tag:blogger.com,1999:blog-6334453475526523597.post8638374812377950275..comments2023-11-07T10:31:25.370+00:00Comments on CoreGenomics: One of our bases is missing: where's the G in NextSeq chemistryJames@cancerhttp://www.blogger.com/profile/02825715598810395734noreply@blogger.comBlogger9125tag:blogger.com,1999:blog-6334453475526523597.post-55244818233111865702014-03-10T21:53:17.029+00:002014-03-10T21:53:17.029+00:00Actually it's worse than that. A as I understa...Actually it's worse than that. A as I understand is dual labelled so significantly sterically larger than label-less G. So this could also lead to the preferential misincorporation of G over A.Anonymousnoreply@blogger.comtag:blogger.com,1999:blog-6334453475526523597.post-80799204771403976652014-03-08T04:01:56.445+00:002014-03-08T04:01:56.445+00:00In the absence of a C nucleotide, G will preferent...In the absence of a C nucleotide, G will preferentially pair with T and visa versa. Since the steric bulk of the label-free G has been decreased relative to the labeled C this then could increase the probability of G:T and T:G mismatches and misreporting. <br />Anonymousnoreply@blogger.comtag:blogger.com,1999:blog-6334453475526523597.post-17396638441296706402014-02-05T01:19:50.999+00:002014-02-05T01:19:50.999+00:00The four original dyes were blue, green, red and y...The four original dyes were blue, green, red and yellow. The only issue was possible FRET under certain circumstances and this was covered in the choice of frequencies and design so not an issue.Anonymousnoreply@blogger.comtag:blogger.com,1999:blog-6334453475526523597.post-85434714226143527292014-02-05T01:13:56.157+00:002014-02-05T01:13:56.157+00:00http://glennklockwood.blogspot.co.uk/2014/01/the-1...http://glennklockwood.blogspot.co.uk/2014/01/the-1000-genome-computational.html?showComment=1389835567115<br />Anonymousnoreply@blogger.comtag:blogger.com,1999:blog-6334453475526523597.post-42910212613142776612014-02-03T21:45:44.841+00:002014-02-03T21:45:44.841+00:00$72M comes from $1000/genome, 18k genomes over 4 y...$72M comes from $1000/genome, 18k genomes over 4 years (72k genomes)<br />The $1000 cost breaks down as follows:<br />$800 for sequencing reagents<br />~$135 for instrument amortization<br />~$65 for library prep, labor and automated analysis (alignment)Shawn Bakerhttp://www.allseq.comnoreply@blogger.comtag:blogger.com,1999:blog-6334453475526523597.post-82537471700371380712014-02-03T16:54:56.549+00:002014-02-03T16:54:56.549+00:00Correct me if I'm wrong, but previous SBS chem...Correct me if I'm wrong, but previous SBS chemistries had four dyes, A and C in the red channel and G and T in the green. As two dyes exist in each channel, there is some spectral overlap which needs correcting for algorithmically - which would surely contribute towards sequencing error. One advantage of the new system is that now there are two dyes, each can exist in its own channel, minimising spectral overlap, removing the need for matrix calculations and, in theory actually reducing error rate....just saying, the new 2-dye system could improve error rates.Unknownhttps://www.blogger.com/profile/14869462307678967101noreply@blogger.comtag:blogger.com,1999:blog-6334453475526523597.post-50481656696046181242014-02-03T12:35:49.864+00:002014-02-03T12:35:49.864+00:00I'm pretty sure the 72 million figure was not ...I'm pretty sure the 72 million figure was not the bioinformatics cost - could anyone chime in? I tought that was all-in and amortized over the amount of genomes expected to be run you get pretty close to $1000?Anonymousnoreply@blogger.comtag:blogger.com,1999:blog-6334453475526523597.post-51770930337638346792014-02-03T01:56:25.993+00:002014-02-03T01:56:25.993+00:00Perhaps, but at least you can see all of the four ...Perhaps, but at least you can see all of the four bases to some extent.<br /><br />Pretty much most things about Sanger sequencing are different so I'm not sure the comparison is valid. Although slow, Sanger is still the gold standard.<br /><br />Illumina don't have all the time in the world. Fluorescence-based sequencing is not the end game by a long way. It's just too expensive. They’re in a race. And if you want proof of Illumina's desperation look no further than the HiSeq 10 X with the 72 million dollars needed for bioinformatics costs over four years to claim the $1000 genome, which, let's face it. is complete b/s. Irrespective of bioinformatics, their not even close to achieving the $1000 genome. <br /><br />And it’s not just the absence of G-signal and confirmation of incorporation, it’s what else is happening that’s not being reported.<br />Anonymousnoreply@blogger.comtag:blogger.com,1999:blog-6334453475526523597.post-47103928678952079082014-02-03T00:34:37.861+00:002014-02-03T00:34:37.861+00:00There is actually one easy way to locate clusters ...There is actually one easy way to locate clusters while getting away from the "no G" signal -- you could just sequence either one of the indexes first. I haven't looked at anything in the sequencing process on the NextSeq, but it seems to be an easy workaround. <br /><br />It's not a great analogy to compare it to Sanger sequencing though. The lack of a signal in Sanger is complemented by a signal in another channel -- that is, if you did everything correctly there will be a signal somewhere. In the 2-channel SBS system, a G means there is no signal whatsoever on that cluster. It's especially disconcerting when you are dealing with homopolymers. At least when you have a signal you can make a ballpark estimate of what's happening, but when you have nothing it's really hard. Also, the way the "G" Q-score is calculated will be completely different and to me seems kind of sketchy.Anonymousnoreply@blogger.com