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1. Thread: RaTG13 Real or Fake?
A Norwegian Scientist @Vehuardo recently claimed RaTG13 is an "authentic virus"
https://t.co/tAXMNTwdhA
This failed to take into account the work of our DRASTIC colleagues & other published analyses.
This thread is a polite attempt to educate.

 2. https://t.co/tw606vgHwp
which says RaTG13 doesn't make sense on how it was sequenced, or how the data got so messed up in the lab as to include copy paste mistakes, bat dna, pangolin dna, rice dna, mice dna, & nobody should use it to form conclusions until it's properly vetted
2 (cont)
"sequence is incomplete & provided segment doesn't function. The identified markers suggest partial match basis & actual virus itself as described by the sequence can't functionally exist due to incoherent structures that appear to be the result of sample contamination"
3. RaTG13 integrity is further attacked here:
The Validity of critical pieces of evidence for the natural origin of SARS-CoV-2 is Dubious, and needed to be reconsidered
https://t.co/yTNYcsbees
4. and here:
Major Concerns on the Identification of Bat Coronavirus Strain RaTG13 and Quality of Related Nature Paper
https://t.co/o3jWQWwJFQ
5. With reference to the levels of bacteria found and certain other anomalies, we have:
https://t.co/qVPC1HTxcA
by @MonaRahalkar and @BahulikarRahul
6. and another analysis focusing on contamination:
https://t.co/DUvBGawcso
Anomalies in BatCoV/RaTG13 sequencing and provenance
from @flavinkins
7. Then we have the anomalies regarding the amplicon sequences, which also remain to be answered:
https://t.co/dYpqvcebuo
and
https://t.co/QUxsxcmnAo
initially proposed by @interne41914499 #DRASTIC
8. Some more recent work which investigates the authenticity of RaTG13 can be found here:
https://t.co/GGIX4UH06b
by @MonaRahalkar and @BahulikarRahul
9. We should not forget the original attack on RatG13 from
@nerdhaspower which can be found here:
https://t.co/U1F1f8p9A6
much hated by the virology community ;)
10. I believe @Vehuardo addressed some of the concerns in this paper in the recent https://t.co/SpcUDqQuBX note, but in my opinion the challenges were insufficient to overthrow the main claims in the paper.
https://t.co/XmPEVP20f5
11. Finally, another anomaly discovered by @notoriousFIL
Discovery of 2 sites in the raw data that don't match the published RaTG13 genome, also noted by
@shingheizhan who found this mismatch & 2 bases which are identical to their counterparts in SARS-CoV-2,
"kind of bizarre"
12. "The SRA data doesn't appear to agree with the reference genome, there are gaps, different consensus and different bases" They could not explain these discrepancies:
https://t.co/deLDt5kUsu
How about you, can you explain them?
13. Is RaTG13 a viable virus?
Apart from some ambiguous results from computer models & pseudoviruses, the consensus is that RaTg13 is not a viable virus, supported anecdotally by Beijing Scientists who failed to recreate and infect animals with it.
https://t.co/AwUdiocb6f
14. Not surprising, as @flavinkins pointed out, since last 4 amplicons of RatG13 clearly display signatures of DNA synthesis assembly errors, meaning they were synthetic, in-vitro materials designed to “fill gaps” for a contiguous assembly to join sequences never found together
15. Traces of Human ERV and HIV-1 derived material was seen in RaTg13, pointing to it being a classical cell culture product. The HIV was was apparently from the lentiviral plasmids and the nucleotide in question was a part of the Gag gene.
16. Back to Viability:
We know that RaTG13 does not even result in infection in bats due to weaker affinity between its S-domain & bat ACE2 receptor compared to SARS-CoV-2 & human ACE2. So, if this is the case, how could it have even emerged from bats?
https://t.co/QbFpgWXYWk
17. Despite similarities between RatG13 & SARS-CoV-2, there is little evidence that RatG13 could infect a bat, let alone a human
https://t.co/R1WDjxzvvf
The question arises, if Scientists are so sure RaTg13 is viable & natural, why haven't more experiments been done to test it?
18. A withdrawn paper used a computer model to analyse potential RaTG13 binding to different animal ACE2, suggested binding would be highest to common lab experimental animals, Ferret & Tree Shrew
https://t.co/z9LNvUzoR4
However, no further in vivo work has been attempted since.
19. In an experiment in July, pseudoviruses bearing spike protein derived from bat raTG13 allegedly could use ACE2 of a diverse range of animal species to "gain entry"
Just "gain entry"?
We have no results about infectivity, so why no in vivo experiments?
https://t.co/3FcszCgk4B
20. An earlier paper in April claimed that
"a wide range of ACE2 orthologs support binding to RBD proteins of Bat-CoV RaTG13"
https://t.co/neyH77DnAr
It was later revealed that they used the wrong species of bat for ACE2 sequence (Rs instead of Ra) thus invalidating their claim
21. our DRASTIC team member @Rossana38510044 also finds it "very unusual that the RBM of RaTG13 has no affinity for any other sequence in NCBI. It is indeed a great mystery how it has evolved."

I would suggest that it may have been the result of "directed evolution"
22. To summarise so far, a few flawed in-vitro experiments using RaTG13 pseudoviruses or ACE2 orthologs have revealed that the only class of ACE2 that can bind RaTG13 is ungulate ACE2
@flavinkins points out that despite high in-vitro activity, it does not have in-vivo infectivity
23. This is likely due to ungulate factors that block entry of Sarbecoviruses:
1 https://t.co/vELGSA8qO1
2 https://t.co/boEKPr2gKt
3 https://t.co/S8glmOJafa
This raises questions:
Where are the in-vivo experiments with RaTG13?
Can it even be recreated?
Why has nobody even tried?
24. Finally, there are the phylogenetic tree inconsistencies when RatG13 is used, clearly elucidated by #DRASTIC team member @AntGDuarte here:
https://t.co/OjJLMpZNZa
25. An example here that claimed to everyone's great surprise, that when the phylogenetic tree is rooted in RatG13, Guandong was the proximal origin in September 2019!
https://t.co/jWniyY9WRI
26. More Evidence of errors distorting Phylogenetic Trees
https://t.co/Vazefx6lz6
The PDF has more complete data and is essential reading:
https://t.co/qghoWdlMDV
27. There is ample evidence that RatG13 is a "construct" rather than a viable virus & little evidence to the contrary
@flavinkins suggests that the amplicons could have been generated by simulators such as MetaSim & short fragments synthesised & sequenced
https://t.co/zKT2Mmm4HW
28. @CZilcho concludes:
TG doesn't add credit to the RaTG13 story
Sanger amplicon only released in May to fill a gap in seqs
Complete SRR full of eukaryotic contamination that didn't even match its alleged host R. affinis
BALF or feces?
Smells like a mineshaft full of guano!
29. RaTG13 has been investigated and almost invariably the researchers are either befuddled, confused, shocked or surprised at the anomalies they found.
For example:
https://t.co/iofBxkQUXq
See Table S1. The whole genome nucleotide similarity https://t.co/DBINNuE35h
30. A further example can be found here:
SARS-CoV-2 & bat RaTG13 spike glycoprotein structures inform on virus evolution and furin-cleavage effects
https://t.co/ilMmjCLjzu
They were indeed forced to take extreme measures to obtain viable RatG13 for their experiments!
31. In fact @Rossana38510044 discovered RaTG13 has the dubious honour of being proposed as the basis for a vaccine against SARS-COV-2!
Due to its inability to infect anything more than Nature Papers crafted by Zhengli Shi and her desperate lab members!
https://t.co/30aqZqDb7i
32. "The mutation between SARS-CoV-2 & RaTG13 is unique across coronavirus species"
Comparative genomic analysis revealed specific mutation pattern between SARS-CoV-2 & Bat-SARSr-CoV RaTG13
https://t.co/F2fHoGWMfN
Note the bizarre mention of "5-FU" in the conclusion.
Anyone?
33. Above reference to 5-FU is "5-fluorouracil":
a drug used for lethal mutagenesis, a broad-spectrum antiviral strategy that increases viral mutation rates & burdens viral populations with mutations reducing infectious progeny
https://t.co/hWedAMiMH2
and
https://t.co/I8iqsFIAsE
34. Assymetric Mutations leave Researchers Puzzled 😲
Excessive G–U transversions in novel allele variants in SARS-CoV-2 genomes (RaTG13)
https://t.co/hQgsE7aDVs
35. The Puzzling Mutation Bias
(underlying divergence between RaTG13 & SARS-CoV-2)
Mutation Patterns of Human SARS-CoV-2 and Bat RaTG13 Coronavirus Genomes Are Strongly Biased Towards C>U Transitions
https://t.co/AelHag0Z12
36. Inconsistencies in RaTG13 Data noted by many!
An Update on the Origin of SARS-CoV-2: Though
Closest Identity, Bat (RaTG13) and Pangolin Derived
Coronaviruses Varied in the Critical Binding Site and
O-Linked Glycan Residues
https://t.co/SEZIrPJjM4
37. Confusion leads to a bizarre hypothesis
Synonymous mutations & molecular evolution of SARS-Cov-2 origins
https://t.co/JrNOJzeob7
"Another possibility is that RaTG13 has been maintained under conditions which have allowed it to continue to evolve after its initial sampling."
38. Reactions?
Recently, Dr. Yan published 2 "reports" on SARS-COV-2, alleging it is a CCP bioweapon.
Whether or not you agree, her discussion of RaTG13 was accurate and clear, but was attacked in a "review":
https://t.co/XN9Q1BmUfo
See the image for their pathetic "arguments"
39. Conclusion?
RatG13 is not what it seems
unroll @threadreaderapp
40. Update regarding specific claims made by @K_G_Andersen and @Vehuardo here:
https://t.co/tAXMNTwdhA
answered by @flavinkins
1. The so-called “amplicons” were published long after the problems with the ILLUMINA were exposed, so no assembly can be made for RaTG13 using the SRA
41. These amplicons have strange things hanging off the ends and are artefacts from gene synthesis assembly errors
2.
They have a publishing date almost a month after the problems with the raw data were found in April.
42.
3. They were made up in May, synthesised on 2 plates, 1 for forward reads, 1 for reverse reads. They then had a problem with S1 & had to quickly make the last 4 amplicons
The result was these amplicons had random, non-vector & non-Biological sequences hanging off the ends
43.
4. Also, the real problem is that the “fecal swab” is anything but a “fecal swab”. You can look with your own eyes and perform a search on “bat feces” on NCBI. Whenever bacteria drops below 2.5% in total cellular organisms, all RNA viral reads are gone.
44. via @flavinkins
5. Bacterial 16S rRNA is a lot tougher than Eukaryotic and viral RNA. When 16S is destroyed, viral RNA will already be obliterated and no assemblable sequences will be possible.
45.
6. In fact, it took them over 3 months to publish the amplicons—“gap filling PCR data”—the timeline does not match up at all for a real sequencing project—those “amplicons” appeared just the same time as you would expect for a gene synthesis company from order to shipment.
46. via @flavinkins
7. The RaTg13 Pipeline is completely inverted by date and time:
Assembly comes first and ILLUMINA comes 2 weeks after assembly, which will be easy as a point-to-point fabrication is easy.
Most rRNA was degraded beyond repair, no mRNA properly matches anything
47.
8. “mRNA” matches match the clone more than
Predicted RNA, & the “virus” had far higher quality than anything else. They could easily get fake data, for example, CoV2 data. Manually alter the base calls by alignment & change the basecall without changing the quality score.
48.
9. They could have merged this into a fake “sample”.
For example, the WGS of the bat, filtered coarsely to generate an exome.
Then after 3 months they published the “amplicons” which is suspicious as 3 months is the time for a gene synthesis company to deliver their order.
49. via @flavinkins
10. Here you can see the Bat and Viral Metagenome:
every dataset is mostly bacteria.
(lowest bacteria in all cellular organisms = 30%)
https://t.co/nADnfIUPzZ
50. Unroll @threadreaderapp
51. Update
https://t.co/uORg2OzuPf

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