I'm a bit puzzled how the chemistry will work without ZMWs (if indeed ZMWs are not being used). If you have ~100nm evanescent depth and ~300nmx300nm pixel, that gives a volume of ~0.01um^3 = 10^-20 m^3 = 10^-17L. If you have 1uM nucleotides then you'll have 10^-23 moles or 6 molecules in the volume at a given time, which will contribute quite a bit of noise. You can drop the nucleotide concentration of course but there's a speed cost to that. Unless the goal is to use synchronized reversibly blocked ncuelotides like ILMN wherein the excess can be washed out.
Pretty interesting nonetheless, thanks for surfacing!
Great question and this is the problem that lead us to TCPA as a solution.
Attempting to do real-time sequencing with many labelled nucs in solution inevitably gets you to start collecting noise instead of signal which leads to saturation of sensor and a whole mess that you cannot untangle. One way around this is volume filtering and using ZMW like PacBio and to restrict the number of fluorophores in the volume of interest. We do some of that with TIRF. We also use TCPA as a temporal filter. The fluorophore that spends enough time, but not too long a time, in the volume of interest will trigger a change event. Anything that is transient spends orders of magnitude less time in volume of interest to contribute to the detection. Likewise anything that lingers and emits will not generate change. We can detect single molecules incorporation events in concentrations greater than 1uM.
Using TCPA may just solve one of the big problems with CMOS arrays. Since TCPA's only report when they detect signal, they run asynchronously so each spot basically reports at whatever rate it is running at. Now!!!! if they have a chemistry that runs continuously, so the reaction goes dye-ligated base binds emits signal and then the next dye-ligated base binds after ligation is completed (in other words) if all bases are present in mix and they have a way of displacing currently bound dye and adding the next base, then TCPA could just start reporting the sequence continuously as soon as rxn gmish is present.
Each spot would be continuosly reporting sequence.
I think I found this vide throu ASEQ. Was it was discussed in this channel. It is mind blowing!!! How much integration they achieved on a single chip!!!!
Thanks for the writeup Nava.
I'm a bit puzzled how the chemistry will work without ZMWs (if indeed ZMWs are not being used). If you have ~100nm evanescent depth and ~300nmx300nm pixel, that gives a volume of ~0.01um^3 = 10^-20 m^3 = 10^-17L. If you have 1uM nucleotides then you'll have 10^-23 moles or 6 molecules in the volume at a given time, which will contribute quite a bit of noise. You can drop the nucleotide concentration of course but there's a speed cost to that. Unless the goal is to use synchronized reversibly blocked ncuelotides like ILMN wherein the excess can be washed out.
Pretty interesting nonetheless, thanks for surfacing!
Great question and this is the problem that lead us to TCPA as a solution.
Attempting to do real-time sequencing with many labelled nucs in solution inevitably gets you to start collecting noise instead of signal which leads to saturation of sensor and a whole mess that you cannot untangle. One way around this is volume filtering and using ZMW like PacBio and to restrict the number of fluorophores in the volume of interest. We do some of that with TIRF. We also use TCPA as a temporal filter. The fluorophore that spends enough time, but not too long a time, in the volume of interest will trigger a change event. Anything that is transient spends orders of magnitude less time in volume of interest to contribute to the detection. Likewise anything that lingers and emits will not generate change. We can detect single molecules incorporation events in concentrations greater than 1uM.
Thanks for the explanation Bojan, sounds super cool. Would you be able to share some data? Very keen to see how it compares to PacBio.
Also, Nava has a very vibrant sequencing discord channel (https://discord.com/channels/1072719341247799336). You'd be amongst very cool likeminded people!
Using TCPA may just solve one of the big problems with CMOS arrays. Since TCPA's only report when they detect signal, they run asynchronously so each spot basically reports at whatever rate it is running at. Now!!!! if they have a chemistry that runs continuously, so the reaction goes dye-ligated base binds emits signal and then the next dye-ligated base binds after ligation is completed (in other words) if all bases are present in mix and they have a way of displacing currently bound dye and adding the next base, then TCPA could just start reporting the sequence continuously as soon as rxn gmish is present.
Each spot would be continuosly reporting sequence.
See this amazing video about 20 years of PacBio development: https://vod.video.cornell.edu/media/2022%20CNF%2045th%2C%20Video%202%3B%20Dr.%20Mathieu%20Foquet%2C%20PacBio/1_tvaaaph6
I think I found this vide throu ASEQ. Was it was discussed in this channel. It is mind blowing!!! How much integration they achieved on a single chip!!!!