Data presented at Microbiology Society Annual Conference, Belfast, 4-7 April 2022
UK genomics company, RevoluGen Ltd. (RevoluGen or the Company), welcomes data presented by scientists from its collaborator the Quadram Institute at this years’ Microbiology Society Annual Conference being held in Belfast. The Quadram’s team results demonstrate the advantages of Fire Monkey high molecular weight (HMW) DNA extraction with multiplexed Oxford Nanopore sequencing for cost effective complete hybrid assemblies of pathogen genomes in resolving genomic rearrangements important in acute and chronic Salmonella infections.
“Whilst small nucleotide-level variations in bacteria that can affect antibiotic resistance and entire metabolic pathways can often be resolved by short-read DNA sequencing, bacteria can also exhibit genomic rearrangement, where large genome fragments shift position and/or orientation around long-repeat sequences to produce different unique genome structures without necessarily affecting the underlying nucleotide sequence. Such variation cannot be identified by short-read sequencing as long-repeat sequences require reads of thousands of base pairs for resolution,” explained Dr Emma Waters, senior postdoctoral scientist within the Microbes in the Food Chain group at the Quadram Institute, Norwich.
At the conference, Dr Waters presented a poster1 showing the ability of multiplexed, long-read DNA sequencing to investigate genome structure relationships between strains of Salmonella enterica serovar Agona associated with acute and chronic infection. Extraction of long length HMW DNA using RevoluGen’s Fire Monkey technology facilitated 48-plex sequencing and the successful generation of complete assemblies for samples with ~30X theoretical coverage. “The quality of HMW DNA we’re getting from Fire Monkey has supported our efforts in driving up the number of samples we can multiplex on the Oxford Nanopore MinION, whilst still getting good outcomes in terms of genome assembly,” said Dr Waters.
In a separate poster2, presented by Luisa Bautista de Carvalho, the Quadram team were able to resolve and investigate structural chromosome rearrangements in Salmonella enterica serovar Typhi (S. Typhi), the causal agent of typhoid fever, again using DNA extracted with RevoluGen’s Fire Monkey Kit and long-read sequencing on the MinION. “In S. Typhi, these rearrangements can impact on the metabolic ability of the bacteria, promoting survival in their human host under certain environmental conditions. Long-read sequencing of other medically important bacterial pathogens also could benefit from this approach,” concluded Ms Bautista de Carvalho.
In an oral presentation3, Dr Gemma Langridge, a Group Leader at Quadram and an early adopter of RevoluGen’s Fire Monkey technology summarised the benefits and opportunities for long-read sequencing of bacteria, “To date, scalable techniques have not been applied to genome structure identification, so it has remained unclear just how variable this is and the extent of its impact on gene expression. However, the emergence of multiplexed, long-read sequencing overcomes this problem, as reads of several thousand bases are routinely produced that can span long repeat sequences to identify the flanking chromosomal DNA, allowing genome structure variation (GSV) identification. GSV may provide a mechanism through which bacteria can quickly adapt to new environments and warrants routine assessment alongside traditional nucleotide level measures of variation.”
RevoluGen’s patent-protected technology is derived from a spin-column based protocol to extract HMW-DNA using a high g-force that does not break the long and fragile DNA molecules. Fire Monkey produces DNA fragments that are not too short and not too long for the long-read sequencing technologies. This size-tuned DNA fragment extract improves the overall sequencing results from the technologies by not wasting sequencing resources on either reading the less useful small fragments or reducing sequencing throughput by blocking the reading pores with fragments that are too long. It also facilitates hybrid sequencing from a single extraction, saving valuable DNA samples as well as reducing time and cost. In addition, for PacBio HiFi users, Fire Monkey offers a parallel fragmentation/extraction protocol option that removes the need for time-consuming and costly post-extraction fragmentation workflows that are needed to achieve a very tight DNA lengths distribution required for optimal results.
Quadram and RevoluGen have been working together for some three years across a number of DNA sequencing projects and RevoluGen is industrial partner in a joint PhD studentship grant awarded to Quadram. RevoluGen has recently transferred its automated Fire Monkey system robot to Quadram to enable automation of HMW DNA extraction across two high volume bacterial sequencing projects: the first will be processing Salmonella samples collected by a UK Health Security Agency (UKHSA) lab; and the second will be processing Salmonella and Campylobacter samples from the Norfolk area looking at antimicrobial resistance (AMR) amongst other things.
RevoluGen has invested over 12 months in automating its Fire Monkey HMW DNA extraction into a standard 96-well filter plate format, the world’s first automated extraction of library ready long HMW DNA. The extraction protocol has been validated to extract and purify DNA from bacterial and mammalian samples, and the collaboration with Quadram is the Company’s first customer roll-out.
“We are delighted to be collaborating with Gemma and the teams at Quadram in their world-leading projects in DNA sequencing and pathogen genomics,” said Dr Georgios Patsos, inventor of the Fire Monkey technology and CSO at RevoluGen.
Fire Monkey is easy to use and as fast as a spin column kit. Now that it can be multiplexed and automated it can be routinely adapted to support large, short-read genomics programmes, which require easy and adaptable extractions, and still go back to the same extract for supplemental long reads, with no need to locate the original biological sample and re-extract for HMW DNA.
In addition to its use at Quadram, Fire Monkey is being used by scientists at the UK’s Health Security Agency. “HMW DNA extraction is the critical first step in the DNA sequencing workflow. Rapid growth of sequencing worldwide has driven the need for automation to handle the volume of samples needed for applications such as population genomics, epidemiological mutation screening and antibiotic microbial resistance gene monitoring,” Dr Patsos said.
Presentations and posters as follows:
- Poster: 48-plex Long-Read Sequencing to Generate Complete Assemblies and Determine Genome Structure in Salmonella Agona, Emma V. Waters, Winnie W. Y. Lee, Amina Ismail Ahmed, Marie A. Chattaway and Gemma C. Langridge, 4 April 2022, 6.30-8pm
- Poster: Screening and phenotypic characterisation of Salmonella Typhi colonies that have undergone genomic rearrangement, Luisa Bautista de Carvalho, Emma V Waters and Gemma C Langridge, 4 April 2022, 6.30-8pm
- Presentation: Impact of Salmonella genome rearrangement on gene expression, Emma V. Waters, Liam A. Tucker, Jana Haase, John Wain and Gemma C. Langridge, 5 April 2022, 10.15am
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