In the first post on Reticula I described my fund raising adventures and in part 2 I described the motivation for low cost ($1 COGS, $500 instrument) sequencing.
Hi Nava, this is a very interesting minimalist idea.
Some thoughts: I assume you'll try to keep the nucleotide concentration very low so that simultaneous incorporation of multiple bases is minimized (the first being bleached well before the second is incorporated), as well as the background fluorescence from bulk nucleotides?
Assuming you're capturing the templates on a random n-mer oligo lawn, is there a possibility that more than one n-mer will bind and prime the same template, be simultaneously extended, creating mixed signals?
Thanks Saurabh, it's possible but I don't think this has been a show stopping issue for other single molecule optical approaches (Helicos). Overall, you will need to control the template density anyway because templates which are too close will result in mixed signals. Mixed templates should however be detectable, they would I imagine be showing incorporations on average faster than other templates among other artifacts
Hi Nava, this is a very interesting minimalist idea.
Some thoughts: I assume you'll try to keep the nucleotide concentration very low so that simultaneous incorporation of multiple bases is minimized (the first being bleached well before the second is incorporated), as well as the background fluorescence from bulk nucleotides?
Assuming you're capturing the templates on a random n-mer oligo lawn, is there a possibility that more than one n-mer will bind and prime the same template, be simultaneously extended, creating mixed signals?
Thanks Saurabh, it's possible but I don't think this has been a show stopping issue for other single molecule optical approaches (Helicos). Overall, you will need to control the template density anyway because templates which are too close will result in mixed signals. Mixed templates should however be detectable, they would I imagine be showing incorporations on average faster than other templates among other artifacts
Another head-scratching question: why are people paying ~$20K for sCMOS cameras if cheap consumer ones will suffice? What's the catch?
sCMOS cameras have better noise characteristics, so the images look better, you can use shorter exposure times etc.
See e.g. https://www.nature.com/articles/s41598-017-14762-6 for a comparison.
Interesting, thanks.