Yan's appendix at the end of the first CoV2 sequence. 616 residues found to be identical to RP11-173E24. a Rho-independent terminator have been observed at the very last of this sequence, just after a stretch of As. could this be the remnant of a vector? that is then quickly lost
at subsequent isolates as CoV2 returns to the normal length (an isolate at BeiJing 2020 was found to carry an appendix of this exact fragment of hgDNA. How?) The isolates have only been occasionally found with this sequence at 3' end, and it was not in WuHu-1. RNA "Telomere"?
Since this is not where RdRp normally initiate at, This sequence is quickly worn away--a rapid shortening of this 3'-end at the earliest isolates can be clearly seen. Could this fragment of hgDNA genuinely exist in the very first isolates? Source?
THA-03/18. DEU-04/08. USA-05/27. newer isolates don't seems to contain this any more. Beijing isolate at 01/03.
Open for interpretation. signal not clear.

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More from @flavinkins

20 Sep
@GentleMutt @BidoliNicola @MonaRahalkar @BillyBostickson @angoffinet @TheSeeker268 zenodo.org/record/4008432…
The real issue is that the raw data itself does not support the existence of that assembly. The rest of the SRA is a complete mess. Everything that have a match to a bat matches the genomic DNA instead of the mRNA. There were no bacterial reads which is
@GentleMutt @BidoliNicola @MonaRahalkar @BillyBostickson @angoffinet @TheSeeker268 Impossible for a “fecal swab”—every fecal sample on NCBI, if you search “bat feces” on SRA, with the Bacterial reads reduced below 5% in total cellular organisms, lacked any (confirmed) reads that can be traced to an RNA virus. There are other reads, including nearly 60% of a
@GentleMutt @BidoliNicola @MonaRahalkar @BillyBostickson @angoffinet @TheSeeker268 Non-homopolymer repeat sequence, which have no known biological mean of origin. It preprints.org/manuscript/202… have only ever been observed in significant quantities in 2 other datasets—the first one is a completely destroyed dataset (from tissue samples) with one amplicon of a
Read 19 tweets
16 Sep
Wreck the KG anderson “data”.

zenodo.org/record/4007845…

preprints.org/manuscript/202…

zenodo.org/record/4008432…

drive.google.com/file/d/1LrYiFr…
RnYN02 is almost 1/10 human(the 3’-ETS not found in bats or non-humans).
No evidence of an insertion event ever happening in RmYN02. FauI utility is RFLP and HR1 cloning.
Esp3I site and BamHI sites in RaTG13 is entirely different from CoV2, and is far harder to clone with no see’ms technology. Could still be Esp3I, since you can cut it out.
Read 4 tweets
14 Sep
@ale_battini @seven7ins @LynnFynn3 @TravelLightP1 @chartreader2 @CovidMillion Just to double check on the consensus that The Human Coronavirus HCoV-NL63 have no furin site. There is no evidence of a Propeptide convertase acting on hCoV-NL63 S, and western blot of this S did not reveal any cleaved S. Confirming that there is no FCS in NL63.
@ale_battini @seven7ins @LynnFynn3 @TravelLightP1 @chartreader2 @CovidMillion jvi.asm.org/content/92/3/e…
Entry of NL63 require TMPRSS2, which the only exposed cleavage site occurs on the S2’ location. Confirming that this S is not cut by furin(inhibiting TMPRSS2, or stopping endosomal acidification, completely stopped virus entry). NL63 can not enter cells
Read 18 tweets
14 Sep
Interestingly—there are no Esp3I sites in CoV2 after ORF1ab—considering that the Esp3I recognition sequence is cut out in no see’ms technology, it’s absence indicate that this sequence may have been optimized to be cloned using this enzyme.
In addition, a BamHi site is found in the end of the S, a BspE1 site is found at the end of the ORF3a site. an Age1 is found at the end of the E gene, in the middle of the M. The Pro in the PRRA is cut by BsaXi, used in RFLP and detects the integrity of the first R in the RRAR.
the BamHi, BspE1 and Age1 sites are unique in the entire genome.
Read 26 tweets
10 Sep
RaTG13: Loss of Integrin binding ruined the ACE2 affinity even more.
Multiple sequence alignment using all available Spike sequences from Sarbecovirus on NCBI, have identified 3 unique sites on the RaTG13 RBM/RBD: T403, Y493 and D501.

sciencedirect.com/science/articl…
Site T403 was found out to disrupt a critical motif conserved across all Sarbecoviruses that have an “full-length” RBM that will bind ACE2 353-354. Removal of R403/K403 results in the reduction of Expression by 9X, and reduction of ACE2 binding by 5X when soluble ACE2-Fc was used
According to the western blot of the Spike proteins expressed by the “PRRA insert” paper, biorxiv.org/content/10.110… they have likely mixed up their plasmids—and the “RaTG13 S” and “RaTG13 S+PRRA” have switched places. This is found to be the case in their pACE2, which have no
Read 26 tweets
6 Sep
Thread🧵
Debunking the recent pangolin PCR paper claiming a CoV detection by PCR from (the same batch of GD) samples.

threadreaderapp.com/thread/1302264…

The Toilet paper🧻 below:
conbio.onlinelibrary.wiley.com/doi/full/10.11…
@BillyBostickson
ncbi.nlm.nih.gov/pmc/articles/P…
PCR negative lung tissue for bacterial 16S is simply impossible. Healthy lungs contain 10^3 to 10^5 CFU/g bacteria.
Read 10 tweets

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