Sample preparation for Oxford Nanopore DNA sequencing

[Smriti]

Hi All,

I am preparing a review on sample prep. steps needed for different Sequencing workflows (Illumina, PacBio, Oxford Nanopore...). In this regard, I am trying to gather information about workflow of Oxford Nanopore Technologies. The company claims that "Making electrical measurements of unmodified DNA removes the need for complex samples prep and the high-performance optics found in today's sequencing systems."

Can anyone help me in knowing the sample prep. steps of Oxford Nanopore workflow. Do they also shear DNA, repair ends, ligate adapters and then sequence some particular length of fragments in one time?

Please provide your inputs.
Regards,
Smriti

[xxqtony]

If I correctly understand what ONT tries to convey in its vague description, its sample prep should be super easy, with only 5 eppendorf tubes. They say in the future it would be one-step preparation.

[gringer]

I recall something like a 5' overhang with an attached modified sequence. The modifications are to make it easier for the pore to find tagged sequences, and stop the passage of the sequence through the pore until the machine is ready to process the sequence.

[AllSeq]

Yaniv Erlich has a really nice explanation of the ONT sample prep on his blog: http://erlichya.tumblr.com/post/6487...epresentatives

[gringer]

Yaniv Erlich has a really nice explanation of the ONT sample prep on his blog: http://erlichya.tumblr.com/post/6487...epresentatives

Right, that's the one I read. Here's the text:

The transposase is pre-loaded with two types of DNA fragments: a loop and special 5’ and 3’ adaptors. The loop is just a short DNA sequence to connect between the two strands of the DNA fragment of interest into one long molecule. The 5’ adaptor has two purposes: first, it creates an overhang that can later fit into the nanopore. Second, it has a special sequence motif (or chemical modification - they were quite secretive about it), denoted by *, that stops the entire DNA fragment from slipping through the other side of the nanopore. The 3’ adaptor is attached to a hydrophobic molecule (denoted by “H”). The hydrophobic molecule is attracted to the nanopore layer, guiding the DNA library to the nanopore area, and reducing the search space from 3-dimensions to 2 dimensions.

...

The structrual hindrance * on the 5’ adaptors prevents the passage of the molecule. Once the run starts, electric potential is applied, generating sufficient force to overcome the hindrance and get the molecule through the nanopore.

According to OxNano, the 3’ end is expected to break and release the entire fragment of interest. They didn’t give specific details, but one option is that the 3’ end is composed of two overlapping DNA molecules that are attached by hydrogen bonds. One molecule is covalently linked to the DNA fragment of interest and the other is covalently linked to the hydrophobic molecule. Pulling the DNA fragment generates enough force to break the 3’ part in two.

Future plans: OxNano is working to reduce the sample prep into one step. One idea is to get rid of the hydophobic molecule and instead create a nanopore surface that directly attracts the DNA molecules.