How do they pull the DNA through the pores? That just seems impossible to me.
I think the sequences are first prepared by attaching a motor protein at the ends of the strands.
After Long Delays, Breakthrough Nanopore Sequencer Finally in Labs
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Re: After Long Delays, Breakthrough Nanopore Sequencer Finally in Labs
#12How do they get the DNA unwound and free from tangles? Isn't it normally wrapped around his tones ?
There are a number of ways to handle that problem. You could melt the DNA or denature it in a basic solution. In some nanopore designs, the diameter of the nanopore is so small that when the DNA is pulled through, only a single strand can pass which forces the DNA to unzip and untangle. I believe the Oxford Nanopore approach is to use an enzyme to cleave single nucleotides off of the end of the strand one by one. That way the signal is only coming from a single base, which is helpful in the decoding process.
Re: After Long Delays, Breakthrough Nanopore Sequencer Finally in Labs
#13Talk about serendipity, I was just reading this article from September 6th:
http://www.economist.com/news/technology-quarterly/21615029-...
"One of these, Genia, is commercialising a process called nanopore sequencing that Dr Church first devised in 1988. Distinct polymer tags are attached to each of the four nucleotides poised to contribute to a single molecule of replicating DNA. As they react, the tags are released near a protein layer full of tiny holes called nanopores. Each tag blocks the flow of electrical ions across the layer in a different way. Because it relies on electronics rather than optics, nanopore sequencing promises faster, cheaper sequencing. Dr Church holds up a fingernail-sized chip containing 128,000 nanopores that he reckons will bring the cost of sequencing down to $100. In June, Genia was acquired by Roche, a Swiss pharmaceuticals giant."
Re: After Long Delays, Breakthrough Nanopore Sequencer Finally in Labs
#14I like that it is called a Minion. Presumably there is a gene sequencer market for evil megalomaniacs who live under volcanoes.
Re: After Long Delays, Breakthrough Nanopore Sequencer Finally in Labs
#15How do they get the DNA unwound and free from tangles? Isn't it normally wrapped around his tones ?
There are a number of ways to handle that problem. You could melt the DNA or denature it in a basic solution. In some nanopore designs, the diameter of the nanopore is so small that when the DNA is pulled through, only a single strand can pass which forces the DNA to unzip and untangle. I believe the Oxford Nanopore approach is to use an enzyme to cleave single nucleotides off of the end of the strand one by one. Tha…
The DNA would have to undergo purification beforehand anyway, to extract it from the cell and separate it out from the cellular proteins and RNA. So histone removal would be through modified salt concentrations and protease action.
Also I think the method you are describing is their other sequencing approach - this one, as far as I know, passes an intact strand through each pore and examines the electrical conductivity of overlapping 6 base pair sequences.
Re: After Long Delays, Breakthrough Nanopore Sequencer Finally in Labs
#16Researcher working on a different approach to nanopore sequencing here. The Minion is really interesting technology, but early reports basically indicate that it's essentially useless in it's current form. One of the issues is that the basecalling algorithm relies on a noisy, two bit signal. Apparently it works okay on trained sequences, like lambda DNA (that's where the 60-85% accuracy comes from). But when used to…
It sounds like they are reading several DNA strands in parallel at the same time, and each output-sequence has noise. It seems to me the problem then becomes one of finding the most probable "signal sequence" given all those noisy output-sequences. Oh, and it also sounds like you wouldn't know which letter is number 1, which is number 2, etc. Is that right?
It seems like a fun problem in information theory. Can you point us to some articles or papers about current approaches to solving it?
Re: After Long Delays, Breakthrough Nanopore Sequencer Finally in Labs
#17Researcher working on a different approach to nanopore sequencing here. The Minion is really interesting technology, but early reports basically indicate that it's essentially useless in it's current form. One of the issues is that the basecalling algorithm relies on a noisy, two bit signal. Apparently it works okay on trained sequences, like lambda DNA (that's where the 60-85% accuracy comes from). But when used to…
It sounds like they are reading several DNA strands in parallel at the same time, and each output-sequence has noise. It seems to me the problem then becomes one of finding the most probable "signal sequence" given all those noisy output-sequences. Oh, and it also sounds like you wouldn't know which letter is number 1, which is number 2, etc. Is that right? It seems like a fun problem in information theory. Can you p…
Winston Timp has done some interesting work in this area (http://www.sciencedirect.com/science/article/pii/S0006349512...). Basically, by training a hidden Markov Model, you can get the most likely sequence from a noisy source.