Automated Fire Monkey HMW-DNA Extraction Validated on Tecan Robot Supports Rapid Growth in Multiplexed DNA Sequencing

• Proof of concept for automated Fire Monkey High Molecular Weight (HMW) DNA extraction/size selection process in multi-well filter plate format validated by sequencing
• Hands-free automated protocol on a bespoke Tecan Fluent 780 robotic liquid handler integrated with a Rotanda 460 robotic centrifuge
• Extraction produces library-ready homogenous, long-length DNA (average 106kb) for short- and long-read DNA sequencing
• Resultant sequence quality equivalent to manually-extracted DNA: >30Gb at N50 longer than 30kb
• Automated DNA extraction enables further growth in high-throughput DNA sequencing applications with estimated market to >$50bn over the next 10 years

London, UK – 4 March 2021 – The UK genomic tools developer, RevoluGen Ltd. (RevoluGen or the Company), today announces the technology validation of the world’s first automated extraction of library-ready High Molecular Weight (HMW) DNA in a multi-well filter plate format.

Under its collaboration agreement with Tecan, announced in 2020, RevoluGen has completed the configuration and validation testing (by DNA sequencing) of a bespoke, fully-automated workflow that integrates an adapted Tecan Fluent 780 robotic liquid handler with an integrated Rotanda 460 robotic centrifuge using laboratory standard multi-well plates, with patented Fire Monkey chemistry and matrix, enabling automation of the spin-based process.

The new automated Fire Monkey HMW-DNA extraction protocol has been validated to extract and purify bacterial samples. Further optimisation of the multi-well filter plates is ongoing in RevoluGen’s R&D laboratories with commercial launch of the automated Fire Monkey HMW-DNA extraction kit anticipate in the coming months.

“RevoluGen's automation of DNA extraction, the critical first step in the DNA sequencing process, comes at a good time as structural variant surveillance could drive a sequencing boom” says the company’s CEO, Pieter Haitsma Mulier. A recent report in leading trade publication BioCentury concluded that the push to keep COVID-19 variants at bay could be the point that turns genomic sequencing from a sophisticated technique into a widely accessible genomic tool, with leading companies focused on automation.

In the validation run to demonstrate quality of sequencing output, eight E. coli cell suspensions were transferred to a 96-well plate and, after a fully hand-free process, the output DNA delivered sequencing results equivalent to manually-extracted Fire Monkey spin-column based HMW-DNA. The average strand length achieved was 106kb with sequencing on the Oxford Nanopore (ONT) MinION LSK110 platform delivering more than 30Gb at N50 longer than 30kb, with good pore health (Figure 1).

RevoluGen’s Fire Monkey/Fire Flower nucleic acid isolation and purification (NAIP) technology is based on a spin-based protocol to extract HMW-DNA using a high g-force that does not break the long and fragile DNA molecules. It produces DNA that contains DNA fragments that are not too short and not too long, as this improves the overall sequencing process by not wasting sequencing resources by either reading the less useful small fragments or killing the reading technology parts by jamming the ONT pores with fragments that are too long.

“Extracting HMW-DNA based on a laboratory standard spin-column process was revolutionary in itself. Given the demand for automation, we have worked with Tecan to achieve a working robotic system that fully automates the process for DNA extraction of long length DNA using an adapted multi-well plate process. This is a truly disruptive innovation and will support massively high throughput, high quality, cost effective DNA sequencing” says Dr Georgios Patsos, inventor of the Fire Monkey technology and CSO at RevoluGen.

The rapid growth of sequencing worldwide will clearly need automated NAIP and sequencing processes to handle the volume of samples needed for applications such as population genomics, epidemiological mutation screening and antibiotic microbial resistance gene monitoring.

Dr Gemma Langridge, a Group Leader at the UK’s Quadram Institute, who has been an early adopter of RevoluGen’s Fire Monkey technology added: “With the automation that is coming from RevoluGen, what we are looking for is to be able to prep hundreds of bacterial strains at a time, then the scope for our research increases quite dramatically that way.”

Continuing, “We look forward to continuing our relationship with RevoluGen to explore the role of their technology in metagenomic analysis and to explore automation.”

Whilst the COVID-19 pandemic has highlighted the benefits of DNA sequencing based viral genomic epidemiology, global surveillance is also critical to track genes for antibiotic resistance to tackle the global problem of Antibiotic Microbial Resistance (AMR). For example, Quadram teams have published positive results using Fire Monkey with Illumina Whole-Genome Sequencing to identify drug-resistant (XDR) Salmonella typhi as well studies as to identify mutations related to ciprofloxacin heteroresistance i.e. population of bacteria with different sensitivity to an antibiotic using Fire Monkey to extract DNA from Salmonella enterica isolated from chicken meat.

Other high volume DNA sequencing applications include the population genomics studies nearly every country is beginning to adopt, and emerging approach to genomic screening of cancer patients for more accurate diagnosis and management of treatments. These applications all have in common the need for automated processes that can handle the large number of patient samples that will be required.

Currently every sequencing run requires each DNA sample to be extracted individually. A validated and automated Fire Monkey process for multiplexed automated HMW-DNA extraction will further lower the overall cost of sequencing and thus accelerate the uptake of sequencing in many high-volume dependent applications. The DNA sequencing market is currently predicted to grow from $4bn to some $50bn over the next 10 years.

“Automation represents a game-changing differentiator and will allow us to rapidly scale sequencing at a lower cost per unit. Now that we have validated the automated protocol, we believe that Fire Monkey is poised to become the DNA extraction technology of choice for high volume applications. Our technology unites short-read and long-read sequencing, enabling both from the same sample. It provides flexibility to revisit the same original sample for more detailed long-read sequencing after a first run with cheaper short-read sequencing” concludes RevoluGen CEO, Pieter Haitsma Mulier.