Hadfield, UK – 27 April 2023 – UK genomics company, RevoluGen Ltd. congratulates its collaborators from the Quadram Institute, Norwich, UK on new data demonstrating the use of its manual and semi-automated Fire Monkey high molecular weight HMW-DNA Extraction Kits in microbial genomics studies.

The data was presented in three posters and an oral presentation by Quadram Institute scientists during the recent Microbiology Society’s Annual Conference held 17-20 April 2023 in Birmingham, UK.

Dr Georgios Patsos, inventor of the Fire Monkey technology and CSO at RevoluGen said, “Our work with the Quadram Institute has demonstrated the value and advantages of both our manual and revolutionary semi-automated HMW-DNA extraction in microbial DNA sequencing with important applications in public health and antimicrobial resistance surveillance.”

“Fire Monkey HMW-DNA extraction can lower the overall cost of multiplexed sequencing and accelerate the uptake of sequencing in many high-volume dependent applications” he added.

Steven Rudder, Bilal Djeghout, Ngozi Elumogo, Nicol Janecko and Gemma Langridge presented a poster1 on the development of a stool metagenomics long read pipeline for Salmonella1.  Culture-based methods are laborious with inherent biases towards specific species or subtypes and often generate results after an outbreak has become established.  Although routine sequencing is conducted for salmonellosis cases, for other enteric pathogens rapid culture-independent testing (CIT) is the predominant choice for diagnostic laboratories, yielding no isolates for sequencing.  As laboratories continue to adopt CIT-only testing strategies, a gap in pathogen genomic epidemiology is created. One potential solution is sequence-based metagenomic approaches to identify bacterial pathogens during an outbreak.  To aid sequence-based long read metagenomic approaches improved high molecular weight (HMW) DNA extraction processes are needed.  This method is now in development and was used in Oxford Nanopore (ONT) long read metagenomic sequencing leading to the identification of the subspecies, serovar, and antimicrobial resistance (AMR) profile of the targeted pathogen in line with results from a pure culture.

In another poster2 Cailean Carter presented his work on Uropathogenic Escherichia coli (UPEC).  UPEC are the main etiological agent for urinary tract infections (UTIs) in Norfolk and are increasingly difficult to treat due to multi-drug resistance associated with clonal groups.  The spin column and Resolvex A200 Fire Monkey protocols were used to extract HMW-DNA from 217 isolates which were sequenced on the Illumina and ONT platforms for in silico multi-locus sequence typing and antibiotic resistance determinant detection.  This work is also published as a preprint3.

In a third poster, by Alice Nisbet, Emma Waters, Marie Chattaway and Gemma Langridge4, the scientists characterised genome rearrangements within Salmonella Paratyphi A isolates from using long read DNA sequencing alongside corresponding short-read sequencing data from isolates collected by the UK Health Security Agency (UKHSA) between 2004 and 2013 from patients with enteric fever. In these studies, HMW-DNA was extracted from a subset of acute and carrier isolates using the RevoluGen spin-column Fire Monkey kit and multiplexed 96x on the PromethION. Understanding genomic differences between S. Paratyphi A isolates provoking acute infection and bacterial carriage provides insights into the role genome structures (GS) play in human carriage.  Sequencing cost per isolate on a 96-plex run on the PromethION cost approximately £25, only 50p more than MinION 48x multiplexing but at 3x more coverage.  

Quadram Institute’s Emma Waters further expanded on these studies during a talk5 entitled ‘Investigating the role of Genomic Rearrangements in Transmission, Infection and Chronic Carriage of Salmonella Typhi’. 

“Genomic rearrangement where large genome fragments shift position and/or orientation around the long repeat sequences of the seven ribosomal operons producing different unique genome structures (GSs) plays a role in the development of S. Typhi carriage” she said, concluding “Genome rearrangement impacts the physical folding of the chromosome, expression of genes on rearranged fragments, carbon-source utilisation and growth phenotypes. Accompanying patient metadata has thus provided insights into the roles GS plays in carriage development and transmission.”

RevoluGen’s patent-protected technology is applied to a filter-column based protocol to extract HMW-DNA using a high g-force or positive pressure, but which does not break down the long and fragile DNA molecules as much as standard spin-column or positive pressure technologies. Fire Monkey produces DNA fragments of an average 100kb in length that are not too short and not too long and are found to be in a relatively tightly defined range of lengths. The Fire Monkey extract has so few small fragments that separate size selection steps are usually not required and so few very long fragments that separate fragmentation steps are also generally not required. This selectivity improves the overall sequencing results by not wasting as much sequencing resources on either reading the less useful small fragments or blocking the pores or wells with fragments that are too long. 

The Fire Monkey technology can also be size tuned into producing DNA fragments in very tight bands of smaller lengths below its standard 100kb average DNA extract length. This tuning can be dialled down to produce the average DNA extract length most suitable for optimal throughput performance of all sequencing technologies. 

In addition to the benefits brought about through automation, Fire Monkey technology unites sample preparation for both short-read and long-read DNA sequencing by providing flexibility to revisit the same original sample for more detailed long-read sequencing after a first run with lower cost short-read sequencing. This can reduce sample handling and storage significantly by only needing to do one extraction rather than two for producing hybrid assemblies cost effectively. 

Dr Gemma Langridge, a Group Leader at Quadram, said, “With the automation using the A200/Fire Monkey combination we have been able to prep tens of bacterial isolates at a time. This increases our research capacity and capability quite dramatically.” 

Caption: RevoluGen’s Fire Monkey HMW DNA extract kits in use at the Quadram Institute, Norwich 

Copyright: Quadram Institute

1.      Salmonella typing directly from stool using long read metagenomics

Steven Rudder, Bilal Djeghout, Georgios Patsos, Ngozi Elumogo, Nicol Janecko, Gemma Langridge

View online HERE

2.      Uropathogenic Escherichia coli population structure and antimicrobial susceptibility in Norfolk, UK

Cailean Carter, Alexandra Hutchison, Steven Rudder, Elizabeth Trotter, Ngozi Elumogo, Gemma Langridge

View online HERE

3.       Preprint:  Uropathogenic Escherichia coli population structure and antimicrobial susceptibility in Norfolk, UK

Cailean Carter, Alexandra Hutchison, Steven Rudder, Elizabeth Trotter, Emma Waters, Ngozi Elumogo, Gemma Langridge

View online HERE

4.      Characterising Genome Rearrangements within Salmonella Paratyphi A isolates associated with the Bacterial Carrier State 

Alice Nisbet, Emma Waters, Marie Chattaway, Gemma Langridge

View online HERE

5.      Talk: Investigating the role of Genomic Rearrangements in Transmission, Infection and Chronic Carriage of Salmonella Typhi

Emma Waters 

11:00 - 11:15 Tuesday 18 April