Fire Monkey/Fire Flower

Spin-column kit for both Short and Long read sequencing.

RevoluGen’s Fire Monkey / Fire Flower (FM/FF) product is a spin-column based Nucleic Acid Isolation and Purification (NAIP) kit that performs High Molecular Weight DNA (HMW-DNA) extraction & size selection. FM produces pure DNA in a narrowed band of lengths with average DNA strand lengths of >100kb in under an hour from animal and bacterial cells utilising a parallel extraction/size selection process. The FF size selection protocol alone can deplete DNA fragments under 10kb in length from any third party extraction process, all within approximately 15 minutes. (Figure 1).

The main technical advantage of HMW DNA extraction is that when used with long-read sequencing, this combination can provide much important structural genomic information that low molecular weight DNA extraction combined with short-read sequencing struggles to provide. A major commercial opportunity therefore exists in the diagnosis of infectious diseases where the combination of FM and the long-read sequencing of Oxford Nanopore Technologies (ONT) has already been shown to rapidly sequence the whole bacterial genome plus all its plasmids. This analysis will prove vital to uncovering all Antibiotic Microbial Resistance (AMR) genes as well as very accurately identifying all bacterial strains, even those that cannot be cultured in the laboratory. RevoluGen is convinced that long-read sequencing of bacteria will therefore become an industry standard for microbiology in the near future. HMW DNA extraction also serves the short-read sequencing technologies equally well. 

This FM/FF technology has been externally and internally validated on a series of sequencing platforms including Illumina, 10X Genomics, Oxford Nanopore Technologies (ONT) and PacBio. The Quadram Institute work validation; Extensively drug-resistant (XDR) Salmonella Typhi causes Typhoid fever that is endemic in Pakistan and shows widespread resistance to first line drugs such as ampicillin, co-trimoxazole, chloramphenicol and fluoroquinoles. XDR patient isolates were processed using the FM protocol and whole genome sequencing (Illumina) and were found to harbor the same resistance determinants as the outbreak strain (Rasheed et al 2020).

Although Illumina sequencing is the gold standard for large scale screening programs there is a growing appreciation that supplementary long read sequencing (10X Genomics, ONT or PacBio) in some cases can provide crucial information in areas that short read sequencing could struggle with. This is known in academic circles but also is highlighted by Illumina’s effort to acquire PacBio. Long read sequencing can support analyses of repetitive regions and large structural variations, since long read lengths are more likely to contain more copy number variations and identify translocations and inversions more accurately then short-read sequencing. It is a great benefit to all sequencing screening programs to be able to extract HMW-DNA with FM for short read sequencing as shown by Harvard with the 10X Genomics publication (Tourdot & Zhang, 2019). Using FM also enables the extracted sample to be used at a later date for long read sequencing of interesting sample cohorts.

Antibiotic Microbial Resistance is one of the biggest challenges and opportunities for all long-read technologies. The starting DNA from extraction needs to be long enough for long-read sequencing but also the nucleic acid mixture should contain as few of the smaller fragments as possible, since they tend to be preferentially sequenced over the longer fragments by both ONT and PacBio systems. FM extracts the long DNA and depletes the short DNA in parallel; but because of FM’s highly innovative column/chemistry combination it does so in a user-friendly spin-column format, which has, up to date, been known in the industry as a no-go process for HMW DNA isolation. As a result, validation of an earlier version of FM performed by ONT revealed that FM produced significantly higher N50 values than Qiagen’s Genomic Tip product (25-35kb vs 20-30kb) in a fraction of the time (Figure 2). The latest FM version can generate N50 values of 56 to 60kb+ at a high throughput (Figure 3) and assemble bacteria genomes in a single contig with a 50x coverage threshold at >122kb (Figure 4). This protocol has also been adapted for stool samples supporting a, Quadram institute Campylobacter Jejuni burden trial currently running in Bangladesh, funded by the Gates Foundation (Figure 5).  Using FF alone on already extracted DNA that is rich in small fragments can double N50 and long reads at a high throughput (Figure 6).

External PacBio validation of both FM and FF is currently ongoing and subject to academic journal publications. Current RevoluGen R&D efforts focus on adapting the spin-column process to an automated format in order to satisfy the commercial need for large volume sample extraction for either short, long or a combination of short and long read sequencing programmes. 

 Figure 1. Fire Monkey / Fire Flower performance summary.

Figure 2. Fire Monkey early version (v1) external validation on ONT Community site.  FMv1 generates N50 values of 25-35kb vs Genomic Tip’s 20-30kb in a fraction of the extraction time (1hr vs 6hrs).  Sequencing kit used was LSK109 (MinION)

Figure 3. a. Latest FM version (v6) generates N50 of >60kb at 15.5Gb with 2.6Gb in 100kb+ reads. b. Throughput can be pushed to 21.1Gb with 2.8Gb in 100kb+ reads at N50: 56kb (genome size: 2.7Gb).  Single R.9.4.1 flow cell (LSK109/MinION).

Figure 4. Complete genome assembly and plasmid recovery with 1000x coverage.  50x coverage with >122kb fragments.  Single R.9.4.1 flow cell (LSK109/MinION).

Figure 5. Gates Foundation Campylobacter burden trial in Bangladesh (stool samples).

 Figure 6. Fire Flower doubles long reads at a high yield (14Gb), after a 15mins of spins and ~30mins of evaporation.

References:

Rasheed et al (2020) Emergence of Resistance to Fluoroquinolones and Third-generation Cephalosrporin in Salmonella Typhi in Lahore, Pakistan. medRxiv https://doi.org/10.1101/2020.02.12.20020578

Tourdot RW & Zhang CZ (2019) Complete Haplotype Determination and Single-Chromosome Analysis. bioRxiv https://doi.org/10.1101/629337