Kevin McKernan 🙂 Profile picture
Cannabis Genome Project,2011. Developed the SOLiD sequencer. R&D lead Human Genome Project at MIT/WIBR
Karen Horton Profile picture Κασσάνδρα Παρί پری Profile picture KM aka 3TW Profile picture Lolos Mom Profile picture Hal Sear Profile picture 29 added to My Authors
14 Jan
1\But but... you ran it to 40-45 cycles?
This is an argument that needs dissection.

The protocols you see in EUAs are speaking to the Camera time not the Call time. To call at 37Ct... you need to see the slope of the curve out to 40. It doesn't mean they are calling at 40.
2/Summertime positivity rates cannot be used as a proxy for wintertime false positive rates.

The virome in the winter is rich in HCoVs not present in the summer. I would assume the background microbiome is as well.
But we tested the Primers against HCoV in our exclusion analysis!

Sure.... but Just 4.

We just witness the S-gene amplicon drop out in the second season of C19 and we should expect HCoVs to have similar diversity.
Read 15 tweets
13 Jan
Kannapedia.net is getting a good face-lift.
Samples now have variant tables with annotation on their impact to the gene.
There is also an IGV integration. You need IGV installed locally but the Start|Jump links will open up you BAM file in IGV.
You must have IGV open and then click the start link.
The sample's BAM will show up for viewing in IGV.
Once open, you can use the jump link to move swiftly around to each variant. This is a variant in THCAS.
You can jump to other variants in the same gene. In this case we have one non-synonymous variant in phase with a synonymous variant in THCAS.
Read 9 tweets
11 Jan
We have written another 60 page dissection of the Drosten PCR protocol.

This should end all of the criticism of the initial retraction request not having enough “wet-lab” proof.

20 peer reviewed papers showing catastrophic problems.

@Eurosurveillanc

cormandrostenreview.com/downloads Image
Ironically, The origin of this quote is even debated but rightly describes the problem in society of Fake News.

It starts with Fake Science.

We will never end fake science if don’t address the lack of transparency in peer review. It must be decentralized and subject to audit. Image
The largest fatality in C19 was the public trust in science.

They have every right to be pissed.

For too long peer-review has remained ancient, ossified and secret.

It is not fit for our current world of rapid communication and transparency.

🦕 thrashing in tar pits Image
Read 10 tweets
11 Jan
I have finally read this in full.
Have not hit 255 references.

Anyone who tries to debunk this in a day... sorry. This would take many weeks to fact check

The PCR section is on point.
However, there is not enough attention paid to the significant false negatives also at play
While a lot has been written about false positives, the nasal swabs can vary 1,000-10,000 fold in their collection efficiency (Dahdouh et al) and many jurisdictions are using human gDNA internal controls to normalize for this large variance.
This variance implies many false negatives mixed in with many false positives.

More sensitive PCR tests won’t fix false negatives that are a result of poor swabs.

They aggravate the problem as track and trace becomes a completely futile boondoggle.
Read 4 tweets
11 Jan
We spend a lot of time calibrating our qPCR assays to replication competent organisms.

When we do this, we don't simply use a single culturing system as the results can vary significantly depending on the media used.

Here is DRBC(left) vs PDA (right) with Aspergillus niger. Image
You can see many more colonies on PDA.
This is likely due to DRBC having Chloramphenicol in it.

Chloramphenicol is an antibiotic with know inhibitory effects on Yeast and Molds.
ncbi.nlm.nih.gov/pmc/articles/P…
ncbi.nlm.nih.gov/pmc/articles/P…
pubmed.ncbi.nlm.nih.gov/25497707/
This begs the ? of which plating system to calibrate qPCR to. Also begs the ? of what a "gold standard" is.

95% of microbes cant be cultured, leaving DNA based methods as the only universal gold standard for absolute detection of wild samples.
See...
osf.io/vpxe5 Image
Read 4 tweets
9 Jan
🙏🏼 for the follows.
My goal is to simply share what I know about genomics with the public when it matters most.

I rarely get technical refutations but embrace the errors so as to correct.

The best they seem to be able to fire back is ad hominens and associative fallacies
From 1996 to 2000 I worked tirelessly to get as much as human genome public as possible.

Our team then built a DNA sequencer that dropped the price of DNA sequencer several orders of magnitude.

Published open access.

genome.cshlp.org/content/19/9/1…
In 2000 we started a genomic company called Agencourt.
It became the largest commercial seq shop at the time.
Turned profitable in 18 months from start-up.

We also built DNA and RNA purification kits for viruses using SPRI

Some of these kits are in use today for C19 prep.
Read 19 tweets
8 Jan
If you are in the UK, this is the serial dilution of the test being run on you for C19.

For each 10 fold drop in Copies/ml, you should see a 3.3 increment in Ct.

What do you see?
Why does a 5000 copies/ml sample have a lower Ct than a 10,000 cp/ml sample? Image
The paper does speak to a spike in MS-2 control but this isn't a true internal control like RNaseP.
Spike-in internal controls are important for measuring assay drop out. They prove the PCR was functional.

But RNaseP controls which amplify human DNA/RNA harvested on the Swab..
These ICs are more important as you really cant quantitate viral load and make claims about S gene knock outs being higher copy number if you don't normalize the Swab collection and sample prep issues. These can vary 10-16 Cts according to Dahdouh et al.

medrxiv.org/content/10.110…
Read 6 tweets
3 Jan
Here is an example of the benefits of public Ct data and tax funded data should but public for the tax payer see.
Particularly if said data is going to be weaponized against the tax pay payer.

This is ONS data showing %positivity using 2 or more gene targets.
Let’s focus in on a few jurisdictions.

Notice the Southwest has zero tests light up with 2+ genes in September?
All tests are positive for ORF1ab?

Other jurisdictions don’t share this pattern. I don’t think a virus can do that. That looks like a lab artifact.
We see a similar anomaly in October for East England.
Only 35% of the test are positive for 2+ genes. That’s an outlier at that time compared to all other labs.

I suspect that’s a lab artifact.
Read 8 tweets
3 Jan
With over 88,000 papers published and a 160K SRA submissions in 2020 on SARs-CoV-2 something has happened in science that has probably never happened before....
Never have we had some much data generated on a single disease in such short time.

No person is capable of integrating all of this data.

Social media has cartelized and forked the scientific field into different schools of thought such that each looks like this to other.
This divide wont be addressed with calls for censorship of the other side.

However, you do not owe anyone your attention and I discount anyone who argues for censorship.

The excuse is always in the name of protecting some other fool.
Read 4 tweets
2 Jan
I've been known to make a stink about public Ct data.
This is helpful. Tabs 6a, and 6b.

A few concerns.
1)Weekly not daily releases
2)Starts in Sept so its difficult to compare to 1st wave
3)No Ct by amplicon. Some info on amplicon fails but Ct are means
ons.gov.uk/peoplepopulati…
What do we see in Tab 6b?
Take the Mean and chart it across time by jurisdiction.
Keep in mind the Ct is LOG 2.
So Yorkshire went from a mean Ct of 21.5 in Sept to 30.8 in Nov 30th. This is 512 fold drop in viral load from Sept-> Dec. Did you hear that in the news?
How could this happen?
First thing I look for is internal control data? Do they have a human gene in their assay like RNaseP?

This can be used to normalize sampling bias in the nasal swab. This has been reported contribute 10-13Cq variance in Dahdouh et al.
Read 6 tweets
1 Jan
When respected Scientists resign over the false narrative... Take notice... and thank them.

Google Translated Letter from

Prof. Dr. Thomas Aigner,
Department of Geosciences,
University of Tuebingen, Sigwartstrasse 10,
D-72076 Tuebingen Germany
Dear colleagues,
It was with the greatest astonishment, deepest concern, and indeed bewilderment, that I took note of the "7th ad hoc statement" from the National Academy of Sciences Leopoldina on December 8th, 2020.
In my opinion, this paper is not worthy of an honest, critical-
weighing science oriented towards the service and well-being of man. I do not have any medical expertise. However, as a scientist committed to nothing but the pure truth, I take the liberty of speaking up. I feel very alarmed by several points:
Read 25 tweets
31 Dec 20
OK Koch heads... bring your hurt.

I’m glad you folks exist as we need people questioning everything.

However, I have found the “virus doesn’t exist” arguments unconvincing.

The virus isn’t the only cause of disease may have some ground.

I remain open to new papers on this. Image
Set some rules.
This was established in 1884. Watson & Crick discovered DNA structure in 1953.
He did not contemplate unculturable organisms... the vast majority of organisms are not culture-able.

Viruses by definition cannot be cultured. They require a host cell to culture. Image
This is where the debate gets dirty.

In order to culture the virus you need to find cells/organisms it can infect but not be a pathogen to those cells.

But Koch wants to demonstrate the pathogen creates symptoms of the disease which be counterproductive to culture. Image
Read 19 tweets
30 Dec 20
This is what 140% Bullshit looks like.
Empty parking lots.
See if you can find the hospitals website and if you can find any mention of COVID on it. Certainly if they were overrun, they should have something on their cite to help fix this?
The ratings on the hospital might explain the empty parking lot.
Read 8 tweets
27 Dec 20
Metagenomes from RNA sequencing datasets from bronchoalveolar lavage fluids (BALF) obtained from patients diagnosed with COVID-19.

Look Mom... No Vero cells.

advances.sciencemag.org/content/advanc…
Many folks voice concerns over the viral isolation being incomplete. It's not my field so I tend to ask these folks what methods would they use to perfectly isolate a virus that opens up human tissue without opening up the capsid? We have new tools now. Why not use them.
This paper comes to mind. Samples directly from sick patients (not vero cell cultured with antibiotics). Whole Transcriptome sequencing (not PCR targeted so hypothesis free) reveals C19 coverage maps that correlate with Ct scores.
ncbi.nlm.nih.gov/pmc/articles/P…
Read 6 tweets
23 Dec 20
Cool paper on which assays target ORF1a and which target Spike Protein. The Pfizer mRNA is of spike.

pubmed.ncbi.nlm.nih.gov/32872332/
This paper has them compared to Corman primers and they have lower Ct's which isn't surprising. I dont see the primer sequences anywhere to confirm they hit Pfizer mRNA of Spike. Anyone know if the Altona primers are public?
sciencedirect.com/science/articl…
Misfire on the CTRL C.
mdpi.com/2075-4418/10/6…
Read 9 tweets
20 Dec 20
I've been asked this many times. So tweet thread it is.

Do the Drosten primers hit the human genome?
Why do some people claim they hit 100%?

TL/DR: Here is the output from BLAST

mega.nz/file/tBpQjLrY#…

Here are the alignments from BLAST
mega.nz/file/0A5QgTBI#… Image
So the first thing people get excited about is that portions of the primers will hit with 100%with 17/17 residues hitting the human genome.

But you will notice the 3' end of the primer doesn't match and that is the business end of the primer. Image
That 17 bases represents the middle of the primer highlighted below and doesn't include the 3 prime end. Not ideal but not lethal. Image
Read 16 tweets
20 Dec 20
Opiate replacement therapy is an important 2020 topic.
Cannabinoids have proven to be very effective here and like HCQ and Ivermectin, they are restricted by pharmaceutical regulatory capture.

We cover this quite frequently @CannMedEvents

nypost.com/2020/12/18/us-…
To help liberate these renegade drugs we have been sequencing the genomes of many of the plants and fungi that can enable people to grow their own medicine. There is certain empowerment and agency over addiction and disease that occurs when the patient is vested in the solution
While cannabinoids are exceptional for opiate replacement therapy, over 6M people in the country resort to Kratom. This is from Mitragyna speciosa.

Related to the Coffee plant, Mitragyna synthesizes mitragynine and 7-hydroxymitragynine.
Read 7 tweets
15 Dec 20
With all the new followers since October it’s worth revisiting the Mask story since this popular thread was posted.

Now consider the Danish study and be sure to read about the Fogen affect.

docs.google.com/viewerng/viewe…
It’s an intuitive hypothesis
Since the mask doesn’t kill virus, it just collects them reversing your natural defenses of expelling virus with large droplets that hit earth.

The mask accelerates evaporation through capillary action making smaller droplets 2 allow deep inhalation.
In effect, the masks are a viral trampoline making the virus exponentially more infective and reaching deeper into more thrombotic tissue.

Some evidence Kanas jumped on this trampoline in the summer.
Read 4 tweets
13 Dec 20
This is the Final nail in the coffin of the current qPCR testing strategy.

We have to get smarter about what we are targeting and stop harassing asymptomatic people.

Now, let’s imagine if these genome integration events can seasonally express.

Permanent PCR Purgatory
Looks like the higher the copy number Subgenomic RNA, the higher frequency of human genomic integration.

Testing with the N gene is a disaster.
Testing with anything that subgenomic is a disaster.
This is nuts!
The integration is most common for the N gene.
That’s the most unfortunate news as most qPCR kits target the N gene.

They demonstrate the integration events can be expressed.
Stress can induce LINE-1 expression.

@PatrickSSte
Read 17 tweets
13 Dec 20
Sethuraman et al.

Its like every paper you read on C19 qPCR has a Charite' primer disclaimer...

Add it to the long list seen here:


Hello @Eurosurveillanc

jamanetwork.com/journals/jama/…
Let's toss in some Vogel et al.
Note the Charite' primers are in bold due to the error that would have been picked up if a reviewer simply BLASTed these primers (5 mins).
Wait... 99.8% of RdRp_R primer has the wrong base in it for C19 amplification?
Let's add to the chorus.
Kuchinski et al. has a whole section of the paper dedicated to the errors in the Charite' primers.

reader.elsevier.com/reader/sd/pii/…
Read 8 tweets
12 Dec 20
Let's walk through the much more complicated problem of identifying pathogens on Cannabis flower. These are inhaled products in an industry that just experienced EVALI.

This will provide a few examples of which steps in the process are prone to producing illusions with qPCR. Image
The first problem is that Cannabis microbes can be both Endophytes and Epiphytes. These live inside the plant or on the surface of the plant. This changes how you survey them. If you don't lyse open plant cell walls, you cant see the Endophytes. You need proof of lysis. Image
We accomplish this by targeting a conserved cannabis sequence in qPCR. Every PCR assay must have a qPCR target that proves you lysed open the plant cells.

Many SARs-CoV-2 assays accomplish this by targeting a human gene known as RNaseP. Image
Read 41 tweets