How does a nanopore sequencer work?

You may have come here from my other Nanopore post, if not then this is a follow-up to that and is meant to outline Nanopore sequencing technologies.

How does a nanopore sequencer work: A nanopore is a very small hole, the generally under 1nM in width in a membrane of some kind. It can be made from a biological molecule or ‘punched’ into a solid surface using an electron beam. Nanopore sequencing has a very simple basic principle, DNA strands or single nucleotides are driven through a nanopore electrophoretically. As each nucleobase passes through the pore the current is affected and this change allows sequence to be read out. Each base has a characteristic change in current and, perhaps just as importantly a specific dwell time in the pore. One of the first publications of the idea was from George Church in a 1995 patent (Church et al), this was a year or two before Shankar Balasubramanian and David Klenerman invented the SBS chemistry and formed Solexa. Nanopore sequencing has been around as an idea for a while!

The earliest demonstrations of the technology used α-haemolysin or Mycobacterial porin A (MspA) biological nanopores. Biological and solid-state pores have now been demonstrated and hybrid systems have also been discussed.

Biological nanopores: cells are very good at making biological nanopores like α-haemolysin, they also make many other similar molecules and it is possible to design pores with specific characteristics using site-directed mutagenesis. These can be checked at the atomic level with X-ray crystallography to verify their structure. α-haemolysin pores have been widely used as the hole in the middle is only wide enough for single-stranded DNA to pass through. Unfortunately DNA moves through these pores very rapidly making detection of each base almost impossible. Slowing its translocation down has been a goal of nanopore research. DNA can be coupled with enzymes like DNA polymerase and “ratcheted” through the pore. Oxford Nanopore have been working on “sequencing-by-digestions” where an exonuclease sits above the pore cleaving individual bases from a strand of DNA which pass through the pore allowing sequence to be read out. Biological pores also offer the promise of detecting not just DNA sequence but also interacting DNA:protein molecules and possibly protein sequence. A big challenge for biological nanopores is that they are often embedded in fragile lipid bi-layers and can be affected by physical conditions such as temperature, pH, etc. It can also be difficult to make large arrays of nanopores and we would need many thousands of pores to sequence a Human genome.

Solid-state nanopores: Many groups are working on solid-state systems using ion or electron-beam sculpting of pores in Silicon nitride membranes. Making the pores and the membranes is challenging as spacing and thickness need to be carefully controlled but these systems are much less affected by physical conditions and may also be coupled to electronic or optical read-out systems. The latest advancement appears to be the use of graphene, a two dimensional sheet of carbon atoms as the membrane of choice. The use of solid-state nanopores is not as advanced as the biological approach and similar challenges for controlling the speed of DNA translocation are as yet unmet.

How will my sequencing be affected: Nanopores offer the promise of sequencing a base per millisecond with high accuracy and detection of base modifications like methyl-C. At these speeds one million base pairs can be read-out in about 20 minutes. With 1000 pores in an array a Human genome might be sequenced in an hour or so.

Nanopores also offer a major advantage over current methods even if speeds don’t quite approach this, no labelling is required and single molecules are analysed. This means there are few reagents and possibly no sample prep other than extracting DNA, so it should be easier and cheaper than current methods!

Church et al. Characterization of individual polymer molecules based on monomer-interface interactions. US patent 5,795,782 (1995).
Venkatesan and Bashir. Nanopore sensors for nucleic acid analysis. Nature NanoTech 2011
Branton et al. The potential and challenges of nanopore sequencing. Nat Biotechnol. 2008