Fire Monkey is a HMW-DNA extraction kit that, in a fairly simple procedure, using standard laboratory equipment, can within one hour, extract and purify long-fragments of DNA from mammalian and bacterial cell samples.
The two major disruptive features of Fire Monkey are that firstly, it generates average DNA fragment lengths of between 100kbp – 130kbp and secondly, it significantly reduces the amount of small DNA fragments shorter than 10kbp in the final product. This combination, where significant amounts of the DNA fragments have intact lengths of over 165,000 base pairs and up to several hundred thousands base pairs in length, combined with the absence of most of the smaller DNA fragments, through its inbuilt size selection capability, massively improves the eventual long-read sequencing of the sample. Fire Monkey improves the N50 measure of sequencing quality by 100% and yet it takes a sixth of the time to do so compared to the main competing NAIP kits.
The Fire Monkey result is therefore much better than the present competition both in ease of use, speed and sequencing results. Both internal and external validation of the improved effect on long-read sequencing is illustrated below using N50 results as a proxy for sequencing performance.
Due to Fire Monkey’s in-built size-exclusion aspect very few fragments shorter than 10kb will be extracted. As a result, post-extraction size-exclusion steps that are time-consuming and could break long DNA fragments are not needed. This leads to more user-friendly sequencing protocols and better quality sequencing results. For example, N50 values of greater than 50kb can be achieved without using the ONT recommended 0.7x SPRI step for LSK109 sequencing.
(A) 2 Fire Monkey horse blood Fraction B extracts (B1, B2) were evaporated at 65oC for ~1hr (Eppendorf lid open) reducing the eluates volume to under 50μl. Full concentrated volumes (no post-extraction size selection) were used on a 48hrs R9.4.1/LSK109/MinION run, including a nuclease flush and library re-load at 24hrs (0-24hrs: B1; nuclease flush; 24-48hrs: B2). DNA Repair-End Prep were performed for 10/10mins at 20/60oC, and ligation for 30mins at RT. This generated ~15.5Gb at an N50 of ~61kb and ~2.6Gb in reads of 100kb+ for a 2.7Gb genome. (B) 48μl of a single Fire Monkey horse blood Fraction A (no post-extraction size selection or evaporation) were used on a 72hrs R10/LSK109/MinION run without a nuclease flush (all library enzyme incubation times as on A), generating an N50 of ~62kb and 4.4Gb.
In the table of results above, ~1500ng of Fire Monkey E coli DNA extracts (600 million or 1 billion cells; plus RNase A) were sequenced according to the ONT LSK109 protocol without a 0.7x SPRI post-extraction step (MinION, FLO-MIN106D R9 Version). The 600 million cells sample produced overall better results. It is essential that cell loading is optimized for every sample type.
Complete Genome Assembly & Plasmid Recovery of bacteria at ~1000x coverage.
Results from a Fire Monkey extract of a sample of 600 million E coli cells sequenced by LSK109/MinION
Fire Monkey results in a combination of long DNA fragments and high yields which facilitates complete genome assembly plus full plasmid recovery (plasmid genes rep, tra in smaller contig).
The initial assembly at 50x coverage was made with fragments larger than 122kb (Flye, Q7) and one polishing step then pushed the assembly to 1000x coverage (Flye, Q7).
Fire Flower is a user-friendly spin-column protocol with a built-in size depletion ability included in the kit, and can be used with any other extraction protocol. Fire Flower can be used to size-select up to 200microLt of extracted DNA despite the sample being rich in small DNA fragments. The duration of the Fire Flower protocol is 15mins and this results in an extract which will effectively double most long read metrics on the LSK109/MinION technology.
Fire Flower produces an Increase in the molecular ratio; High Molecular Weight vs Low Molecular Weight DNA. The chart above shows the Fire Flower effect on the number of molecules at various lengths
In the above experiment, the highly biased synthetic input sample consisting of lots of Low Molecular Weight DNA mixed with a relatively small amount of High Molecular Weight DNA (total mass: ~3µg) was used as the starting point for the experimental results shown here. The above results show the mass of both the short and long DNA components from each of the input sample, the SPRI bead treated sample and the Fire Flower treated sample. Fire Flower was compared for size-selection against 0.7x SPRI beads. After these size exclusion processes, the Nanodrop and Agilent Femto Pulse analysis of the DNA outputs were used to calculate the number of nanograms below and above the 10kb measurement threshold. Both SPRI beads and Fire Flower reduce the mass of the DNA fragments over 10kb at a similar rate. However, Fire Flower had a much greater efficiency at reducing the mass of DNA fragments under 10kb in length than the SPRI beads did.