Seqanswers Leaderboard Ad

Collapse

Announcement

Collapse
No announcement yet.
X
 
  • Filter
  • Time
  • Show
Clear All
new posts

  • #16
    Originally posted by gringer View Post
    If this is a raw per-molecule error (as I assume with a "single molecule" technology), then I consider it acceptable. Illumina and SOLiD technologies use hundreds of identical sequencing reactions per cluster to generate a consensus read, and it's unlikely they would have the same accuracy with single molecules per cluster..
    Thank you for spotting that, it's nice to meet someone with insight.

    C.

    Comment


    • #17
      Originally posted by gringer View Post
      The bias would increase the chance of two strands having an error at the same location, which would probably mean there'll be some tricky regions that still can't be sequenced accurately, regardless of how many times it is done in parallel.
      You sure about this? With the complement strand the sequencer would see not only a completely different series of bases, but bases from the "other" direction, I think. if anything I think the error profile would be remarkably different for complementary strands.

      Comment


      • #18
        Originally posted by clivey View Post
        Thank you for spotting that, it's nice to meet someone with insight.

        C.
        Was the 4% error rate for the consensus sequence from hairpin reads or for single strands?

        Comment


        • #19
          Originally posted by nxgsqq View Post
          The bigger unknown is the true, customer usability of their pores. I would assume they are Poisson loading the pores before they ship to users.
          The information on the website suggests that the pores (in solution) are loaded onto the chip at the start of a run:

          Sorry, this page has been moved or deleted Are you looking for any of the following? Pu


          The sensor chips are stored dry, but on starting the experiment, the flow cell is automatically exposed to the relevant electrophysiological and other fluids required to create nanopores in bilayers in experimental conditions.
          I presume the system will be set up so that the reported shelf life of the cartridges will give a reasonable proportion of active nanopores up to the use by date.

          They intend to use solid-state pores in the future, which I guess would make the cartridges last a whole lot longer.

          Sorry, this page has been moved or deleted Are you looking for any of the following? Pu


          How many pores are still active after an hour of use? I know the bilayers can be made stable and inert, I can accept the error profile can be made length independent (especially if they tether the motor to the pore, else Brownian motion of long DNA can act to pull the complex off, even against the electric field), but how many pores are sequencing at a given time and how does that number drop off over time?
          The occupancy/speed is customisable. Faster movement has a quality reduction and greater chance of missed bases. From reading one of the press releases (I forget which one), it sounded like the USB MinION would run for no more than 10 hours, but the GridION cartridges could keep going for a few days. Samples can be ejected during a run and shifted to other cartridges, cartridges can be replaced, runs can be started at any time, and stopped due to a number of different desired factors, so any drop-off in occupancy probably won't have too much impact on result output (as long as there's still money to burn).

          Comment


          • #20
            Originally posted by Heisman View Post
            You sure about this? With the complement strand the sequencer would see not only a completely different series of bases, but bases from the "other" direction, I think. if anything I think the error profile would be remarkably different for complementary strands.
            I can only guess at the nature of the error that they are talking about, but the "tricky regions" may not necessarily be an obvious class of sequences. Maybe there'll be an issue with only A/T homopolymers advancing a bit slower (or quicker) than expected. Perhaps there would be particular palindromic sequences (appearing the same in reverse complement) that have a high likelihood of read errors even when sequencing both strands.

            Even if it would be possible to reduce error by doing more sequencing, they will still need to try their hardest to increase the single-strand accuracy (and reduce bias) given that it's reported as a single-molecule sequencing technology.

            Comment


            • #21
              Originally posted by larissa View Post
              Even with 4% error rates? Will they deliver in improving that? That's not acceptable for clinical use. It may be very useful for a lot of other stuff.
              I wouldn't say ONT is really going for that market yet.

              I don't even think it competes with the current sequencers. This is something that complements them very nicely, with some applications it can do uniquely.

              Comment


              • #22
                Originally posted by SeqAA View Post
                That's not acceptable for clinical use. It may be very useful for a lot of other stuff.
                I wouldn't say ONT is really going for that market yet.
                The website is surprisingly useful for answering questions about many things related to their method and intentions (hence my numerous references to it). For example, they are definitely considering clinical uses for their nanopore system:

                Sorry, this page has been moved or deleted Are you looking for any of the following? Pu


                Use of the GridION platform in Personalised Healthcare
                The GridION platform is an electronic analysis system that can be tailored for the analysis of DNA, RNA, protein and other analytes. This novel technology has applications across personalised healthcare. this may include the analysis of a patient's DNA, discovery and validation of new protein biomarkers or an electronic diagnostic test for discovered biomarkers.
                In the clinical setting, it looks like they're putting a bit of effort into a more direct protein identification (via aptamers), which is likely to be more useful for diagnostic and monitoring purposes when compared to the mostly unchanging DNA.

                Sorry, this page has been moved or deleted Are you looking for any of the following? Pu

                Comment


                • #23
                  Finally! (I am being optimistic and assuming the legitimate concerns about accuracy will be adequately addressed over time).

                  I have always thought the true definition of "Next generation" was not amount of data but rather read length. I have been disappointed that so-called "Next gen" technologies from ILMN and Life would deliver shorter reads than Sanger but just boat-loads of them. It has always left a bad taste in my mouth.

                  Now with PacBio, GnuBio and Nanopore, it seems like we are finally focusing on the true Next Nex-gen. 100 Mega-base reads, anyone? Of course it drastically changes the jobs of bioinformatics folks like me. And makes life kinda fun!
                  Kamalakar Gulukota,
                  Director,
                  Center for Bioinformatics and Computational Biology
                  NorthShore University Health System, [email protected]

                  Comment


                  • #24
                    It seems to me like the acceptability of a 4% error rate would depend on the sample type. One advantage of sequencing clusters (or beads) is that each read is a pretty accurate determination of sequence derived from a single template. I am a bit of a statistics moron, but it seems like if your starting material is impure (e.g. a tumor sample), that it would be easier to distinguish normal sequence from minority-contributor sequence (say 5%) if you are 99.9% sure of each base in a read than if you are 96% sure of each base in a read. While 200x coverage might be sufficient for the former, would it be sufficient for the latter?

                    Comment


                    • #25
                      Originally posted by joss211 View Post
                      It seems to me like the acceptability of a 4% error rate would depend on the sample type. One advantage of sequencing clusters (or beads) is that each read is a pretty accurate determination of sequence derived from a single template. I am a bit of a statistics moron, but it seems like if your starting material is impure (e.g. a tumor sample), that it would be easier to distinguish normal sequence from minority-contributor sequence (say 5%) if you are 99.9% sure of each base in a read than if you are 96% sure of each base in a read. While 200x coverage might be sufficient for the former, would it be sufficient for the latter?
                      That's one of the reasons why 4% error rate is not acceptable in the clinic, for guiding treatment/dosing and inclusion/exclusion criteria. It will be acceptable for lots of other things, all R&D oriented, in academia and private sector.

                      Comment


                      • #26
                        Originally posted by nxgsqq View Post
                        I too agree the error is acceptable, IF, it is indeed the typical error they see and not a 'best case' presented for effect and advertisement. I am suspicious of resolving 64 levels (3 base read) electronically considering how small the differences will be. I don't completely buy the algorithmic deconvolution either, especially if they are still using a polymerase. If it is a non-stochasitic transport, a Viterbi/HMM algorithm might give 94% accuracy.

                        The bigger unknown is the true, customer usability of their pores. I would assume they are Poisson loading the pores before they ship to users. How many pores are still active after an hour of use? I know the bilayers can be made stable and inert, I can accept the error profile can be made length independent (especially if they tether the motor to the pore, else Brownian motion of long DNA can act to pull the complex off, even against the electric field), but how many pores are sequening at a given time and how does that number drop off over time?
                        They are certainly not using a polymerase; they have developed their own 'motor-protein enzyme' by screening 300+ mutations of some natural enzyme (possibly a helicase?).

                        Their nanopore array is possibly the most innovative feature of their system - they have developed some sort of a synthetic polymer that replaces the lipid bilayer. So they are able to embed the protein in the polymer and array them on the chip overcoming the Poisson distribution. According to Brown, 80% of the chip is still functional after 3 days (I assume 3 days after activation). They also exposed the chip to blood and sewage and it retained functionality. The extreme stability of the synthetic polymer is what's enabling the MINIon technology really.

                        Comment


                        • #27
                          Originally posted by Nanoporous View Post
                          Their nanopore array is possibly the most innovative feature of their system - they have developed some sort of a synthetic polymer that replaces the lipid bilayer. So they are able to embed the protein in the polymer and array them on the chip overcoming the Poisson distribution.
                          .

                          If he specifically mentioned overcoming Poisson, did he say how successful that was? I would say you can tweak it some, but that you'll always have a bit of the old alea iacta stuff going on, with some no-shows and some doubles. It might make sense to ship dry and have some priming program run after activation. You clearly have to be recording in order to array the pores so you can follow them go in and it would take some time, I guess, until you have a sizeable portion of membranes with exactly a single pore. Fascinating stuff.

                          Comment


                          • #28
                            If I recall correctly from his talk he mentioned 25% of the wells (pore binding sites?, array positions? not quite sure what to call them...) had a single pore.

                            edit : one could call them membranes, I suppose.

                            Comment


                            • #29
                              Originally posted by kgulukota View Post
                              Finally! (I am being optimistic and assuming the legitimate concerns about accuracy will be adequately addressed over time).

                              I have always thought the true definition of "Next generation" was not amount of data but rather read length. I have been disappointed that so-called "Next gen" technologies from ILMN and Life would deliver shorter reads than Sanger but just boat-loads of them. It has always left a bad taste in my mouth.

                              Now with PacBio, GnuBio and Nanopore, it seems like we are finally focusing on the true Next Nex-gen. 100 Mega-base reads, anyone? Of course it drastically changes the jobs of bioinformatics folks like me. And makes life kinda fun!
                              Is there any evidence that either PacBio or GnuBIO could reach ultra long reads? PacBio is making improvements in read length, but at the pace they're going, it doesn't seem like they'd ever reach anything approaching 1Mb. As for GnuBIO, how large of an n-mer would they need to be able to sequence a whole human genome with their SBH approach?

                              Comment


                              • #30
                                Originally posted by GW_OK View Post
                                If I recall correctly from his talk he mentioned 25% of the wells (pore binding sites?, array positions? not quite sure what to call them...) had a single pore.

                                edit : one could call them membranes, I suppose.
                                25%? Not exactly overcoming Poisson (ideally 36,8% if I recall?)

                                Comment

                                Latest Articles

                                Collapse

                                • seqadmin
                                  Strategies for Sequencing Challenging Samples
                                  by seqadmin


                                  Despite advancements in sequencing platforms and related sample preparation technologies, certain sample types continue to present significant challenges that can compromise sequencing results. Pedro Echave, Senior Manager of the Global Business Segment at Revvity, explained that the success of a sequencing experiment ultimately depends on the amount and integrity of the nucleic acid template (RNA or DNA) obtained from a sample. “The better the quality of the nucleic acid isolated...
                                  03-22-2024, 06:39 AM
                                • seqadmin
                                  Techniques and Challenges in Conservation Genomics
                                  by seqadmin



                                  The field of conservation genomics centers on applying genomics technologies in support of conservation efforts and the preservation of biodiversity. This article features interviews with two researchers who showcase their innovative work and highlight the current state and future of conservation genomics.

                                  Avian Conservation
                                  Matthew DeSaix, a recent doctoral graduate from Kristen Ruegg’s lab at The University of Colorado, shared that most of his research...
                                  03-08-2024, 10:41 AM

                                ad_right_rmr

                                Collapse

                                News

                                Collapse

                                Topics Statistics Last Post
                                Started by seqadmin, Yesterday, 06:37 PM
                                0 responses
                                10 views
                                0 likes
                                Last Post seqadmin  
                                Started by seqadmin, Yesterday, 06:07 PM
                                0 responses
                                9 views
                                0 likes
                                Last Post seqadmin  
                                Started by seqadmin, 03-22-2024, 10:03 AM
                                0 responses
                                50 views
                                0 likes
                                Last Post seqadmin  
                                Started by seqadmin, 03-21-2024, 07:32 AM
                                0 responses
                                67 views
                                0 likes
                                Last Post seqadmin  
                                Working...
                                X