under the broad umbrella of "plant".
Library prep: how to generate ultra-long reads
If Vânia's presentation was the main course then Simon Mayes, a Principal Scientist in the Applications team at Oxford Nanopore, was providing the truffle-fries side dish. Nanopores can process fragments of any length, and due to this represent the only technology which can sequence truly ultra-long fragments to aid in genome assembly, resolve challenging regions such as centromeres, or enhance phasing. Generating those fragments and keeping them intact right through until sequencing was the focus of Simon's talk, from tissue homogenisation all the way through to re-loading your flow cell to get the most sequencing data from your library. Recent optimisations and experiments in-house have also demonstrated the preference of using the NEB Monarch HMW DNA Extraction kit for extraction of ultra-high molecular weight DNA, a natural pre-requisite for ultra-long nanopore reads. Complete with videos of the key steps, this was a golden resource for how to best interact with your samples to ensure they retain their length.
Library prep: how to choose the right nanopore sequencing kit
Continuing the theme of choosing the right tools for the job, our Field Applications Scientist Marta Verdugo took over next to guide watchers through choosing the right nanopore sequencing kit for their purposes. Simple sample preparation options exist for a number of applications, with specific categories for DNA or RNA sequencing, as well as targeted approaches. Ligation or rapid chemistries (the latter of which is transposase based, and also utilised in the ultra-long sequencing kits) make up the core DNA kit offerings, while RNA sequencing has available a number of options including, unique to nanopore sequencing, a direct RNA sequencing kit. Marta finished up by covering your options if you wish to carry our targeted sequencing, such as 16S for microbial samples, or Cas9 for PCR-free, cost-effective sequencing for specific regions of interest. Also included here for targeted sequencing was Adaptive Sampling, a sequencing-software-based method for amplification of sequences of interest on your flow cell in real time: no sample prep required for this one.
Sequencing: how to load a Flongle Flow Cell
Giving viewers the opportunity to get hands-on, our next session saw the return of the interactive Flongle Flow Cell loading demonstration. Those users who had pre-registered for the demonstration received everything required to follow along with Mark Wyatt, the Production Sequencing and Kit Release team lead at Oxford Nanopore. In the first half of the presentation Mark covered the layout of the Flongle flow cell before demonstrating the prime, flush and load steps as they would be carried out in the lab. For comparison he then showed the similarities and differences of these same steps with the MinION and PromethION flow cells. With that covered it was time for viewers to have a go themselves, using the provided Flongle flow cells, syringes and buffers to mirror the process they would carry out with the real thing.Our hope is that this is our last virtual flow cell loading, and we see you back in person in 2022!
Analysis: how to basecall and detect methylation
Oxford Nanopore provides a full-stack solution - we have options for preparation, sequencing and analysis. With the first two stages now covered in these masterclasses, it was time for Jessica Anderson of the Technical Services team to cover how to basecall your sequencing data and, excitingly, detect methylation directly from your native DNA data. Unique to nanopore sequencing, FAST5 files containing the raw data are your starting point for sequencing and are generated by the MinKNOW software. Once basecalled, a user will have their familiar FASTQ files. Jessica took a moment to reflect on the very information-rich signal contained within the raw data, and how the accuracy improvements demonstrated by Oxford Nanopore over the past 2 years can all be realised if you re-basecall old data. The signal remains just as rich, but the neural network algorithms employed by Oxford Nanopore are becoming better and better at identifying all the context contained within that signal. Detailing Guppy, the production basecaller from Oxford Nanopore, Jessica outlined basecalling of canonical bases before talking about methylation calling and the impressive results achieved using the R9.4.1 nanopore and native DNA, showing greater uniformity of depth of coverage and lower GC bias when compred to bisulfite sequencing.
Analysis: how to generate assemblies and call variants
To complete our analysis and also bring a close to the day, Anthony G. Doran, a Technical Services Scientist with a Bioinformatics focus, took us through how to generate assemblies and call variants from basecalled nanopore data. Highlighting the wealth of resources available to inform about, as well as provide solutions for, late-stage analysis, he touched upon the range of options available for all levels of bioinformatics expertise with a brief look at EPI2ME Labs tutorials and EPI2ME Labs Workflows. He then went into detail on genome assembly, comparing and contrasting reference-guided assemblies against de novo assemblies, as well as workflows for small or large genomes and the importance of polishing. This was backed up with examples from the literature where nanopore reads had improved existing assemblies, from viruses right through to large plant genomes. To finish Anthony covered variant detection with nanopore sequencing, from reference alignment to SNV detection (using Medaka, PEPPER/DeepVariant with WhastHap, or Clair3 with WhatsHap) and structural variant calling using cuteSV.
Tune in tomorrow for the start of our talks, presentations, and more!