tag:blogger.com,1999:blog-36768584.post8614158602551911880..comments2024-03-03T18:49:34.382-05:00Comments on Omics! Omics!: Paying a Painful 75% Secrecy TaxKeith Robisonhttp://www.blogger.com/profile/04765318239070312590noreply@blogger.comBlogger16125tag:blogger.com,1999:blog-36768584.post-2793327525964973022011-09-20T19:15:48.154-04:002011-09-20T19:15:48.154-04:00here's a possible solution??
1) make the 5...here's a possible solution?? <br />1) make the 5' primer to contain the adaptor. the 3' primer should not<br />2) do PCR of any size<br />3) shear the DNA <br />4) select the size (up to 150bp)of sheared DNA you want<br />5) ligate only the 3' adaptor. Only those fragments that contain a 5' adaptor will bind the bead (so you will likely need a higher starting DNA conc than you normally would use), but all your reads should contain the first 150bp sequenced from the 5' end, so you could align that portion and compare these for the snps etc.<br /><br />haven't tried it... just theorizing this could work? let me know if it does! :)monique_haakensenhttps://www.blogger.com/profile/04311779274348626325noreply@blogger.comtag:blogger.com,1999:blog-36768584.post-27783768636179652832011-06-03T22:42:56.278-04:002011-06-03T22:42:56.278-04:00The one touch won't help, droplet size is plen...The one touch won't help, droplet size is plenty big enough to drive copies to the bead. People really don't understand how it works. It is all about how many copies on the bead you can get with longer pieces of DNA. This property is well documented for solid surface DNA amplification.<br /><br />When sequencing amplicons, do you need to also trim the PCR primer read? I always assumed since the PCR primers are in molar excess and extension occurs off the 3' end, the PCR primer sequence is related to the synthetic oligo and not the sample.<br /><br />Subtracting the PCR priming sites from the amplicons would really hit their usable read length.Porihnoreply@blogger.comtag:blogger.com,1999:blog-36768584.post-27770125986536145422011-06-03T22:24:28.365-04:002011-06-03T22:24:28.365-04:00Ion's primers have biotin to make the enrichme...Ion's primers have biotin to make the enrichment faster. If you designed your primers on the older protocol, then you will get no enrichment. 20% enrichment with 16% polyclonal pretty much shows that.<br /><br />They sequenced your sample with no enrichment pretty much.Anonymousnoreply@blogger.comtag:blogger.com,1999:blog-36768584.post-9620224315604626152011-06-03T07:53:20.030-04:002011-06-03T07:53:20.030-04:00He didn't have a machine and wanted to see the...He didn't have a machine and wanted to see the reads, haven't you read his blog post ? Now, on the other hand I do have to agree that scientists are now into saving every penny possible and Ion Torrent's usage price is attractive. Why you know, Life will be very quick to tell you that Ion Torrent was used by BGI for that super killer cucumber bug sequencing. If you read BGI carefully they basically mentioned that they used what they had, which was a previously well known genome template to work from. Ion Torrent really didn't offer a huge difference.<br /><br />Does all this really matter ? Everyone here knows that sequencing is going the oligo way, there will be tons of houses who will offer NGS sequencing at pennies, and soon we will buy pennies and companies will sell pennies and the bubble will crumble again and we will be making wallmart science with wallmart quality.Anonymousnoreply@blogger.comtag:blogger.com,1999:blog-36768584.post-56046323508341147932011-06-02T20:06:54.320-04:002011-06-02T20:06:54.320-04:00Should have bought a GS Junior for Amplicon Sequen...Should have bought a GS Junior for Amplicon Sequencing...<br /><br />Sorry but it is your own fault for not looking into the technology properly and for believing Life Tech reps instead of doing what scientists should do....RESEARCHAnonymousnoreply@blogger.comtag:blogger.com,1999:blog-36768584.post-56391868171977712172011-05-31T17:15:14.546-04:002011-05-31T17:15:14.546-04:00Your service provider should have run the samples ...Your service provider should have run the samples out on a bioanalyzer to verify the sizing before ever doing the run. This is generally included in the charge. Who did you use as some are better than others. I would stick to the CSP providers if at all possible.Anonymousnoreply@blogger.comtag:blogger.com,1999:blog-36768584.post-78592835424262065342011-05-31T16:59:25.958-04:002011-05-31T16:59:25.958-04:00Keith, thanks for posting the data. really interes...Keith, thanks for posting the data. really interesting.<br /><br />You can estimate the representation of each amplicon in the sequence library experimentally by qPCR with the original primers used for the amplification. We've found this to be a useful way of estimating representation before sequencing, and relative copy numbers calculated from delta Cq correlate pretty well with the observed relative depth of coverage in sequence data (for 454 at least). This approach should help assess the relative contributions of library generation and (emPCR+sequencing) to representation in these data.Andynoreply@blogger.comtag:blogger.com,1999:blog-36768584.post-56751083863324886682011-05-31T16:47:46.808-04:002011-05-31T16:47:46.808-04:00GasStation- Ion Torrent posted a similar accuracy-...GasStation- Ion Torrent posted a similar accuracy-vs-length analysis of their latest data in Figure 4 of the "performance overview" pdf on their website. This analysis indicates they are now getting 99% accuracy at 100 bp read length. Does this mean that they've improved their accuracy to be equivalent to Illumina, or are they doing something sneaky that isn't obvious to a non expert?Wraithnotnoreply@blogger.comtag:blogger.com,1999:blog-36768584.post-91007958165513635682011-05-31T16:33:03.431-04:002011-05-31T16:33:03.431-04:00Keith,
Thanks for the additional info. Hopefully...Keith,<br /><br />Thanks for the additional info. Hopefully a recent post on SEQanswers indicating that the One Touch system will help with this size limitation is correct. It also sounds like Ion needs to tweak their enrichment protocol since the data you showed included this step.Wraithnotnoreply@blogger.comtag:blogger.com,1999:blog-36768584.post-18320611374402961822011-05-31T13:41:50.822-04:002011-05-31T13:41:50.822-04:00I posted some data (not mine, so I can't give ...I posted some data (not mine, so I can't give more details) showing how read length and error rate varied with quality trimming for the Ion Torrent:<br />http://gasstationwithoutpumps.wordpress.com/2011/05/31/a-use-for-an-ion-torrent/Anonymousnoreply@blogger.comtag:blogger.com,1999:blog-36768584.post-71218934806607920122011-05-31T11:38:00.986-04:002011-05-31T11:38:00.986-04:00Wraithnot: I checked with the provider and an enri...Wraithnot: I checked with the provider and an enrichment step was performed.<br /><br />Yes, it does seem that shorter amplicons show up more frequently in the data, though the results are not clean. We didn't normalize the input amounts, and poor amplifiers in the original PCR definitely fared worseKeith Robisonhttps://www.blogger.com/profile/04765318239070312590noreply@blogger.comtag:blogger.com,1999:blog-36768584.post-68676255585000621882011-05-31T09:03:33.475-04:002011-05-31T09:03:33.475-04:00Let me know when N>1. What's the p-value o...Let me know when N>1. What's the p-value of this blog?Anonymousnoreply@blogger.comtag:blogger.com,1999:blog-36768584.post-17315226404671991192011-05-28T13:56:41.297-04:002011-05-28T13:56:41.297-04:00Your read length estimates are falling prey to the...Your read length estimates are falling prey to the same error Ion Torrent makes in their ads: ignoring the quality of the reads. Now that you have your own data, plot the read length distributions after quality trimming. <br /><br />I think that you'll find that the quality drops rapidly after about 60 bases, so you don't have as much data as you think.<br /><br />Ion Torrent suppressed a paper that had that info in it, with the threat of a lawsuit.Anonymousnoreply@blogger.comtag:blogger.com,1999:blog-36768584.post-83837211397982060712011-05-27T16:41:04.075-04:002011-05-27T16:41:04.075-04:00Thanks for posting this info- we're trying to ...Thanks for posting this info- we're trying to decided whether or not to buy a PGM for amplicon sequencing and there's very little independent information out there. <br /><br />I also had a few quick questions:<br />1. If the amplicon size was indeed the issue, your shorter amplicons should have been over-represented in the usable data. Did you observe this?<br />2. The sales rep described a streptavidin bead enrichment step to remove "ion spheres" that didn't get a template molecule (and therefore didn't incorporate the biotinylated A adapter) during the emulsion PCR. But the ratio of "live ISPs" to total ISPs and the ratio of multiclonal ISPs to live ISPs are consistent with Poisson statistics (at least if I did the math properly and if I am interpreting live ISPs correctly). Did your sequencing provider run the enrichment step?Wraithnotnoreply@blogger.comtag:blogger.com,1999:blog-36768584.post-47426820235697221992011-05-27T12:27:34.940-04:002011-05-27T12:27:34.940-04:00Some knew, some didn't care.
Fact: Reps at L...Some knew, some didn't care. <br /><br />Fact: Reps at Life Tech are constantly being told they might get fired. Reps at Life Tech get a Rolex for a certain number of PGM sold. <br /><br />So you end up with a spectrum of actions: honest reps and other reps, who are stuck between feeding their kids or lusting for that ugly power watch of the 90s.<br /><br />I smell a good MiSeq advert right there.<br /><br />Don't blame the rep, blame the culture.<br /><br />NB Pretty sure it's the same at other companies...Anonymousnoreply@blogger.comtag:blogger.com,1999:blog-36768584.post-20114624705345764272011-05-27T05:07:57.445-04:002011-05-27T05:07:57.445-04:00The amplicon length limit was disclosed to us by L...The amplicon length limit was disclosed to us by LifeTech salespeople back in January. I'm surprised that they don't make this very very obvious, because it is THE major reason why not to buy a PGM yet.Douglas Yuhttps://www.blogger.com/profile/01581746537475599763noreply@blogger.com