Flavinkins (@Flavinkins)

https://gab.com/Flavinkins/posts/108946058120800199 How would you go onto constructing a synthetic consensus backbone that would be 1: guaranteed to be rescuable. 2: easy to manipulate as fragments. 3: easy to manipulate even after being ligated? You would need to choose fragments with type IIS restriction sites such that 1: each individual type IIS site must have a precedent in its presence of absence in the wild in at least 1 Sarbecovirus (distance doesn’t matter here. Can be as distant as BM48-31. Abundance matters statistically (rarer individual sites are less likely to show up at spillover) but does not matter if you are making a clone. As long as you find your desired presence/absence of a site at a specific location in even just one genome, you can use it and be confident that it won’t break your consensus genome. This is governed by both the availability of individual side precedence in sampled genomes and by the location of the site, whatever that doesn’t break the genome and is optimal for cloning gets chosen) to ensure that highly conserved RNA structural motifs aren’t disrupted. 2: there must be no BsaI sites between two BsmBI sites and no BsmBI sites between two BsaI sites to minimize the need of double digestion. 3: the number of overly small fragments should be minimized while no overly large fragments should be present at all. That is, the Standard Deviation (S.D.) Of the length of the fragments shouldn’t be too high. 4: the number of fragments should be kept to as low as possible with the restriction that you can easily manipulate each individual fragment within a M13/pUC vector backbone, as an excess number of fragments are more difficult to keep track of or manipulate. The type IIS restriction sites in the SARS-CoV-2 genome matches all the requirements above. Other genomes in its clade (or even any that is found in Asia)? Not that much. Unfortunately since clonability does not translate to spillover potential (without a lab), the fact that easily clonable Sarbecovirus genomes are rare in general also translates into the possibility that one that ended up spilling over just happen to be one that is easily clonable as SARS-CoV-2, being low. Since the fragments are what that is being chosen for the consensus genome, site selection influences ReCCA especially for the segments that were located near restriction sites—the segments surrounding them are what that were originally chosen in the consensus construct, where one of the requirements used is that the pattern of type IIS sites within the selected set of fragments should make the final genome assembled from them easy to clone. Finally, how could all the non-SARS-CoV-2 genomes you use in the ReCCA graph have a BsaI site in the first position of the F3 fragment (this site is highly conserved in Sarbecoviruses found in Asia) , but the final ReCCA, supposedly generated from related sequences sampled in the wild, not having that site? Either the ReCCA algorithm itself have considered these sites as part of the synonymous sites that was used to infer the “highly conserved segments”, and included SARS-CoV-2 itself into the analysis (making the algorithm tautological), or it is the result of the consensus-generation process where the choice of segments and sites to be used for the consensus included an requirement for the ease of cloning and manipulation for the final genome—something that would matter if you are synthesizing and rescuing it in the lab, not so much for anything that “spills over from the wild”. https://archive.ph/VypuD