DNA sequencing: cheap or accurate?
DNA Sequencers: precision versus simplicity and cheapness
Kirill Stasevich, CompulentaAlthough DNA sequencing has been learned for a long time, researchers do not stop looking for new methods and optimizing old ones.
The purpose of such work is to reduce the cost of the process, make it fast, simple and accurate, so that you can be sure of the DNA sequence you read the first time.
And, as in any scientific matter, success here is not complete without failures, more or less severe. It happens that researchers simply fail to take the bar that they have set themselves: for example, the accuracy of the method increases, but not as fast as we would like. Something similar happened with Oxford Nanopore Technologies, which two years ago announced a rather unusual sequencing method from which very large chunks of DNA could be read at the same time.
Speaking about DNA sequencing, you need to understand that this does not mean continuously reading the entire DNA molecule that makes up the bacterial or, especially, the human chromosome. There are many methods of sequencing, but in each of them, the output is a set of short read sequences that fit into the original large DNA. These small fragments can be combined due to their mutual overlap, that is, it still depends on how accurately the small fragments were read and how accurately they were combined.
A MinION sequencing device connected to a laptop (photo by Oxford Nanopore Technologies).
The essence of the procedure proposed by Oxford Nanopore was as follows: DNA was stretched through a pore, while DNA changed the ion flow in this pore, and such changes depended on which nucleotide was currently passing through the pore. In theory, such a method would allow thousands of nucleotides to be read at a time, without subsequent manipulation of sequence fragments. The idea is already twenty years old, but it was technically possible to implement it only in 2012.
The authors of the method immediately gathered to put it on stream. But, firstly, it turned out that the membrane through which they passed DNA in experiments was difficult to manufacture in large quantities - and she had to look for a replacement. Secondly, seeing the enthusiasm that their method aroused in the biomedical community, the scientists tried to make a portable device accessible to everyone, in which it would simply be necessary to change consumables.
For two years there was no word about it. And now at the Advances in Genome Biology & Technology conference http://agbt.org/about.html which takes place in Florida (USA) and where they report on the latest advances in genomic technologies, David Jaffe from the Broad Institute (USA), collaborating with Oxford Nanopore, made the news about the new method. As it turned out, the expectations regarding the "nano-holes method" turned out to be somewhat overstated: with its help, it was possible to read pieces with a length of 10,000 nitrogenous bases (really a huge length), however, errors inevitably appeared in the read pieces that did not allow them to add up a complete genome (experiments were carried out with Escherichia coli DNA with a length of 4.6 million base pairs and with DNA of Scardovia wiggsiae with a length of 1.55 million base pairs). To combine the fragments into a genome, we needed data obtained using another sequence method – for example, the one used in Illumina machines.
The sequencing machine of the company Illumina (photo Illumina).
The Illumina method (more precisely, Illumina/Solexa) consists in synthesizing a complementary chain of nucleotides with a fluorescent label on the DNA template to be read. After embedding a nucleotide into a growing chain, its glow is read, and thus it is possible to find out what kind of nucleotide has taken the next position (all four nucleotides have different luminous labels). Here, we can say, the sequence is literally read, and the overall picture consists of a set of read fragments about a hundred nucleotides long.
As we remember, one of the important criteria of the sequencing method is its cheapness (the cheaper the procedure, the more clinics will be able to afford it). This year, Illumina stated that it managed to reduce the "cost" of one human genome to $ 1,000 (which includes both reagents and operator labor) – however, the problem with its machines is that they themselves are extremely expensive, and in order to pay off, such an aggregate must process 1,800 genomes per year. Not every medical organization deals with such a scale, so the company is still working for biomedical giants and large-scale projects.
But let's return to the Oxford Nanopore device, which was named MinION. Its authors are not discouraged, offering everyone to try it for $ 1,000, which, if they don't like the result, they will return back. It is believed that it will be useful for a rough analysis of bacterial microflora (for example, in soil) or for analyzing DNA contained in food. However, the whole question is how useful its capabilities will be in each case: many researchers have questioned the prospects of this portable sequencer, since its data still needs to be backed up by another method.
Well, the moral that can be deduced from this story is, apparently, the following: DNA sequencers will not soon become as accessible as an iron or washing machine. If, of course, we want exhaustive and accurate results from such devices.
Prepared based on ScienceNOW: DNA Sequencing Firm's Second Act Gets Mixed Reviews.
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