Tomasz Dobrzycki, a Technical Applications Scientist from Oxford Nanopore, presented at London Calling 2022 a masterclass on “Preparation: selecting the right library prep method for your experiment.” This is the second session I watch featuring Dobrzycki. This session began with the benefits of nanopore sequencing: real-time information-rich data. Oxford Nanopore Technologies (ONT) offers several options for library preparation. the nanopore is the biological structure that is central to Nanopore’s sequencing system. The passing of nucleic acids through the nanopore causes changes in current… at a speed of 400+ bases per second for DNA. Library prep for nanopore sequencing requires a sequencing adapter with a motor protein that is pre-bound to the adapter. The adapter then facilitates tethering. Dobrzycki mentioned that there are two main chemistries: ligation sequencing and rapid kits. The ligation sequencing kit is optimized for throughput. The two enzymatic steps are end-prep and ligation of sequencing adapters. Dobrzycki noted that this kit is highly versatile, as it can use genomic DNA, cDNA, and amplicons as input. The rapid chemistry is optimized for “simplicity and speed.” Fragmentation of the DNA occurs through a transposome complex that fragments and tags the fragmented ends. This protocol is compatible with barcoding as the barcodes can be introduced in the transposition step. Dobrzycki noted that this kit is best for high-molecular weight DNA. The ultra-long DNA sequencing kit starts with high amounts of ultra long DNA. With this kit, Dobrzycki explained that you can generate N50s >50 kb and 10-20 Gb yields on MinION. They recommend the NEB Monarch kit for extraction. They also recommend loading the flow cell three times and and washing. Dobrzycki then spoke about RNA sequencing and how nanopore sequencing can be direct RNA or cDNA sequencing. With both approaches, information about isoforms is obtained. They recommend the PCR-cDNA for highest throughput, the direct cDNA to avoid PCR bias, and the direct RNA kit for RNA methylation detection. The direct RNA sequencing approach requires 50 ng of polyadenylated template. The process includes an optional RT step, though the cDNA strand is not sequenced. The motor protein for RNA sequencing processes at 100 bps. The cDNA sequencing kit is optimized for high-throughput sequencing of full transcripts and requires 4 ng of poly-A RNA or 200 ng of total RNA. For targeted sequencing, Dobrzycki explained that the 16S barcoding kit is a comprehensive workflow. The Cas9 sequencing kit allows for sequencing of a region of interest. You can design your own probes and sequence the region(s) of interest. Dobrzycki mentioned that you can target up to 100 targets. Adaptive sampling does not require a kit: it is a software-based approach. I want to explore adaptive sampling more carefully and also try cDNA and direct RNA sequencing sample prep workflows. I hope to find a video/session about bacterial transcriptomics.
