A nanopore is a hole in a material that is approximately a nanometer in diameter, a little bit wider than a single strand of DNA. In nature, often the only way to get across a cell membrane is to travel through a pore created by a protein, and there are many, many different types of proteins that allow different types of molecules to pass through. In nanopore sequencing, an electric current is passed across a synthetic membrane that contains a protein nanopore. The membrane is resistant to the electric current, meaning the only way for current to flow across the membrane is to travel through the pore. Because DNA and RNA are charged molecules, the electric current also pulls DNA and RNA through the pore with it, in the same way a current pulls DNA or RNA through agarose in gel electrophoresis. The pore, however, is only large enough for a single strand to fit through, one nucleotide at a time. Because the nanopore is so small, when a nucleotide is in the pore, current passing through the pore is partially blocked, each nucleotide blocking the current in a distinctive way. By measuring the change in current caused by the nucleotide, scientists can use algorithms to figure out which nucleotide caused the change. This is much like how the light passing through a projector is blocked by each frame of film in a distinctive way, allowing us to see the picture.

To sequence a DNA or RNA sample, the end of each DNA or RNA molecule is first attached to a “sequencing adapter”, a small piece of DNA with an enzyme motor wrapped around it. The sample is then put on the nanopore membrane. When an electric voltage is applied to the top and bottom of the membrane, the DNA is pulled into the nanopore until the enzyme motor sits on the nanopore like an oversized cork that is too big to fit into a bottle, and the strand of DNA dangles into the nanopore. The enzyme motor will then work as a helicase, unwinding the DNA double helix. As the DNA is unwound, the enzyme motor allows a single strand of nucleotides to pass through the nanopore at a rate that is slow enough to measure the change in ionic current, nucleotide by nucleotide, until the entire strand has passed through. This process allows for very long sequences to be read. Where other sequencing technologies typically only read a few hundred nucleotides at a time, nanopore sequencing can regularly read sequences that are tens of thousands of nucleotides long. These long sequence reads can be a huge advantage when repetitive elements and other types of sequences can make it difficult to assemble many short sequences into one long one. Once the entire nucleotide strand passes through the pore, a new nucleotide strand will be pulled in, and the nanopore will start sequencing again. And because nanopores are so small, thousands of nanopore membranes can fit on a grid the size of a postage stamp, each simultaneously sequencing a different strand from the same original sample.

Better, cheaper sequencing in a pocket-sized machine

Nanopore sequencing does have its disadvantages. It tends to be error prone; error rates in nanopore sequencing can be as high as 15%. If sequencing large amounts of the same sequence, this error rate can be tolerable because multiple copies of the same sequence will allow the user to recognize and eliminate mistakes. When looking to study small or rare sequence differences, however, such error rates can be a big problem. Changes to the proteins involved in the sequencing process have dramatically reduced this error rate over time, and in the future, it is only expected to become more accurate. And while nanopore sequencing is around the same cost as other sequencing technologies, current nanopore membranes have a limited shelf life before they need to be replaced at a cost of several hundred dollars. So-called solid-state nanopores that are in development will no longer use protein channels, instead using pores made directly in synthetic materials. This could increase the lifespan of the membranes, allowing membranes to be stored for much longer periods of time, and potentially fitting many, many more nanopores into an array, reducing the cost of sequencing even further.

A major advantage to nanopore sequencing machines is that they can be very small, use few reagents, and need very little power. While other sequencing technologies use machines that can be the size of a dishwasher, current nanopore sequencers can easily fit in your pocket and are powered by a USB connection to a laptop computer. Scientists are now bringing mobile laboratories to previously inaccessible places, collecting organisms and sequencing their DNA, all without leaving the field, and in 2016, the International Space Station added nanopore sequencing to its scientific capabilities. What and where scientists will sequence next is anybody’s guess, but we do know this: it is likely to be where no sequencer has gone before.

• Yong, Ed. “A DNA Sequencer in Every Pocket.” The Atlantic. 28, April 2016. Link.

• Oxford Nanopore: How it works. Link.

Review

Critical thinking

Discussion