One Pot Sequencing
And from the prior post we can see the degree of color chaos going on in a cluster. If I understand the note on the sequence of the templates, this should read TCAGG which appears to map to red, blue, green, yellow, yellow -- but the majority color here is red red green yellow yellow. So it would appear the deprotection of the cycle 1 T (red) did not go very well (again, I'd prefer to see a log plot of the intensities!!) as we see intense red in cycle 2 and still a bunch in cycle 3 and a bit in 4 and 5 -- and the next T in the template isn't until position 10! I'd be tempted to design the molecules at this point to go a really long ways before the first base shows up again, so could measure very carefully the degree of lagging phasing. We see the yellow prephasing as a blip in 2 but significant in 3 -- again, it would be interesting to design some libraries where there are more positions before the first instance of a given base. If I were working on this, I'd probably be designing a whole library of templates to use in given runs -- complexity of four in each run but various choices of design to stress-test various aspects of the chemistry. Okay, I'm getting sucked into the challenge here...
What's The Market?
The low end sequencing market has neither gotten much love nor been very successful. Ion Torrent pretended to go for this market, but an all-in upfront cost of $100K isn’t hobbyist territory. Genapsys officially launched their $20K solution, but I never saw one in the field nor ever heard from a customer - and they’re gone. Illumina has iSeq, but that’s instrument has never seen upgrades and is all but ghosted by Illumina management
But the market does have MinION, a fully functional sequencing device you can get for $2K (not a typo!) that’s really works now. In theory the Flongle (another $ for the adaptor) gets run costs in the low double digits. In the home computer market, the arrival of fully featured machines was the death knell of the kit computers, only much later revived with concepts like Raspberry Pi
So how many people will buy in to the 454.bio concept early? During the MAP, ONT saw significant attrition in the user base because the platform was still very buggy and reagent availability was erratic. Others were hooked; for me it was seeing in our first run one very noisy but alignable 48 kilobase read with the entire lambda phage genome. Maybe some novices who build a 454.bio will have a similar epiphany with their first sequence data, but I'm skeptical it will be a wide-spread phenomenon -- though it would make be happy to be proved wrong on that point.
Unconventional operations, an innovative technology at proof-of-concept and appealing to tech hobbyists -- 454.bio should be fun to watch even if they don't succeed in carving out a major presence in the sequencing technology landscape.