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Super high throughput testing using Next Generation Sequencing

B: One to watch

This is not a method for RNA isolation but rather the replacement of testing by qPCR by a far more high throughput method – millions of tests in one experiment. When RNA isolation problem stops being a bottle neck in testing, the method proposed below will remove the need for qPCR, which, in turn, will become the new bottle neck.

The scheme of the process is shown in attached Figure.

- The reverse transcription primer contains sequence standard for Illumina flow cell (blue), followed by a unique known sequence, barcode (white), for a sample from each patient. 12 nucleotide long barcode has 16777216 variants. The 3’ end part of the primer is the standard complement to the viral RNA (magenta).

- After reverse transcription, the cDNA now carries unique identification barcode for each patient.

- Short PCR (6-12 cycles) on the cDNA is performed with standard Illumina forward primer (blue), and reverse primer containing sequence standard for Illumina flow cell (blue), and the standard complement to the viral RNA at the 3’ part (magenta).

- PCR fragments carrying unique barcodes from different patients are mixed together and applied to NGS by Illumina. Millions of different barcodes can be sequenced in one go.

- Analysis of the number of reads associated with each unique barcode will reveal the presence or absence of viral RNA in corresponding patients.

Have you validated this method, if so, how and what were the results of the validation?​

No

How quickly could this be deployed and what are the dependencies?​

NGS library preparation is a routine nowdays, so it can be implemented very soon. Major dependancy is the preferred method for RT, which may require some adjustments for the NGS library preparation.

What is the likely production volume?​

The RNA preparation will be the bottle neck of the whole testing process. After RT of individual samples, theoretically all nation can be tested in one run of sequencing

What are the risks and barriers to using this at scale?​

can't see any

Who are you already partnering with on this?

Nobody, it is just an idea

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Theo Sanderson Apr 14, 2020

The general concept of NGS-based quantification is sound but there are a number of important additional features for a method that is robust across a wide range of viral titres. These can be seen in the Octant SwabSeq protocol: https://www.notion.so/Octant-SwabSeq-Testing-...038aa80a5a901fd

There are a number of differences from the procedure outlined here, the most important of which is that the PCR reactions are all run separately to saturation, with an internal spike-in of 10 molecules of synthetic RNA. If you don't do this then the dynamic range is limited to a few logs, because a sample with very high viral load can swamp a sample with a very low (but still >0) amount of virus.

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Jose A. Carrasco Lopez Apr 16, 2020

This method has few botlecknecks. The RT step, library prep and multiplexing must be carried out individually for each sample. Once those steps are completed, then the NGS run can process all the samples at the same time. Automation platform can handle 384 and 1536 well plates to create libraries for NGS, and a effcient pooling system will be requiered to mix all the samples to be mixed on the step before the run.

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Sonia Gandhi Apr 19, 2020

Status label added: B

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Bev Matthews Apr 25, 2020

The idea has been progressed to the next milestone.

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Bev Matthews Apr 25, 2020

Hello Nikolay, our reviewers would be interested to hear more about your solution. Please could you share additional data and information when it becomes available. Bev

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Bev Matthews Apr 25, 2020

The idea has been progressed to the next milestone.

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Tom Jordan Jun 27, 2020

Hi Nikolay,

It will be great to hear how you have been able to progress this idea - this will allow our reviewers to consider the proposal in more detail and consider how to progress it. Please do share details if you can.

Kind regards,
Tom
Testing Methods team

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