Increased yield can drive disposable costs for MinION to $35/Gb and for PacBio to <$10/Gb

• Fire Monkey version 8 (FMv8) record-breaking 30Gb+ yield of raw DNA data from an E. Coli sample on a single Oxford Nanopore Technologies (ONT) MinION flow cell (Fig. 1)
• Increased yield boosts DNA recovery and improves sequencing (Fig. 2)
• Based on achieved yield, Fire Monkey v8 could cut disposable costs of DNA sequencing: on ONT MinION to $35/Gb; on PacBio to under $10/Gb (Fig. 3)
• Fire Monkey v8 extracts DNA suitable for both short and long-read sequencing and upgrades the sequencing options for Illumina users 
• High yield of homogeneous High Molecular Weight (HMW) DNA maximises results with long-read sequencing technologies 

London, UK – 30 November 2020 – The UK genomic tools developer, RevoluGen Ltd. (RevoluGen or the Company), today announces new results from its improved Fire Monkey/Fire Flower version 8 (FMv8) protocol, demonstrating a record breaking 30Gb+ yield of long DNA reads raw data of an E. Coli bacterial sample on a single Oxford Nanopore Technologies (ONT) MinION flow cell. The increased yield offers significant potential cost and performance advantages across all sequencing platforms, including PacBio and Illumina.

RevoluGen’s game-changing Fire Monkey/Fire Flower nucleic acid isolation and purification (NAIP) technology extracts a homogeneous range of long-length DNA, the important first step in both short-read and long-read DNA sequencing.

The FMv8 protocol introduces a needle-aspirate based, cell-resuspension step prior to cell lysis that boosts DNA recovery. The spin-column has been optimised to deliver homogenous High Molecular Weight DNA (HMW-DNA) extracted and purified from bacterial and mammalian samples. For E. Coli, the FMv8 protocol allows the ONT MinION to deliver optimal sequencing throughput at an impressive N50 of 36.7Kb. The raw data yield of 30Gb+ DNA coverts to 28.5Gb post the Q7 cut off (see Figure 1).

“This is a remarkable record-setting performance that approaches the maximum reported from a ONT MinION flow cell” added Dr Georgios Patsos, inventor of the technology and CSO at RevoluGen.

Dr Patsos has been focused on improving the outputs from Fire Monkey since its first prototype was introduced in 2018 (Figure 2). New patents filed in Feb 2019 dramatically improved the performance of the FMv8 over the FMv5 protocol. The FMv8 protocol now includes a cell re-suspension step that de-clumps the cells before the lysis stage to enable better exposure of the individual cells to the lysis chemicals.

“These results also make the ONT MinION capable of sustaining a much greater multiplexing potential because the greater the throughput the more you can multiplex.  Facilitating delivery of more than 30Gb of data per ONT MinION flow cell is a remarkable record-setting performance. This is close to the maximum possible from an ONT MinION flow cell. In theory, such a result is capable of handling the multiplexing of up to 60 bacteria samples at a time to a level of 100 times coverage. Such yields could improve the error rate of the bioinformatics assembly and possibly reduce it to near zero” he said.

RevoluGen is now concentrating on automation and improving the overall $/Gb sequencing costs that its NAIP kit produces. The $/Gb cost is likely to be one of the key determinants to uptake of both short and long-read sequencing by the market in future. Automation of the Fire Monkey process is underway in a 96 well plate format using a Fluent 780 system supplied by Tecan. The FMv8 protocol has at least doubled the extraction yields of the ‘not too short and not too long’ homogeneous HMW DNA it produces, with average DNA fragments of about 100kb length, all ready for immediate library preparation. The ONT MinION flow cells respond to the material presented to it from this process by wearing out much more slowly and generating much larger data yields. The reduced amounts of smaller fragments mean that they do not monopolise the early reads from the pores and the lack of massive DNA lengths reduces the jamming effect they have on the pores. 

Dr Patsos has been invited to present a poster at the Oxford Nanopore Community Meeting, 1-3 December and will be presenting these data as well as progress on automation.

Dr Patsos said “I am really excited by these results. The Fire Monkey development has been a long story of constant improvement for the world’s only spin column kit that extracts and purifies homogeneous HMW DNA. Our latest results enable a bit of a virtuous circle; doubling data yields at high N50 values reduces the $/Gb sequencing costs significantly whilst at the same time, these bigger yields promise near error free assembling of whole bacterial genomes from multiplexed runs.”

The markets for the Fire Monkey technology are multiple and fast growing, across bacterial and mammalian sequencing, for both short and long-read applications. There is external validation that the DNA extracted using Fire Monkey can be used on any DNA sequencing platform including ONT’s MinION flow cells, PacBio’s SMRT cells and Illumina’s systems.

When Fire Monkey was used independently to extract HMW-DNA for sequencing with a PacBio system it yielded such a high yield of 139Gb that the disposable costs for the PacBio sequencing worked out at less than $10/Gb. Similar improved yields from Fire Monkey v8 produced HMW-DNA that reduced the costs of the disposable components of the ONT MinION sequencing down to $35/Gb. This enhances the trend to bring long-read sequencing costs much closer to short-read sequencing cost (Figure 3).

Using Fire Monkey for DNA extraction would also give Illumina users the option to perform short-read DNA sequencing and to be able to return to exactly the same sample for a long-read sequencing session. This option offers a big reduction in potential handling errors when trying to resolve structural variations or long repeats in addition to the short-read information.

“Our technology unites short-read and long-read sequencing, enabling both from the same sample, offering experimental flexibility and sample-saving. Based on lower cost and superior performance, we believe we can become the DNA extraction technology of choice in the global $4bn/year DNA sequencing market” concludes RevoluGen CEO, Pieter Haitsma Mulier.

Figure 1

Results from a long-read DNA sequencing of an E. Coli bacterial sample on a single ONT MinION flow cell following NAIP with Fire Monkey/Fire Flower v8 protocol. Copyright RevoluGen.

Figure 2   Development of Fire Monkey HMW NAIP 

FM = Fire Monkey. WBC = white blood cells. Source; RevoluGen internal records.

Figure 3   Cheaper $/GB sequencing results from using Fire Monkey

CLR = continuous long-read. Adapted from Logsdon et al 2020 Long Read Human Genome Sequencing and its Applications. Source; Nature Review Genetics (21) 597-614 plus RevoluGen data. All cost estimates exclude the cost of labour, instrumentation, maintenance, and computer resources.