tag:blogger.com,1999:blog-4457216402399127579.post8958789061453577253..comments2024-03-13T11:06:06.997-04:00Comments on Next-Gen Sequencing: Genotyping on MiSeq with overlapping paired-end reads Anonymoushttp://www.blogger.com/profile/14602560263535951430noreply@blogger.comBlogger25125tag:blogger.com,1999:blog-4457216402399127579.post-39138253329553699962021-09-02T11:10:15.005-04:002021-09-02T11:10:15.005-04:00I really want to thank Dr Emu for saving my marria...I really want to thank Dr Emu for saving my marriage. My wife really treated me badly and left home for almost 3 month this got me sick and confused. Then I told my friend about how my wife has changed towards me. Then she told me to contact Dr Emu that he will help me bring back my wife and change her back to a good woman. I never believed in all this but I gave it a try. Dr Emu casted a spell of return of love on her, and my wife came back home for forgiveness and today we are happy again. If you are going through any relationship stress or you want back your Ex or Divorce husband you can contact his whats app +2347012841542 or email emutemple@gmail.com website: Https://emutemple.wordpress.com/<br />Ric Claytonhttps://www.blogger.com/profile/09645806549640826100noreply@blogger.comtag:blogger.com,1999:blog-4457216402399127579.post-91211571440251910442021-05-11T10:13:52.482-04:002021-05-11T10:13:52.482-04:00Great post! I would be very interested in what you...Great post! I would be very interested in what you come up with to update quality scores based on the overlapping reads.<br /><br /><a href="https://www.anacyte.com/blog/publication-highlight-application/" rel="nofollow">RNA Sequencing</a><br /><br /><a href="https://www.anacyte.com/blog/publication-highlight-application/" rel="nofollow">Single Cell RNA Sequencing</a><br /><br /><a href="https://www.anacyte.com/" rel="nofollow">Protein Stabilization</a>Anacyte Laboratorieshttps://www.blogger.com/profile/01088451951906129468noreply@blogger.comtag:blogger.com,1999:blog-4457216402399127579.post-32580566942206807962021-01-23T00:37:25.652-05:002021-01-23T00:37:25.652-05:00Транспортно-логистическая компания "Азия-Трей...Транспортно-логистическая компания "Азия-Трейдинг" - это специализированный таможенный агент, деятельность которого осуществляется в зоне действия Владивостокской и Находкинской таможни и основным направлением компании является организация таможенного оформления импортных грузов.Anonymousnoreply@blogger.comtag:blogger.com,1999:blog-4457216402399127579.post-73806033464899184862021-01-22T07:07:44.791-05:002021-01-22T07:07:44.791-05:00[url=http://albertaland4sale.com/author/stevenlut-...[url=http://albertaland4sale.com/author/stevenlut-stevenlut/]http://albertaland4sale.com/author/stevenlut-stevenlut/[/url]Anonymousnoreply@blogger.comtag:blogger.com,1999:blog-4457216402399127579.post-89264755671778406952014-05-12T15:42:13.131-04:002014-05-12T15:42:13.131-04:00Hi Yev,
The framshifted molecule tagged primers i...Hi Yev,<br /><br />The framshifted molecule tagged primers include an Illumina adapter (which I think is the same thing as the index primer). So when those primers are designed they look something like:<br /><br />[Illumina index primer] [molecule tag with frameshifts] [spacer] [target primer]<br /><br />After all PCR steps the index can be added using the index primer.Anonymoushttps://www.blogger.com/profile/01081006207025463719noreply@blogger.comtag:blogger.com,1999:blog-4457216402399127579.post-71607849542995987912014-05-09T18:33:21.897-04:002014-05-09T18:33:21.897-04:00Hi Scott Yourstone,
Since you use the staggered p...Hi Scott Yourstone,<br /><br />Since you use the staggered primer approach, how do you design the Index sequencing primer (and reverse compliment Read 2) since there is variable number of bases between the reverse primer/linker?<br /><br />Thanks for any insight.Yev Marusenkonoreply@blogger.comtag:blogger.com,1999:blog-4457216402399127579.post-4659984611330785562014-01-31T10:54:49.228-05:002014-01-31T10:54:49.228-05:00Do you have experience in SNP discovery using MiSe...Do you have experience in SNP discovery using MiSeq with polyploid organisms?Anonymousnoreply@blogger.comtag:blogger.com,1999:blog-4457216402399127579.post-73744180190946281412013-12-17T18:00:44.490-05:002013-12-17T18:00:44.490-05:00Seems interesting-
PEAR: A fast and accurate Illum...Seems interesting-<br />PEAR: A fast and accurate Illumina Paired-End reAd mergeR<br /><br />http://bioinformatics.oxfordjournals.org/content/early/2013/10/18/bioinformatics.btt593.shortAnonymousnoreply@blogger.comtag:blogger.com,1999:blog-4457216402399127579.post-43278181561658242672013-08-24T21:58:55.469-04:002013-08-24T21:58:55.469-04:00Hi,
Scott and my paper describing MiSeq paired-end...Hi,<br />Scott and my paper describing MiSeq paired-end sequencing methods with frameshifting primers for great quality without phiX and molecule tags for correcting errors/bias will be coming out online in Nature Methods Sept. 1st. Look for this and also check out a paper by Jeremiah Faith / Jeff Gordon which describes some very similar techniques (they call their LEA-seq) and was published this July in the journal Science. Scott wrote some user-friendly software (optional GUI) that can process paired end reads from either of these and related techniques. Anonymoushttps://www.blogger.com/profile/16519119946853347844noreply@blogger.comtag:blogger.com,1999:blog-4457216402399127579.post-45184598162471596402013-07-29T08:57:15.627-04:002013-07-29T08:57:15.627-04:00Hi Genohub,
Yes, we use the staggered primer appro...Hi Genohub,<br />Yes, we use the staggered primer approach, and because of that we don't need any PhiX. However, I have heard that with the most recent MiSeq upgrades only ~2% PhiX is needed to get high quality sequences.Anonymoushttps://www.blogger.com/profile/01081006207025463719noreply@blogger.comtag:blogger.com,1999:blog-4457216402399127579.post-29292058318545018932013-07-27T23:33:36.205-04:002013-07-27T23:33:36.205-04:00Hi Scott,
Curious to hear about what % PhiX you a...Hi Scott, <br />Curious to hear about what % PhiX you are using now for Amplicon-Seq. Have you tried a staggered primer approach?Genohubhttp://www.genohub.comnoreply@blogger.comtag:blogger.com,1999:blog-4457216402399127579.post-51121121175166661972013-05-21T15:47:00.488-04:002013-05-21T15:47:00.488-04:00http://yeastinfection7.com/ <a href="http://yeastinfection7.com/" rel="nofollow">http://yeastinfection7.com/</a>Anonymousnoreply@blogger.comtag:blogger.com,1999:blog-4457216402399127579.post-55435314510297048392013-05-21T09:43:04.609-04:002013-05-21T09:43:04.609-04:00We just did a MiSeq 2x150 amplicon run with the ne...We just did a MiSeq 2x150 amplicon run with the new software and an extremely small PhiX spike-in and it has effectively doubled our yield! It's great to see the technology continually improving; we're now getting almost 10 times as many reads from each 2x150 MiSeq run as we did just a few months back.Dr. Brendan Hodkinsonhttps://www.blogger.com/profile/07673530922073637396noreply@blogger.comtag:blogger.com,1999:blog-4457216402399127579.post-66485095233714020802013-05-06T15:30:09.836-04:002013-05-06T15:30:09.836-04:00Dr. Brown:
When you said "Our Genomics lab tr...Dr. Brown:<br />When you said "Our Genomics lab tried it with 40% added Phix and got poor results.". Would tell a little detail why you think the results are poor?Anonymousnoreply@blogger.comtag:blogger.com,1999:blog-4457216402399127579.post-69195855495195188422013-04-26T10:51:29.905-04:002013-04-26T10:51:29.905-04:00Thanks for contributing those links! I had not se...Thanks for contributing those links! I had not seen them before. Yes, we are essentially doing the SafeSeq method on the MiSeq. It seems like the duplex tagging is a great idea too.Anonymoushttps://www.blogger.com/profile/01081006207025463719noreply@blogger.comtag:blogger.com,1999:blog-4457216402399127579.post-63692082608715866212013-04-26T01:48:43.144-04:002013-04-26T01:48:43.144-04:00Hi Scott,
Is your molecule tagging like the Verit...Hi Scott,<br /><br />Is your molecule tagging like the Veritag method from Population Genetics (http://www.populationgenetics.com/technology/veritag/) or the SAFE-Seq method (http://www.pnas.org/content/108/23/9530) or the Duplex Sequencing approach (http://www.ncbi.nlm.nih.gov/pubmed/22853953)? It would be nice to see one of these ported to the MiSeq.Anonymousnoreply@blogger.comtag:blogger.com,1999:blog-4457216402399127579.post-50485385950899089202013-04-25T06:40:40.116-04:002013-04-25T06:40:40.116-04:00we had a high AR result for FLT-ITD mut. in AML sh...we had a high AR result for FLT-ITD mut. in AML shall we proceed with direct sequencing or NGS & will your technique with large overlaps be better for clarifying the sequencemagda assemnoreply@blogger.comtag:blogger.com,1999:blog-4457216402399127579.post-22186515007497387282013-04-24T21:14:44.330-04:002013-04-24T21:14:44.330-04:00Thanks for that comment Scott. I will look at the ...Thanks for that comment Scott. I will look at the website. We are running the MiSeq with the long overlaps this week. I will have data on quality scores next week. Anonymoushttps://www.blogger.com/profile/14602560263535951430noreply@blogger.comtag:blogger.com,1999:blog-4457216402399127579.post-56779879397734638212013-04-24T20:49:19.835-04:002013-04-24T20:49:19.835-04:00Great post! I would be very interested in what yo...Great post! I would be very interested in what you come up with to update quality scores based on the overlapping reads. <br /><br />We have been working on some of these exact problems with our MiSeq 16S amplicon sequencing. We developed a method of frameshifted primers that requires no PhiX, and it should be extendable to the HiSeq platform. We also have a new method which we call molecule tagging that can reduce amplicon error rates down to ~0.42 ept.<br /><br />I am working on a website that explains these processes in greater detail and provides the necessary bioinformatic tools (<a href="https://sites.google.com/site/moleculetagtoolbox/description" rel="nofollow">https://sites.google.com/site/moleculetagtoolbox/description</a>). It is still a work in progress and will be until our paper gets accepted (which should be very soon).Anonymoushttps://www.blogger.com/profile/01081006207025463719noreply@blogger.comtag:blogger.com,1999:blog-4457216402399127579.post-42045939114462927342013-04-24T07:34:36.124-04:002013-04-24T07:34:36.124-04:00Our MiSeq past this test running MCS2.2 and RTA 1....Our MiSeq past this test running MCS2.2 and RTA 1.17.<br />Very good quality for low diversity libraries with only 2% Phi-X in a 2x151 PE run.<br />Hurry for Illumina.<br />Unfortunately they do not plan to introduce the new RTA algorithm into the HiSeq RTA. So a control lane remains necessary...Faevaenoreply@blogger.comtag:blogger.com,1999:blog-4457216402399127579.post-59302292804608944952013-03-26T14:37:50.701-04:002013-03-26T14:37:50.701-04:00PhiX is needed nomore!
Illumina's latest RTA w...PhiX is needed nomore!<br />Illumina's latest RTA works equally well on a single amplicon with no PhiX as 20 amplicons and 50% PhiX. Our first test using the new RTA is running now!James@cancerhttps://www.blogger.com/profile/02825715598810395734noreply@blogger.comtag:blogger.com,1999:blog-4457216402399127579.post-32473392316693895452013-03-09T09:08:25.566-05:002013-03-09T09:08:25.566-05:00This was told to me directly on the phone by two d...This was told to me directly on the phone by two different Illumina support specialists (MiSeq operations and Sample Prep). Anonymoushttps://www.blogger.com/profile/14602560263535951430noreply@blogger.comtag:blogger.com,1999:blog-4457216402399127579.post-29124295224489120902013-03-09T05:55:20.233-05:002013-03-09T05:55:20.233-05:00"Illumina tech support insists that a spike-i..."Illumina tech support insists that a spike-in of 50% Phix DNA is necessary for any amplicon assay"<br /><br />Where did you read this information? Is this statement true only for Nextera or every amplicon assay?Anonymousnoreply@blogger.comtag:blogger.com,1999:blog-4457216402399127579.post-71431254219827388642013-03-08T16:30:40.857-05:002013-03-08T16:30:40.857-05:00That's why we want to use long reads on a shor...That's why we want to use long reads on a short amplicon, so they overlap a lot and we don't have to use the sequence at the ends of the read. I will post some overlapped and joined reads with adjusted Q-scores when we get them. <br />Anonymoushttps://www.blogger.com/profile/14602560263535951430noreply@blogger.comtag:blogger.com,1999:blog-4457216402399127579.post-37704896756335640002013-03-08T12:24:01.105-05:002013-03-08T12:24:01.105-05:00It might be a silly question, but what about quali...It might be a silly question, but what about quality, that drops down towards the end of the read?Povilashttps://www.blogger.com/profile/03089114027488985624noreply@blogger.com