Akelia Wauchope-Odumbo, a Technical Services Manager – Americas with Oxford Nanopore Technologies (ONT), led a Masterclass at London Calling 2023. I watched the recording tonight, as the session title aligns with the course goals for the ONT-based lab I am designing: “How to get started with nanopore sequencing and plan your experiment.” The session started with the ONT catchphrase: “from sample to answer” and focused on guidance on planning an ONT-based experiment. Wauchope-Odumbo described the nanopore as a protein that serves as gateway between two systems. The pores have been selected with the best features for sequencing in the ONT platform. The current produced by the ion flow is interpreted by a basecaller to provide sequence data. Wauchope-Odumbo noted that the motor protein helps pass a strand through the nanopore. Wauchope-Odumbo highlighted that ONT allows for PCR-free sequencing, if desired, and is read-length agnostic… including ultra long sequences. Methylated bases can also be captured and interpreted. ONT is not a sequence-by-synthesis platform and enables versatile applications. To start planning an experiment using ONT devices, guidance for extracting DNA/RNA is a good place to start exploring. Wauchope-Odumbo described how the nucleic acid that has been extracted from a desired sample is turned into a prepared library that will be sequenced and produce data, analyzed through bioinformatics to obtain an answer. Considerations for planning an experiment are: the goal, the size of the target, the sequences of interest, and other considerations such as throughput and speed. Wauchope-Odumbo emphasized qualifying and quantifying the extracted nucleic acids obtained from samples. For DNA, there are ligation-based, rapid, and ultra long options as well as rapid PCR, 16S, and Cas9. For RNA, there are direct RNA and cDNA PCR sequencing kits. Native and rapid multiplexing options are available. Without any library preparation modifications, adaptive sampling can be used to enrich for desired sequences. ONT has released a protocol for recovery of a prepared DNA library to load a new flow cell. The instruments to sequence on will also affect the throughput: from Flongles to multiple MinION flow cells on a GridION to PromethION flow cells. In terms of applications, Wauchope-Odumbo shared examples of sequencing in the field because of the portability of instruments. For software, ONT has a decision tree diagram for different data analysis options. MinKNOW can do basecalling, alignments, and some barcoding/demultiplexing. EPI2ME has several near-realtime cloud-based options. EPI2ME labs offers additional often command-line options. Wauchope-Odumbo recommended doing all the hardware and flow cell checks and using the protocol checklists. The Nanopore Learning and Extraction Protocols sections of the Nanopore Community website have resources that are very helpful when planning an experiment. I have watched similar sessions and Masterclasses before… yet every time there is new material and examples of applications.
