Pocket-Sized DNA Reader Used To Scan Entire Human Genome Sequence (arstechnica.com)

An anonymous reader quotes a report from Ars Technica: A few years back, a company called Oxford Nanopore announced it was developing a radically different way of sequencing DNA. Its approach involved taking single strands of the double helix and stuffing them through a protein pore. With a small bit of current flowing across the pore, the four bases of DNA each created a distinct (if tiny) change in the voltage as it passed through. These could be used to read the DNA one base at a time as it wiggled through the pore. After several years of slow progress, Oxford Nanopore announced that its sequencing hardware would be as distinctive as its wetware: a USB device that could fit comfortably in a person's hand. As the first devices went out to users, it became clear that the device had some pros and cons. On the plus side, the device was quick and could be used without requiring a large facility to support it. It could also read very long stretches of DNA at once. But the downside was significant: it made lots of mistakes.

With a few years of experience, people are now starting to learn to make the most of the devices, as demonstrated by a new paper in which researchers use it to help sequence a human genome. By using the machine's long reads -- in one case, nearly 900,000 bases from one DNA molecule -- the authors were able to get data out of areas of the human genome that resisted characterization before. And they were able to distinguish between the two sets of chromosomes (one from mom, one from dad) and locate areas of epigenetic control in many areas of the genome. In light of all the distinct information it can provide, the machine's error rate is seeming like less of a problem.

Comments

Re:Easy Fix

[Pseudonym]

That's what we do now with short reads. It kind of works, but only because we understand in a lot of detail about how errors happen.

For example, 454 sequencing tends to get the number of nucleotides in a repeat sequence wrong. So, for example, CTAAAGT might be read as CTAAAAGT. Illumina sequencing doesn't have that problem, but tends to degrade along the length of the read. So the last few nucleotides are more likely to be wrong than the first few.

And this is just read errors; with short-read sequencing, there are also PCR amplification errors, which is why we think nanopore sequencing will do better. When you start "unwinding" a chromosome, the parts that you unwind first tend to get amplified more than the parts that you unwind nearer to the end. Some sequences are amplified more than others for chemical reasons, and the relative error might depend on the specific revision of reagent chemicals.

We don't really understand enough about nanopore sequencing to be able to develop appropriate algorithms to match long-read sequences together. We don't even know what the right number of multiple passes is yet. And that's important, because genomics and transcriptonomics are important, but the bigger issue for researchers is economics.

Re:Wrong use.

[gringer]

Direct RNA sequencing can be done with the MinION as well, no hardware change needed:

https://store.nanoporetech.com...

Depending on how important it is to sequence all RNA, polyadenylation prior to sequencing might also be needed.

Re:Q&A from a previous time

[gringer]

Some errors are random, some are systematic. The systematic errors tend to be either small shifts in long stretches of the same base, or interesting features of the DNA (e.g. methylation), and there are a few people trying to work out what those interesting features are.

A key obstacle to getting people interested in nanopore sequencing (or other types of observational sequencing) is that we have been locked in for so long to the idea of DNA as a sequence of letters that we forget there are other things attached to it that also have functional roles. Nanopore is more accurate when matching sequences at the signal/electrical level, but almost no one is doing that yet.