Jon's profile

"Detecting SARS-CoV-2 variants with SNP genotyping"

Tracking genetic variations from positive SARS-CoV-2 samples yields crucial information about the number of variants circulating in an outbreak and the possible lines of transmission but sequencing every positive SARS-CoV-2 sample would be prohibitively costly for population-scale test and trace operations. Genotyping is a rapid, high-throughput and low-cost alternative for screening positive SARS-CoV-2 samples in many settings.

We have designed a SNP identification pipeline to identify genetic variation using sequenced SARS-CoV-2 samples. Our pipeline identifies a minimal marker panel that can define distinct genotypes.

To evaluate the system we developed a genotyping panel to detect variants-identified from SARS-CoV-2 sequences surveyed between March and May 2020- and tested this on 50 stored qRT-PCR positive SARS-CoV-2 clinical samples that had been collected across the South West of the UK in April 2020.

The 50 samples split into 15 distinct genotypes and there was a 76% probability that any two randomly chosen samples from our set of 50 would have a distinct genotype.
In a high throughput laboratory, qRT-PCR positive samples pooled into 384-well plates could be screened with our marker panel at a cost of < £1.50 per sample.

Our results demonstrate the usefulness of a SNP genotyping panel to provide a rapid, cost-effective, and reliable way to monitor SARS-CoV-2 variants circulating in an outbreak. Our analysis pipeline is publicly available and will allow for marker panels to be updated periodically as viral genotypes arise or disappear from circulation.

Hi Melanie you need to think like a genotyper now. In SNP genotyping its normal to run multiple SNPs Or in this case Viruses in panels. So when you make you master RNA plate you just simply copy "stamp" out multiple times in a 384 you can have huge combination of sub sets of viruses or sequence targets. The slowest way to run whole process is in singleplex. Multiplex is the easy normal procedure..
http://results.lgcgenomics.com/software/krake...projecttree.htm
The standard SNP line process accommodates both single assays and multiple assays. A single 384 plate can be run as 384 different virus target assays on one patient. Or 384 patients on one RNA target. Or anything in-between. Each well in single Plexiglas though. The biggest criteria is having enough RNA to split. So would use Speedex based his quality RNA extraction and not crude extraction..
All of this is simple and has been done for 20 years. The system that looks after everything is called KRAKEN the Laboratory Information Management System LIMS.
Does that make sense..

Dear all I need an answer to this by Monday thanks..

Hi Sam I’m down your lab with Clare’s and going through the Qnostic SARS cov 2 QC panel. But it does not make any sense to me as this is a test of a whole existing system and does not just test "End Point". Very worrying actually... No design of experiment at all..!!!

The variables pre the "end-point" PCR reaction are huge and if LGC are also testing "end-point" this could result better/worse data only due to the extraction side.
For instance, we could get better looking data tomorrow than LGC, who may actually have better end point chemistry than us - just because the Qnostic samples have been extracted differently.

It is only a useful panel if you are comparing the whole process from start to finish against an alternative which has also been run from start to finish. It does not enable to you to test one individual part of the process, such as the PCR, as it is dependent on the extraction quality of the samples. Just proves little or nothing to be honest.

My strong suggestion based on having optimised commercial mass testing processes for years, is for East Kent to make test plates of known positives/negatives. Then split into multiples so that anyone's end point (or even qPCR) chemistry can be directly compared. The test plate can be a combination of good fresh RNA or poorer quality RNA. Then all data goes back to Claire/yourself blind and for you to score and decide on performance. Twist bioscience make synthetic RNA and you could easily make an exact control dilution plate for all sides to determine LOD CT's Cycling characteristics for all processes.

https://ecommerce.twistdna.com/users/sign_up?...0898.1590695174

Also, as LGC are now carrying out the same tests, would it be good for you to see this and input into their process via Crowdcity as you may want to partner up with them also. I know personally the ability to partner has added considerable value and been its strength. I have offered on many occasions LGC contact yourself and get your real covid samples run in their platforms but offer not taken up. They might be a much better fit than ourselves who knows?

Plus, the best whole process may be a combination of ours and theirs. I have already shared every aspect of ours on Crowd-City site for all to critique plus LGC have had our technical file for weeks. They also need an automation friendly swab/saliva tube and tube handling as well. I also know their extraction side in Berlin are a fab team. Remember every tube in a supermarket has been handled in the same way by the same automation company. No one needs to re-invent this... I would like yourself and Claire to directly compare data from the:

Thermo/Lighthouse End point 2.5ul 5ul 10ul
3CR Mix End point 2.5ul 5ul 10ul
LGC mix End point 2.5ul 5ul 10ul
East Kent clinical End point 2.5ul 5ul 10ul

Correct calling Grouping Distance from NTC NTC movement Cluster tightness Then you can compare same again on extraction chemistry’s and join the best components. Should be a win, win. This gives everyone the best outcome plus can’t be more than a few days to a week's work tops. Plus, if any chemistry is to be used, it is a bloody big thing and would expect more than just a check using this Qnostic plate to be honest.

Phil is very well placed to write a comprehensive design of experiment, as he did this at GSK genetics mass testing labs and KBioscience. He could write then share with all parties. Phil and I are not selling anything so no vested interest. Phil just wants to be able to go down pub and play golf again ASAP. Ta Jon

PS HIV and Ebola are both detected end point, Not exactly new to virology.

Updated full technical file: Nose to tail. Swab to data.


Tube automation by "RNA-Automation" https://www.rnaautomation.com appropriate name and best in the world at this. RNA Auto are also working on US CDC's tube handling. Their design team have validated our approach as best in class.

Updated full technical file. Tube handling process validated by RNA Automation
https://www.rnaautomation.com largest sample handling company in world. Ironic name though.

The ability to data handle 200k-1,000,000 tests per day in rtPCR is well documented to be a considerable task. End point is the reverse, almost zero data handling. Has been tested for 20 years at 1.5 million data points per day with one technician. On Billions of tests >400 genomes and over 1,000,000 various assays.
http://results.lgcgenomics.com/software/krake.../f=overview.htm
KRAKEN LIMS designed specifically for End Point Analysis and processed many Billions of End Point tests since yr2000 Developed by KBioscience now LGC

Mass testing data handling capable of 10,000,000 per day 1 person in house.

Mass testing data handling 10,000,000 ++ per day

Nose to tail. Mass covid testing

Nose to tail submission. Mass Covid testing

Yep FDA approved Q tips. Place under tongue for 30 seconds. Saliva good, see Oragene saliva kits etc. But would have to change whole lighthouse process.