tag:blogger.com,1999:blog-36768584.post1801538195318917917..comments2024-03-03T18:49:34.382-05:00Comments on Omics! Omics!: Nanopore Community Meeting 2018: The Clive ReportKeith Robisonhttp://www.blogger.com/profile/04765318239070312590noreply@blogger.comBlogger3125tag:blogger.com,1999:blog-36768584.post-34702859284574409202018-12-02T10:18:54.171-05:002018-12-02T10:18:54.171-05:00Bertrand: a very unfortunate detail omitted by me!...Bertrand: a very unfortunate detail omitted by me! That’s 200Gbases per flowcell - so many terabases per instrumentKeith Robisonhttps://www.blogger.com/profile/04765318239070312590noreply@blogger.comtag:blogger.com,1999:blog-36768584.post-84431589075489654442018-12-02T09:25:46.116-05:002018-12-02T09:25:46.116-05:00Hi Keith,
Thanks for this very useful summary, ful...Hi Keith,<br />Thanks for this very useful summary, full of information. I have one query: the throghput you indicate for PromethIon (200 G at Oxford Nanopore) is for ONE PromethIon flowcell, not 24 or 48? Or am I hopelessly confused?<br />Thanks for clarifying,<br />BertrandBertrandhttps://www.blogger.com/profile/03186067074163012827noreply@blogger.comtag:blogger.com,1999:blog-36768584.post-71212848811380775012018-11-29T13:52:53.905-05:002018-11-29T13:52:53.905-05:00Hi Keith,
Thanks for the speedy wrap up! Saved me...Hi Keith,<br /><br />Thanks for the speedy wrap up! Saved me wading into the tweets.<br /><br />I wanted to propose an explanation for the shoulders you mentioned for the Cas9 enrichment technique. The approach uses two separate Cas9 RNPs containing (presumably) different guide RNAs, which will likely each cut with different efficiencies. If one RNP cuts better than the other, this would lead to some products with one digested end only. This would presumably still be a substrate for adapter ligation, but the read would not stop at the predicted position as the other RNP did not cut. This would explain the lop-sided shoulders for several of the sites tested (INS, SCA3, FMR1) - one RNP is much more efficient than the other in each case. <br /><br />Shoulders on both sides could be explained by both guides having similar efficiencies, but both not cutting to completion, leaving some single-cut products to be ligated and sequenced from each side. This looks to be the case for the SCA2, SCA6 and HTT sites in the figure.<br /><br />There are a few strange anomalies in the data still: at the MUC1 and INS sites the shoulders themselves seem to stop abruptly. If my explanation is correct, there should be a long, tapering tail that will depend on the natural, random fragmentation of the DNA used to prepare the input sample (as it looks to be at the FMR1 and SCA3 sites). So why the abrupt stops in the MUC1 and INS examples? Could these be due to off-target cut sites by the RNPs? Seems unlikely that they would occur so close to the intended cut sites. Could these be regions that break more often due to shearing? Is that a known phenomenon - non-random shearing? That's all I can come up with right now, but let me know if you can think of anything else!<br /><br />Hope this makes some sense!<br /><br />Cheers,<br />Mike D'Angelo<br />Mike D'Angelohttps://twitter.com/mdangelo32noreply@blogger.com