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Old 11-23-2012, 11:21 AM   #7
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Location: USA

Join Date: May 2011
Posts: 7

Several things that one needs to think about nanopore sequencing even at a layman's level

(1) whether eventually it is possible to have single base resolution in the so-called "strand sequencing" mode

(2) if not, the bigger the foot print of a DNA inside the pore, the many more states one needs to distinguish. There are two related issues, dynamic range of the current measurement and the noise level. One needs to increase the range of the current measurement and reduce the noise to call a definite state. When I was a kid, I was always puzzled by why modern day digital computers do not use more then two states. I think there is a good reason about that. (There are some attractive ideas about new analog/clockless computer, but it is certainly harder to do engineering work.)

(3) Now, if one can not get definite states. Let's say some states are close to a degenerate level. It is in principle possible to use advanced algorithms to improve the final base calls. Have such algorithms already be known well enough such that investing to put them on ASIC/FPGA is worthy already? If not, one will better use a general purpose CPU for such processes. In an academic lab, this is a non-issue. For a commercial product, one needs to consider to the right development paths.

(4) Does a DNA molecule really go through the pore as smooth as shown in the videos/cartoons? Double strand DNA is stiffer but single strand DNA is very flexible. For those who use land-line phone before, one must notice how easy the telephone hand-set coils get tangled. How does such molecule level entanglement affect the ability for reliable signal/data processing pipeline. For example, if one sees the current is stuck at one level for a long time. What is happening? Nothing in the pore? Something in the pore but the DNA is not moving due to entanglement + current level calibration is off? Some modified DNA bases to get itself stuck? The pore is broken? In a cartoon, one does not have to think about all of these issues. In reality, one needs to consider all possibilities.

(5) Is nanopore sequencing robust against any impurities in the sample? Do salt and other unwanted molecules affect the sequencing results? It is hard to believe one can take blood and get high QV non-random DNA sequencing data without any sample preparation. It seems deifying the 2nd law of thermodynamics.

All of these are really interesting problems. Maybe nanopore seq. does not have all these potential issues, however, only some real data can prove it.

By the way, although I have some knowledge about general statistical physics, I have never worked on any nanopore exp/data analysis. I can be totally wrong about nanopore sequencing and I am looking forward to the technological advancement to solve all potential problems. It is indeed an interesting time to live to see how to reveal the mysteries about life piece by piece.

Last edited by seqnextgen; 11-23-2012 at 11:44 AM. Reason: fix grammar
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