Yan Yang from the Oxford Nanopore Technologies Technical Services North America West Coast Team, presented the NCM 2022 Masterclass “How to select the right library preparation workflow for your experiment.” Yang explained how Nanopore DNA/RNA sequencing works as the nucleic acid is passed through a nanopore and electrical signals are interpreted into sequence data. Yang explained that adapters are needed. For DNA sequencing kits, there are two chemistries: ligation and rapid. Ligation chemistry is optimized for high-throughput, while the rapid is optimized for speed. The ligation sequencing kits require about sixty minutes of prep and can take a range of input samples including gDNA, cDNA, and amplicons. There are PCR and PCR-free multiplexing options. The rapid kits use a transposome complex to cleave and tag the fragment ends. There is a field sequencing kit available for cold-chain-free transportation and storage. The rapid chemistry can linearize plasmids, Yang explained. I had not considered this advantage! The rapid chemistry has been adapted for the ultra-long DNA sequencing kit. It is transposase-based and can reliably generate N50s !50 Kb. Yang mentioned that this kit can be paired with the NEB Monarch High Molecular Weight kit we use. There are PCR-based sequencing kits that are optimized for low input. There is a PCR sequencing kit that is ideal for low input amounts (~100 ng) and a rapid PCR barcoding kit that is optimized for lower input and speed. I have used the rapid PCR barcoding kit and it was easy! One must consider that the PCR step does take a couple of hours.
Targeted sequencing options include the 16S barcoding kits for taxonomy classification, the Cas9 targeted PCR-free enrichment. The adaptive sampling option is done thorough the software by enriching or depleting the sequences you select. Yang explained that the 16S barcoding kit is best for enrichment but whole genome sequencing is advised for species-level detection. The Cas9 targeted library prep can help sequence regions of interest through the specific cleavage and ligation.
For RNA, Yang recommended the direct RNA sequencing kit for detecting modifications and sequence. The prep is less than two hours and requires 50 ng of polyA mRNA. The cDNA sequencing kit offers high-throughput sequencing of full-length transcripts. cDNA sequencing couple with PCR offers the highest data output. This kit is also compatible for single cell transcriptomics.
Yang explained that kit 14 promises accuracy of 99% and higher throughput. The adapter is new and offers high capture. Interestingly, it also provides a chance the chance of the second strand being sequenced for duplex sequencing. I am interested in duplex sequencing.
Yang shared that the Voltrax offers automated library prep, though I know they have a limited number of protocols. Yang did explain that ONT offers several automation options for liquid handlers. I wonder if they have scripts that they can share?
Yang highlighted the Nanopore Community website to learn about updates and protocols. The protocol builder tool can help with selection of kits and protocols. I haven’t used the protocol builder tool more than a couple of times. I should revisit it to learn how to use some of the kits/protocols shared in this session.
