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Are you sequencing the whole plasmid or just your inserts? The reason I'd never use a nanopore for my plasmid work is because the reason I sequence is to confir
by vikramkr 6y ago
Are you sequencing the whole plasmid or just your inserts? The reason I'd never use a nanopore for my plasmid work is because the reason I sequence is to confirm assembly and sequence, especially after cloninf protocols, which is going to be one of the more common (if not most common) use cases for plasmid sequencing. I need Sanger for the base pair level accuracy.
- koeng 6y agoWhole plasmid. In my experience, Sanger is less accurate than Nanopore, especially since you’re getting 1x coverage per read, and in order to reliably get good coverage of sequence you need a primer every 600 base pairs. Consensus reads on Nanopore aren’t that bad, and you can automate them way better than Sanger. I haven’t found any issue using them - IMO the “it isn’t accurate” stuff isn’t very accurate. Also, base pair resolution is bad on Nanopores primarily at homopolyer stretches. Guess what you can’t synthesize in the first place? (Edit: I’ve done Sanger sequencing on about 3,000 plasmids at once before for high throughput cloning, and that SUCKED.)