tag:blogger.com,1999:blog-36768584.post5086008806446578077..comments2024-03-03T18:49:34.382-05:00Comments on Omics! Omics!: NGS Saves A Young LifeKeith Robisonhttp://www.blogger.com/profile/04765318239070312590noreply@blogger.comBlogger15125tag:blogger.com,1999:blog-36768584.post-27332958190136104812014-05-27T22:11:51.682-04:002014-05-27T22:11:51.682-04:00PasserBy: thanks for the comment! While I agree ...PasserBy: thanks for the comment! While I agree with you that a good qPCR panel could be an option, the appeals of sequencing are that on the one hand one is empowering detection of a very broad range of possible pathogens and on the other that the cost or sequencing is still on a steady downward drop, whereas qPCR is a pretty mature technology. Shotgun sequencing also has the advantage of not requiring any specialized (for the assay) reagents to be pre-positioned at point-of-care; for this setting time is critical. Keith Robisonhttps://www.blogger.com/profile/04765318239070312590noreply@blogger.comtag:blogger.com,1999:blog-36768584.post-79025743671270026582014-05-27T02:12:39.188-04:002014-05-27T02:12:39.188-04:00What I think the bigger issue is the failure of th...What I think the bigger issue is the failure of the directed PCRs. What this shows is a disconnect of curation of the PCR or similar technique (qPCR, sanger sequencing, fusion NGS) that keeps the sequence design current with the correct specifications. NGS is a great tool and I use it quite often for specific cases like this, however after reviewing what was done and not done it almost always comes down to failure to update the oligo design for current strain and region information that causes a specific assay to fail. Other times, it is the time point of when the sample is collected an analyzed makes it so the target is not present in the sample. NGS is a great tool, but for several hundred/thousand dollars per sample it would be much more beneficial to the patient and cost effective to run an accurately designed and curated qPCR or panel of qPCRs in a half day than spending the effort required by NGS. That said, each molecular analysis tool has its place. The key to successful molecular analysis is rapid, redundant, and repetative bioinformatics curating the assay desings in silico on a regular and ongoing basis. Good to hear the causative agent was identified in this case.PasserBynoreply@blogger.comtag:blogger.com,1999:blog-36768584.post-38592174895237847582014-03-28T14:43:01.304-04:002014-03-28T14:43:01.304-04:00Thanks for the interesting post: a triumph for dia...Thanks for the interesting post: a triumph for diagnostic metagenomics! Readers might be interested in some of our recent publications on this approach:<br />Diagnostic metagenomics: potential applications to bacterial, viral and parasitic infections<br />http://journals.cambridge.org/action/displayAbstract?fromPage=online&aid=9186805<br /><br />A culture-independent sequence-based metagenomics approach to the investigation of an outbreak of Shiga-toxigenic Escherichia coli O104:H4<br />http://jama.jamanetwork.com/article.aspx?articleid=1677374<br /><br />Metagenomic analysis of tuberculosis in a mummy<br />http://www.nejm.org/doi/full/10.1056/NEJMc1302295Mark Pallenhttps://www.blogger.com/profile/06911675151032525386noreply@blogger.comtag:blogger.com,1999:blog-36768584.post-44719952592236475392014-03-28T14:41:03.291-04:002014-03-28T14:41:03.291-04:00Thanks for the interesting post: a triumph for dia...Thanks for the interesting post: a triumph for diagnostic metagenomics! Readers might be interested in some of our recent publications on this approach:<br />Diagnostic metagenomics: potential applications to bacterial, viral and parasitic infections<br />http://journals.cambridge.org/action/displayAbstract?fromPage=online&aid=9186805<br /><br />A culture-independent sequence-based metagenomics approach to the investigation of an outbreak of Shiga-toxigenic Escherichia coli O104:H4<br />http://jama.jamanetwork.com/article.aspx?articleid=1677374<br /><br />Metagenomic analysis of tuberculosis in a mummy<br />http://www.nejm.org/doi/full/10.1056/NEJMc1302295<br />Mark Pallenhttps://www.blogger.com/profile/06911675151032525386noreply@blogger.comtag:blogger.com,1999:blog-36768584.post-61523436333251168882014-03-12T02:29:11.522-04:002014-03-12T02:29:11.522-04:00Thanks for this story - it is particularly interes...Thanks for this story - it is particularly interesting to me having worked on Leptospira genomics since I started in bioinformatics 11 years ago!<br /><br />The k-mer approach for identification is a good strategy, recently implemented in Kraken by Wood and Salzberg: http://ccb.jhu.edu/software/kraken/<br /><br />Leptospira has now been at AGBT, on The Simpsons, on Mythbusters, and on The Big Bang Theory! :-)<br /><br />Torsten Seemannhttps://www.blogger.com/profile/12241185247897084810noreply@blogger.comtag:blogger.com,1999:blog-36768584.post-88026146206531580702014-03-11T15:07:47.691-04:002014-03-11T15:07:47.691-04:00@Anonymous: I was a grad student in the DeRisi lab...@Anonymous: I was a grad student in the DeRisi lab. Before switching over to NGS, the lab did similar pathogen hunting using a custom designed mircoarray called the ViroChip. In fact, they (I say "they" because I worked on a completely different project) helped identify the causative agent for SARS, and there were several projects in the lab on identifying novel viruses in patient samples of unknown etiology from Bay Area hospitals. So, the lab is actually somewhat well-known for this type of thing.klontoknoreply@blogger.comtag:blogger.com,1999:blog-36768584.post-6404028266640770412014-02-28T05:45:29.513-05:002014-02-28T05:45:29.513-05:00The quality of the reads coming from NGS platform ...The quality of the reads coming from NGS platform is a key for diagnostic purposes. The time is fine but the quality is everything. !!!Anonymoushttps://www.blogger.com/profile/09082436817564110733noreply@blogger.comtag:blogger.com,1999:blog-36768584.post-51580948770336532222014-02-27T13:17:00.434-05:002014-02-27T13:17:00.434-05:00Great post Keith - your comment on using the Ion p...Great post Keith - your comment on using the Ion platform for "rapid ID of pathogens" is spot on - some protocols in my lab are <12 hours at the moment (ampli-seq) for biosurveillance purposes. The MiSeq though - is a very capable fast, brute force "metagenomics" platform when it needs to be as well. (speaking from experience) But in a diagnostics/biosurveillance role - you're not _really_ doing metagenomics. The basic question is "Given a list of pathogens (maybe a fairly big list, but smaller than Genbank) - are any of these bugs in this sample?"<br /><br />IMHO - this is where NGS is going for rapid pathogen diagnostics. In a few years, given the rate of NGS innovation, the days of PCR testing samples for pathogen ID will be dead.Jonathan Jacobshttps://www.blogger.com/profile/06133232985480734844noreply@blogger.comtag:blogger.com,1999:blog-36768584.post-40079915572825481602014-02-27T09:16:37.596-05:002014-02-27T09:16:37.596-05:00Thanks for sharing this. When doctors trying to u...Thanks for sharing this. When doctors trying to use culture to identify the pathogen for my 2-month old son one month ago, I strongly feel sequencing should be used for such purposes. <br /><br />I also like your proposal on CASP or Assemblothon style challenge. There was such a challenge organized by DTRA of Department of Defense last year (DTRA Algorithm Challenge, 1 million dollar prize) covering exactly the same problem. We won the challenge by developing a series of new algorithms, such as fast host read filter, fast and sensitive GenBank alignment tool (has to work with reads from Miseq Ion Torrent 454 PacBio), and an accurate taxa assignment algorithm. Hopefully those algorithms will be released soon, and hope sequencing will be routinely used in hospitals to detect pathogens soon. xiechaoshttps://www.blogger.com/profile/11641744834383350858noreply@blogger.comtag:blogger.com,1999:blog-36768584.post-53736163369731300582014-02-27T09:15:23.481-05:002014-02-27T09:15:23.481-05:00Thanks for sharing this. When doctors trying to u...Thanks for sharing this. When doctors trying to use culture to identify the pathogen for my 2-month old son one month ago, I strongly feel sequencing should be used for such purposes. <br /><br />I also like your proposal on CASP or Assemblothon style challenge. There was such a challenge organized by DTRA of Department of Defense last year (DTRA Algorithm Challenge, 1 million dollar prize) covering exactly the same problem. We won the challenge by developing a series of new algorithms, such as fast host read filter, fast and sensitive GenBank alignment tool (has to work with reads from Miseq Ion Torrent 454 PacBio), and an accurate taxa assignment algorithm. Hopefully those algorithms will be released soon, and hope sequencing will be routinely used in hospitals to detect pathogens soon. xiechaoshttps://www.blogger.com/profile/11641744834383350858noreply@blogger.comtag:blogger.com,1999:blog-36768584.post-1888020430754745472014-02-26T14:26:34.284-05:002014-02-26T14:26:34.284-05:00Many thanks Keith for this write-up. (You saved me...Many thanks Keith for this write-up. (You saved me the effort!)<br /><br />There are a few items that I've thought about since that <b>great</b> talk, in order:<br /><br />1) A bit of a custom-tailored situation to demonstrate the power of NGS for a critical-care case. You point out the multiple possible etiologies for encephilitis, and in this case the physicians presumed since the infectious diagnostics came back negative, it was presumed it was autoimmune in nature. And the patient's condition only grew worse. So here was a case where the patient was both immune-compromised and exposed to a possible environmental pathogen.<br /><br />2) DeRisi focused his efforts on the 90 min of analysis and how it could be accelerated, and no detail about how the sequencing could have been sped up. Thanks for the acknowledgement that <a href="http://www.lifetechnologies.com/us/en/home/life-science/sequencing/next-generation-sequencing/ion-torrent-next-generation-sequencing-workflow/ion-torrent-next-generation-sequencing-run-sequence/ion-pgm-system-for-next-generation-sequencing.html" rel="nofollow">Ion Torrent PGM</a> could have been used to shave at least 10h if not 12h from the sample to answer cycle.<br /><br />3) Also Joe did not mention anything at all about the 7h sample preparation. He knew he was working with an unknown causative agent, including fungus and virus. In the Q&A he was asked about it, he just mentioned it was a 'total nucleic acid prep', which presumably meant accounting for both RNA and DNA viruses, along with fungi which could pose problems of its own, along with Gram+ and Gram- bacteria. But DeRisi knew all this, and knew how to prepare separate libraries, equalize/pool them, and sequence.<br /><br />Bonus point 4: Out of 1570 cases in the past 7y, a full 63% went undiagnosed. So there's a real unmet healthcare need here that NGS can solve.<br /><br />Thanks again for the post.<br /><br />Dale Anonymoushttps://www.blogger.com/profile/06793338970769363594noreply@blogger.comtag:blogger.com,1999:blog-36768584.post-89802384218949690012014-02-26T14:25:43.429-05:002014-02-26T14:25:43.429-05:00Many thanks Keith for this write-up. (You saved me...Many thanks Keith for this write-up. (You saved me the effort!)<br /><br />There are a few items that I've thought about since that <b>great</b> talk, in order:<br /><br />1) A bit of a custom-tailored situation to demonstrate the power of NGS for a critical-care case. You point out the multiple possible etiologies for encephilitis, and in this case the physicians presumed since the infectious diagnostics came back negative, it was presumed it was autoimmune in nature. And the patient's condition only grew worse. So here was a case where the patient was both immune-compromised and exposed to a possible environmental pathogen.<br /><br />2) DeRisi focused his efforts on the 90 min of analysis and how it could be accelerated, and no detail about how the sequencing could have been sped up. Thanks for the acknowledgement that <a href="http://www.lifetechnologies.com/us/en/home/life-science/sequencing/next-generation-sequencing/ion-torrent-next-generation-sequencing-workflow/ion-torrent-next-generation-sequencing-run-sequence/ion-pgm-system-for-next-generation-sequencing.html" rel="nofollow">Ion Torrent PGM</a> could have been used to shave at least 10h if not 12h from the sample to answer cycle.<br /><br />3) Also Joe did not mention anything at all about the 7h sample preparation. He knew he was working with an unknown causative agent, including fungus and virus. In the Q&A he was asked about it, he just mentioned it was a 'total nucleic acid prep', which presumably meant accounting for both RNA and DNA viruses, along with fungi which could pose problems of its own, along with Gram+ and Gram- bacteria. But DeRisi knew all this, and knew how to prepare separate libraries, equalize/pool them, and sequence.<br /><br />Bonus point 4: Out of 1570 cases in the past 7y, a full 63% went undiagnosed. So there's a real unmet healthcare need here that NGS can solve.<br /><br />Thanks again for the post.<br /><br />Dale Anonymoushttps://www.blogger.com/profile/06793338970769363594noreply@blogger.comtag:blogger.com,1999:blog-36768584.post-50671825387345991372014-02-26T11:41:48.976-05:002014-02-26T11:41:48.976-05:00Anonyomous: that's how DeRisi presented it in ...Anonyomous: that's how DeRisi presented it in my memory - a desperation call. Whether they were aware of his polar bear work he didn't say; I found that when checking if the story had been published. Not being in an academic medical center, I can't comment on the degree to which this communication occurs -- but it certainly needs to!Keith Robisonhttps://www.blogger.com/profile/04765318239070312590noreply@blogger.comtag:blogger.com,1999:blog-36768584.post-53042034068846745942014-02-26T11:00:05.277-05:002014-02-26T11:00:05.277-05:00"In desperation, the doctors approached DeRis..."In desperation, the doctors approached DeRisi to use sequencing as an unbiased search for an occult pathogen." This part is fascinating to me. Do we know how the doctors knew to contact DeRisi? How did they know who he was, or what he might be able to do? If this were fiction, this would be a deus ex machina. It's that missing link that explains so much of what goes on in the world. Sure, maybe he plays golf with one of the doctors and it was totally serendipitous. Or is there now a consciousness among clinicians of NGS (and knowledge of the people doing the sequencing and bioinformatics) that stories like this actually happen all the time. I'm just curious.Anonymousnoreply@blogger.comtag:blogger.com,1999:blog-36768584.post-43630936750467047202014-02-26T10:16:19.033-05:002014-02-26T10:16:19.033-05:00Thanks for the awesome post. Glad that you beat me...Thanks for the awesome post. Glad that you beat me to do it :) I thought this great story got completely swept away due to Nanopore talk at AGBT. Really glad that you wrote it. I have storified tweets from Joe DeRis's talk here <br /><br />http://nextgenseek.com/2014/02/ngs-in-critical-care-a-feel-good-story/ nextgenseekhttp://nextgenseek.comnoreply@blogger.com