Plasmidsaurus redefine the gold standard: whole-plasmid sequencing with Oxford Nanopore

At the Nanopore Community Meeting 2023 in Houston, Texas, we heard from Mark Budde, co-founder and CEO of Plasmidsaurus, and how he started using Oxford Nanopore Technologies to sequence plasmids.

Plasmidsaurus offer sequencing as a service, specialising in nanopore sequencing of plasmids. There are over 500 Plasmidsaurus drop boxes located around the globe, with overnight plasmid sequencing available in central locations.

Replacing Sanger sequencing with Oxford Nanopore

Mark described how ‘DNA is the language of biological systems’, and it can be studied by modifying DNA with plasmids. Plasmids are small, circular DNA molecules that are easily manipulated and incorporated into genomes; however, they must be verified to ensure that the manipulated regions are correct.

In a recent Nature article, Bruce Lahn, the Chief Scientist at VectorBuilder, a company providing gene-delivery tools, stated that ‘he and other biologists have been noticing problems with plasmid quality for years’, suggesting a lack of plasmid quality control. In their recent analysis of over 2,500 plasmids, they found that nearly half contained errors that would have negatively impacted gene therapy experiments, emphasising the critical need for plasmid verification¹.

Plasmid verification is achieved by DNA sequencing and Sanger is considered to be the gold-standard method but, Mark explained, ‘what we found is that nanopore is a far superior alternative’.

Mark first started investigating Oxford Nanopore Technologies ‘almost exactly 10 years ago’, when he was trying to sequence plasmid DNA with many DNA hairpins. He was investigating a lineage system in which cells recombine their DNA as they divide; however, the recombinase sites created DNA hairpins that Sanger sequencing could not span. Two years later, he received a MinION and was able to easily sequence the whole plasmid — the nanopore reads spanned the hairpins easily, with no evidence of artefacts.

‘That was sort of like an “aha” moment that this is ultimately what’s [going to] replace Sanger’.

Direct sequencing of plasmid DNA

When Mark first heard about Oxford Nanopore, he explained, his initial thoughts were: ‘this sounds too good to be true … I don’t think it’s ever [going to] work, but if it did, it would be the only thing you would ever use’. Mark went on to highlight the benefits of using nanopore sequencing, starting with ease of use because primers are not required. Without the need for primers, there are fewer of the sequencing constraints that are common with Sanger sequencing, meaning DNA hairpins, high-GC content, and repeat-rich regions can all be easily sequenced with Oxford Nanopore.

Furthermore, nanopore sequencing is quicker and easier to scale up because primers do not need to be designed, prepared, or troubleshooted — the plasmid just needs to be cut and the adapters attached for sequencing. Mark reflected on a time when he struggled to sequence a construct with Sanger sequencing. He ended up losing a week of work due to troubleshooting the primer design needed to span a stop codon. With nanopore sequencing, he would have been able to sequence ‘right through the whole thing — no, no troubleshooting’.

‘… when we’re talking about accelerating something by a week, that’s a big deal, right? That has real-world ramifications’.

Tackling repeat-rich and complex regions

GC-rich regions are also no match for nanopore sequencing. A common question Plasmidsaurus receive from their customers concerns CAG promoters: ‘hey, you know your sequence for the pCAG is different than the map I have?’.

The CAG promoter (pCAG) is the strongest mammalian promotor for expression and is very GC rich, meaning it cannot be resolved with Sanger sequencing. Therefore, the sequence maps for pCAG are usually estimates due to sequencing difficulties with legacy techniques. However, because nanopore sequencing can generate any read length, GC-rich regions can be sequenced without difficulty, leading customers to often receive unexpected results for their pCAG plasmids.

Similarly, nanopore sequencing can tackle repeat-rich regions — ‘repetitive DNA is not fun … unless you’re using nanopore’. Mark explained that with Sanger, if the repeat region is longer than the read length, you can’t assemble it. As this is not an issue with nanopore sequencing, Plasmidsaurus often receive customer samples with over a hundred tandem repeats that they can sequence without a problem.

Revealing the whole picture

The final benefit of nanopore sequencing that Mark described was its single-molecule read capability, meaning that monomer and dimer plasmids can be discriminated. Occasionally, nanopore sequencing will reveal that a customer has a dimer plasmid rather than an expected monomer due to plasmids naturally multimerising. Unlike with Sanger sequencing, the whole plasmid is sequenced with long nanopore reads, revealing the whole picture — including any number of plasmid copies in a single plasmid, showing a ladder of sizes in the results.

‘One of the crazy things about nanopore, right, is you’re just getting a single-molecule read from one end to the other.’

Often people send in samples of synthesised plasmids they have ordered and are surprised by the unexpected sequencing results they receive. Mark explained that, more often than not, it turns out that the customer has received the wrong plasmid. Either the wrong plasmid was sent by mistake, or a deletion occurred in the tube during transfer — a very common issue.

As an example, Mark explained that in a lentivirus, the backbone will recombine, and a fragment will ‘pop out’, creating a deletion. However, this deletion will not be noticed by Sanger sequencing due to the primer having been designed specifically to the original strand sequence, missing the strands with the deletion and producing artificially good sequencing results. In contrast, because nanopore sequencing enables direct analysis of the DNA, a mixture of plasmids can be distinguished, providing a true view of the plasmids in the tube.

Advancing sequencing services together

Mark closed out his talk by highlighting his customers’ great work, including plates of bacterial colonies depicting dinosaurs, and emphasised that Plasmidsaurus also provide sequencing services for PCR products, bacterial colonies, and yeast, often within 24 hours — ‘… a game changer for people that are doing microbial genome engineering’.

As of the 23^rd July 2024, Oxford Nanopore Technologies and Plasmidsaurus will collaborate to advance plasmid sequencing to offer superior data quality and speed, further opening access to sequencing services worldwide. Both companies are committed to developing nanopore applications beyond plasmid sequencing to include microbial, gene therapy applications, and many more to serve researchers’ needs.

Find out more about plasmids and microbial genomics with nanopore sequencing