Super, it is so simple when we see image.Excuse my english, We have a Miseq, the optimisation is well when do the exact process of the protocol. But, I think that we must have more document for the interpretation of data of run like density; CPF;Q30
Guys, one thing that I can't get-how to make cluster monoclonal?Because after DNA fragmentation(tagmentation) we will get different sequences(not the same). We have different DNA fragments with same adapters at the ends,so as result-different sequences can link to the nanowell oligos? Please, explain if possible.
Hi, I had the same question, basically they use a method called exclusion amplification (ExAmp): "ExAmp cluster generation: clustering on the HiSeq 4000 and X Ten platforms use Illumina's latest "Exclusion Amplification" chemistry. ExAmp ensures only a single template molecule binds and forms a cluster. Cluster amplification is almost instantaneous and excludes other molecules from binding. Most wells have a single molecule cluster, some wells are polyclonal but excluded from the by the chastity filter and will not appear in your %PF reads. A few nanowells will be empty. Some will be "ExAmp duplicates", which form because a single molecule which formed one cluster is able to hybridise to another nanowell nearby and create a second "ExAmp duplicate" cluster. One obvious part of the exclusion amplification as implemented is the very viscous enzyme mix. Probably the diffusion of the library fragments towards the flowcell is very much slowed down (requiring also higher library concentrations?) giving the molecule that arrives first the chance to become amplified and fill entire nanowells before a second one arrives (www.google.com/patents/WO2013188582A1?cl=en). The viscosity enhanced "drag" also could explain the stronger bias towards smaller inset size reads? The high viscosity buffer together with high library concentrations and "RPA" amplification for the clustering process ("Recombinase Polymerase Amplification" ( www.twistdx.co.uk/our_technology/ )) might be sufficient for the Kinetic Exclusion Amplification on the nanowell flowcells? It seems to me that the other methods described in the patent might not be compatible with the old cBots (these can be used for the Hiseq3000/4000 clustering after a software upgrade)?" Since the science behind it is patented, I think the exact mechanism is confidential. But I hope this helps
@@romiarz Thanks for the patent link. It was very helpful! I think I agree with your assessment. The emphasis on simultaneous binding and cluster amplification is telling. Best.
I still have a question, at this video 55" - 59", a single molecule move in with both adaptor sites double stranded, then a "red" material (enzyme?) removed it. Why they have to do it this way? why not just the single molecule moves in?
Fantastic animation!
Super, it is so simple when we see image.Excuse my english, We have a Miseq, the optimisation is well when do the exact process of the protocol. But, I think that we must have more document for the interpretation of data of run like density; CPF;Q30
Guys, one thing that I can't get-how to make cluster monoclonal?Because after DNA fragmentation(tagmentation) we will get different sequences(not the same). We have different DNA fragments with same adapters at the ends,so as result-different sequences can link to the nanowell oligos? Please, explain if possible.
Hi, I had the same question, basically they use a method called exclusion amplification (ExAmp):
"ExAmp cluster generation: clustering on the HiSeq 4000 and X Ten platforms use Illumina's latest "Exclusion Amplification" chemistry. ExAmp ensures only a single template molecule binds and forms a cluster. Cluster amplification is almost instantaneous and excludes other molecules from binding. Most wells have a single molecule cluster, some wells are polyclonal but excluded from the by the chastity filter and will not appear in your %PF reads. A few nanowells will be empty. Some will be "ExAmp duplicates", which form because a single molecule which formed one cluster is able to hybridise to another nanowell nearby and create a second "ExAmp duplicate" cluster.
One obvious part of the exclusion amplification as implemented is the very viscous enzyme mix. Probably the diffusion of the library fragments towards the flowcell is very much slowed down (requiring also higher library concentrations?) giving the molecule that arrives first the chance to become amplified and fill entire nanowells before a second one arrives (www.google.com/patents/WO2013188582A1?cl=en). The viscosity enhanced "drag" also could explain the stronger bias towards smaller inset size reads?
The high viscosity buffer together with high library concentrations and "RPA" amplification for the clustering process ("Recombinase Polymerase Amplification" ( www.twistdx.co.uk/our_technology/ )) might be sufficient for the Kinetic Exclusion Amplification on the nanowell flowcells? It seems to me that the other methods described in the patent might not be compatible with the old cBots (these can be used for the Hiseq3000/4000 clustering after a software upgrade)?"
Since the science behind it is patented, I think the exact mechanism is confidential. But I hope this helps
@@romiarz Thanks for the patent link. It was very helpful! I think I agree with your assessment. The emphasis on simultaneous binding and cluster amplification is telling. Best.
@@romiarz @user-mf8hf4uk3h
Thanks to my seniors, my curiosity was resolved!!☺
I still have a question, at this video 55" - 59", a single molecule move in with both adaptor sites double stranded, then a "red" material (enzyme?) removed it. Why they have to do it this way? why not just the single molecule moves in?
J Q If only new what you guys are talking about.
Illumina - *ti* ?