About Nanopore Sequencing

Comparison of Sanger sequencing to Nanopore sequencing (whole plasmid sequencing)

Both Sanger and Nanopore can be performed with either linear DNA (Amplicons/PCR products) or Plasmids which are circular "mini genomes" present in bacteria. The primary benefit of sanger in either of these scenarios is that Sanger has a very high level of coverage and a high confidence in accuracy. The main limitation of Sanger is that it can only sequence about 1200 bases in a single run. This is especially a concern with plasmids which are typically bigger than 3000 bases. Sanger is very good for sequencing very small fragments of DNA.

Nanopore can sequence millions of bases at once with almost no size limitation (although the technology struggles with very small amplicons). This means that Nanopore can sequence an entire plasmid at once. This also eliminates the need for primer walking if that is something a customer is using to isolate a sequence. Nanopore also has relatively high confidence but not as high as Sanger. Nanopore is almost always the best technology for plasmid sequencing and is also better when the amplicon/PCR product is larger. Obviously Nanopore can also be used for whole bacterial genome sequencing which is a very very good way to identify a bacteria and its clade.

TLDR:

.Sanger is best for smaller amplicons and samples that need very high confidence.

.Nanopore is better for plasmids, large amplicons and whole genomes.

As far as optimizing sample prep, we recommend bead purification (bead purification is especially good for PCR products) or spin columns. If the sample is purified I would do so as soon as possible after PCR or extraction. Of course we can always perform these services for our customers. If prepping plasmids I would recommend RCA over mini-preps. Ship all samples in TE buffer or DI water. Make sure to submit at least 8ul of samples.