DNA extraction - long-read ratio - N50

Fire Monkey extracts HMW DNA from bacterial and mammalian cells within 1hr

Fire Monkey is a standard spin-column process which extracts HMW-DNA with average strand lengths of 100kb and above from both bacterial and mammalian cells within 1hr. 

Protocols are user-friendly and can be high-throughput.

Only basic laboratory equipment is required

 

 

DNA extraction - N50 long read fire monkey kit
Fire Monkey long-read DNA extraction trace graph

This Agilent Femto Pulse trace shown above is an extended run of a white blood cell sample after a Fire Monkey extraction has produced an average DNA fragment length of 114,378bp.

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 ~50kb can be achieved without using the ONT recommended 0.7x SPRI step for LSK109 sequencing.

Georgios-Revolugen-April FM Blood Table

In the table of results above, 47µl (instead of the ONT recommended 20µl) of a Fire Monkey white blood cell extract DNA extract were sequenced according to the ONT LSK109 protocol without a 0.7x SPRI post-extraction step (MinION, FLO-MIN106D R9 Version).

Georgios-Revolugen-April E coli Table

In the table of results above, 47µl (instead of the ONT recommended 20µl) 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.

Fire Flower is a size selection kit that removes the shorter DNA fragments from extracted samples

Fire Flower is a standard spin-column process that can size select extracted DNA from all sample sources within 15mins.  Protocols are user-friendly and can be high-throughput.

Fire Flower can increase the overall average strand length by 30%, while depleting DNA fragments up to 30kb.

Only basic standard laboratory equipment is required.

Fire Flower produces an Increase in the molecular ratio; High Molecular Weight vs Low Molecular Weight DNA. This chart shows the Fire Flower effect on the number of molecules at various lengths.

DNA extraction - N50 long read fire monkey kit
Fire Monkey long-read N50 molecular DNA ratios

Fire Flower produces an Increase in the molecular ratio; High Molecular Weight vs Low Molecular Weight DNA. This chart shows the Fire Flower effect on the number of molecules at various lengths.

Fire Flower produces a decrease in mass of the shorter DNA molecules than the SPRI beads do

Fire Monkey long-read DNA - N50 ratio

In this 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.

Purchase the Fire Monkey 10 Prep Kit here

DNA extraction - long-read ratio - N50

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