Thread by @Ayjchan

After the first SARS-CoV-2 genome went public, at least 3 separate teams of scientists harnessed 3 different approaches to synthesizing its genome without having to put in any novel cloning or restriction sites. It took each team only a few weeks.

2 of the teams had to put in silent mutations to differentiate the synthetic virus from the natural virus that might've contaminated their lab.

Otherwise how could they know whether the strain they had synthesized was a lab-made virus or a virus from a covid sample?

“Full-genome sequencing showed that the recombinant virus retained the three engineered synonymous mutations with no other sequence changes, demonstrating the rescued virus did not result from contamination by the parental virus isolate​​.”
pubmed.ncbi.nlm.nih.gov/32289263/

"To distinguish our recombinant viruses from the circulating SARS-CoV-2 strains, we introduced a silent mutation (T15102A) into a conserved region in nsp12 to ablate an endogenous SacI site in the molecular clone"
pubmed.ncbi.nlm.nih.gov/32526206/

The other team used a yeast recombination cloning approach.

"Sequencing.. revealed that almost all DNA clones and viruses contained the correct sequence, except for some individual clones that contained mutations.. probably introduced by RT–PCR"
nature.com/articles/s4158…

Seamless cloning of virus genomes has come a long way since the first SARS epidemic 2 decades ago. Back then, scientists had to introduce cloning/restriction sites to make it feasible to clone virus genomes.

For example, to synthesize the 1st SARS virus genome, scientists had to remove + insert restriction sites via silent mutations.

"BglI site at nucleotide position 1577 was removed and new BglI sites were inserted by the introduction of silent mutations"
pnas.org/content/100/22…

When you read these papers on synthesizing virus genomes and introducing novel genetic features to them, there's no rule that your modification must be "in-frame". Basically, whatever helps you to clone the genome and QC it afterwards goes.

It's unclear to me, if one looked at synthetic genetic constructs in the past 5 years, how many insertions/deletions/modifications are in-frame vs out-of-frame.

It depends on the approach the scientist uses and if they rely on specific restriction sites for cloning or QC.

It's possible to ask scientists to get an estimate of how many changes are in- or out-of-frame using public data on synthetic genetic constructs, but more confidence would be gained by looking at each lab's purchases of specific cloning reagents.

An analogy would be like gaining access to someone's grocery receipts to get a better sense of what they might be cooking for dinner.

As opposed to browsing a database of recipes to guess what a particular individual might be most likely to cook.

I likely have also cloned constructs "out-of-frame" or introduced silent mutations several times before. It's not a dealbreaker whether your insertion is in- or out-of-frame. If the cloning produces a functional construct, you choose whichever approach is the most effective.

Some experts are telling people that you can tell from the sequence of SARS-CoV-2 that it's very unlikely to be a lab construct.

How could you estimate the likelihood? Have you got the labs' cloning reagent receipts? Do you know what sequences they had been synthesizing?

We now know, by early 2018, scientists in Wuhan and the US had a pipeline, expertise and everything they needed for synthesizing numerous recombinant SARS-like virus genomes and introducing human-specific novel cleavage sites into the spikes of live viruses for infection studies.

Based on the above knowledge, if you think it is plausible or even likely that SARS-CoV-2 was a lab engineered virus that accidentally reached its pandemic potential and escaped from a lab...

You are not a conspiracy theorist.