tag:blogger.com,1999:blog-36768584.post1690871462613795427..comments2024-03-03T18:49:34.382-05:00Comments on Omics! Omics!: Brown Webcast Note: Corrections and ExpansionsKeith Robisonhttp://www.blogger.com/profile/04765318239070312590noreply@blogger.comBlogger6125tag:blogger.com,1999:blog-36768584.post-41792159901758435752018-02-14T00:58:54.546-05:002018-02-14T00:58:54.546-05:00J Ir:
I'm just as in the dark as you are. Ele...J Ir:<br />I'm just as in the dark as you are. Electrophoresis would be one approach, but I don't believe you could do it without physically modifying the device -- which I assumed isn't something Clive is talking about but that is pure assumption. Keith Robisonhttps://www.blogger.com/profile/04765318239070312590noreply@blogger.comtag:blogger.com,1999:blog-36768584.post-29839711093073193132018-02-13T22:57:11.661-05:002018-02-13T22:57:11.661-05:00I have no idea about anything of this so please do...I have no idea about anything of this so please don't hurt my inner child if you disagree, but might a separation stage using electrophoresis be helpful in separating large and small DANA segments for the nanopore?Anonymoushttps://www.blogger.com/profile/17782724405782294652noreply@blogger.comtag:blogger.com,1999:blog-36768584.post-87800283833641660042018-02-12T15:29:55.843-05:002018-02-12T15:29:55.843-05:00I am planning an experiment using MinION with ~400...I am planning an experiment using MinION with ~400bp fragments as well. The Sales Rep from ONT did mentioned about shorter fragments getting lower performance. I was imagining that I could overload the flowcell (increase my loading DNA library concentration) to compensate for the lagging time between strands at the pore. Not sure will this help yet as I still haven't gotten my MinION! Leeloohttps://www.blogger.com/profile/15043907748265033854noreply@blogger.comtag:blogger.com,1999:blog-36768584.post-75681655975335725872018-02-11T18:48:25.164-05:002018-02-11T18:48:25.164-05:00Clive previously mentioned that the flow cells wor...Clive previously mentioned that the flow cells worked a bit like a battery in that they discharge over time. Pores discharge quicker when they're not actively sequencing, and I expect this has something to do with less electric current being required to zap through a DNA strand compared to an open pore.<br /><br />There is a loading time between one strand finishing sequencing and the next one starting, so anything that leads to an increased loading time in proportion to the run length (with associated open pore state) will lead to quicker degredation of the pores. Assuming this loading time is a fixed period of time, shorter sequences will have a higher proportion of time loading vs sequencing when compared to long sequences, so will degrade the pore quicker.David Eccleshttps://www.blogger.com/profile/11754558756169247029noreply@blogger.comtag:blogger.com,1999:blog-36768584.post-21428554014164468262018-02-11T11:45:40.997-05:002018-02-11T11:45:40.997-05:00Duarte:
I don't believe anyone knows for sure...Duarte:<br /><br />I don't believe anyone knows for sure -- of if ONT knows they haven't been effusive in describing the phenomena. One possibility is that every time a DNA exits (or docks?) it has some potential to damage the pore -- so libraries full of many short fragments represent many such events<br /><br />My belief that short fragments cause faster activity drops is based on experience with several ligation libraries of E.coli preps which had very sharp dropoffs & similar libraries that did not -- and the difference was the fraction of the library composed of short fragments. Now, that difference was due to another variable -- the E.coli genotype -- and the libraries were prepared at different times.<br /><br />So that's why I see this pressing need -- which Flongle helps make more financially practical -- for ONT to have a test kitchen ala Mom's job offer to explore the landscape of potential problems. So take some DNA that behaves well, such as lambda, and then dope in defined amounts of potential problems such as short fragments and measure the effect on productivity vs. time. Perhaps short fragments are a red herring and there is something else going on. And, as you ask, if short fragments are a problem, then how short? <br /><br />Knowing this is important for anyone designing counting experiments or trying to detect targets by PCR or working with degraded DNA or so many other scenarios in which the input DNA will be short. And for some of those, it won't be a deal breaker -- but it is important for users to understand how much less data they can expect if working with such materials.<br /><br /><br />Keith Robisonhttps://www.blogger.com/profile/04765318239070312590noreply@blogger.comtag:blogger.com,1999:blog-36768584.post-39958414741402834022018-02-11T02:21:58.251-05:002018-02-11T02:21:58.251-05:00Why do small fragments kill pores? Any idea? I was...Why do small fragments kill pores? Any idea? I was thinking of testing an application where I wanted to run a library prep consisting of 180 bp fragments (8 of those bp custom barcodes).<br /><br />Clive indicated nanopore can read small fragments ( he suggested it could read even smaller than that - 50 to 60 bp)<br /><br />If small fragments are really that bad together with the much lower number of pores on the flongle flowcell I'm not sure my application will work.Duartehttps://www.blogger.com/profile/13960784528179885911noreply@blogger.com