How does one extract high-molecular-weight firefly DNA?
In order to sequence the firefly genome, we first needed to extract high-molecular-weight firefly DNA (aka super long DNA). But how does one achieve such a feat?
See below for some pics of the process in action! And maybe more information on firefly DNA extraction than you ever wanted to know.
Step 1) Collect fireflies. Collecting July 10th and July 11th 2016, from Mercer Meadows in New Jersey. Left to right: Tim Fallon, Aska Pluskal, Tomáš Pluskal,. Big thank you to Aska and Tomáš (Tomáš = postdoctoral fellow in the Weng lab, Aska = Tomáš’s wife) for taking the drive from MA to NJ to collect with me! A quote from Tomáš, a trained computer scientist who got his PhD in computational biology, on the magic of firefly hunting: “Now I feel like a real biologist!”. Also a big thank you to the Mercer County Parks Commission for the collection permit to collect from this site. Remember folks, if you’re going to do science, you need permission if it’s not your private land.
This is the result. Lots and lots (and lots) of Photinus pyralis fireflies. This is maybe 1/10th of the fireflies that were ultimately collected. After this point a small piece of paper towel made damp with tap water was put in each container to keep the humidity up. Then containers were then placed in a large styrofoam box with 4˚C cooling packs to keep the fireflies cool and happy. The fireflies can survive for several days on the oxygen in such sealed containers, but they need to be kept cool and humid! Nonetheless I would periodically open the containers ~every 2 days and briefly fan it to swap out the air. If you want to feed the fireflies, they seem to like to drink the juice from slices of apple (remove apple slices when browned)
If you’re lucky, and/or diligent, you can even find a female Photinus pyralis firefly (top firefly shown above), which don’t (typically) fly, and instead can be found on the ground responding to the flashes of the flying males. The male & female fireflies above are mating. Notice the difference in the light producing tissue (lantern) morphology between the male & female Photinus pyralis firefly. The female only has a small lantern patch whereas the male has two full abdominal segment surfaces! The eggs from this mating were actually successfully collected and hatched. Did you know that firefly eggs glow? I had some fun with this property of firefly eggs on Twitter: https://twitter.com/photocyte/status/758086167606751233. Unfortunately, the firefly larvae that hatched did not survive long. Rearing North American firefly larvae is still very challenging! See Sara Lewis’s Silent Sparks book blog for more information/photos/videos on rearing fireflies: https://silentsparks.com/2016/08/09/raising-fireflies/
What’s next after collection & successful transport of fireflies back to the lab? Fireflies are sorted by size (roughly by eye), flash frozen in liquid nitrogen (-195˚C), lyophilized (freeze dried under vacuum – shown above), and placed in a -80˚C laboratory freezer until the time of DNA extraction comes. Why store fireflies at all? Why not not extract the DNA immediately? Answer: To keep the DNA in the most pristine state possible! PacBio sequencing requires very high-molecular-weight (aka “long”), high-quality & high-purity DNA. Any damage to the DNA, either in terms of its size distribution (how long it is), or chemical changes along the DNA (oxidation, phosphate backbone nicks, etc.), make the PacBio sequencing work less well. High-molecular-weight DNA should be stored at 4˚C for not more than a week, and freezing the HMW DNA will shear it, decreasing the size-distribution and causing other DNA damage. This isn’t your everyday run of the mill DNA, nor a run of the mill DNA extraction! My strategy for long-term DNA storage is that the DNA is probably safest where it was originally: packed into chromosomes in the nucleus, only without water and stored at -80˚C.
Okay, fast forward 1.5 months, the sequencing is all setup, the funds have been transferred, and the sequencing service provider is ready to accept the DNA samples. Time to extract that DNA!
Next step, sort dried fireflies. As the sizes/masses of Photinus pyralis fireflies can vary by 2 times, and presumably the most DNA can be extracted from the largest fireflies, 20 of the largest fireflies were selected. Before dissection/extraction masses of the dried fireflies were weighed, and documentation pictures were taken.
Next, fireflies were dissected to document & remove any parasitic mites. How does one remove parasitic mites from a dried firefly that crumbles into dust if you handle it too harshly? Very carefully. Shown above are 12 fireflies in the microcentrifuge tubes which have already been dissected and are sitting in 1mL 100% ethanol to help clean off any other microorganisms that might be adhering to the firefly's surface. The next Fireflies to be dissected are seen in the tissue culture plates sitting around. One might notice a large insect(?) sitting in one particular tissue culture plate. This is actually a bioluminescent elaterid beetle (not firefly), Pyrophorus luminosus, but that is a story for another day : )
The ethanol from the previous step is removed by careful decanting and a speedvac (not shown), and then the dried cleaned fireflies are broken into smaller pieces, by cutting with scissors, and transferred to 15mL falcon tubes with 5mL lysis buffer.
Next, the firefly fragments are incubated in lysis buffer @ 50˚C and rotated overnight to mix. Tip: Wrap Parafilm (a laboratory wax paper) around the top of the tubes, as even though these are screw top tubes they will leak at 50˚C, and the parafilm helps prevent that! In this run, even with the parafilm, 3/20 tubes leaked, losing about 1/5th of the solution from the tube when it leaked - bye bye DNA : (
Since I am a biochemist and get a kick out of this sort of thing, I can’t help but comment on the lysis buffer and what is going:
Qiagen G2 Lysis buffer: Water = A generally useful solvent that solubilizes many biomolecules! 800 mM guanidine HCl = Chaotropic salt. Denatures proteins and inactivates DNAses 30 mM Tris-Cl pH 8.0 = Tris is a buffer molecule, which keeps the pH stable over time. 30 mM EDTA pH 8.0 = Metal ion chelator. Inactivates DNAses 5% Tween-20 = Detergent (like soap) solubilizes lipid membranes & hydrophobic proteins 0.5% Triton X-100 = Detergent, solubilizes lipid membranes & hydrophobic proteins Protinase K = Very robust protease. Digests all the protein. RNAse A = Robust RNAse. Breaks down any released RNA.
The idea is, release all the DNA from the cells (combination of detergents & protinase K), inactivate any DNAses in the extract so the DNA doesn’t get degraded once it is released (combination of guanidine HCl & EDTA), degrade all the RNA (RNAse A) so what you’re left with in solution is HMW DNA and a soup of degraded RNA, degraded protein, solubilized lipids, small molecules, and likely polysaccharides like glycogen (probably largely intact, as no enzyme is around to break them down). Left out of solution are the insoluble polymers, like chitin, which make up the exoskeleton of insects. Tip from me: In the past I’ve used ~1-2mM DTT as a reducing agent, and it seemed to help the lysis yield. Be advised DTT and other reducing agents inactivate RNAse A, so you may want to add RNAse A after the overnight lysis with DTT rather than before.
Looking good after overnight lysis. A centrifugation of the tube at 4˚C & 3,500g for 30 minutes ensures all the remaining tissue fragments and anything that precipitated over the lysis process overnight (potentially proteins) are spun down to the bottom of the tube. I’m pretty sure the yellow color is largely from the lucibufagin steroidal defense compounds that fireflies have. Lucibufagins smell pretty bad, so this is a stinky mixture!
Next, the yellow liquid from the above step is transferred to an “anion exchange” liquid-chromatography column, and allowed to drip through. Recall that an anion is a negatively charged thing (-), so the “anion exchange” column is actually a positively charged thing (+) that binds negatively charged things. You may recall that DNA is an acid (DNA = deoxyribonucleic acid). What does that mean? It means that normal DNA in water solution has given up a hydrogen ion to make the solution more acidic (pH = -log10( concentration of hydrogen ions ) ), leaving behind a negatively charged phosphate group on the DNA, so the DNA is a negatively charged thing (anion) that the anion exchange column can bind! See this image for a demonstration of what I mean.
What else is negatively charged and might bind the column? RNA (ribonucleic acid), but remember that the RNA was degraded with RNAse in the last step. Things like fatty acids also have the negative charge, but may be too small / not charged enough to bind strongly to the column. In short, the larger the molecule and the more negative charges that it has, the stronger it will bind to the anion exchange column. Since HMW DNA is very long & very negatively charged, it binds great! Then, the DNA can be eluted off the anion exchange column by increasing the concentration of normal table salt, sodium chloride (NaCl). The Chloride (Cl-) ions are also anions, and when there is enough of them, they shield the positive charges on the column, so the DNA is prevented from binding to the anion exchange column, and comes off into solution.
Before applying the DNA, the columns have to be washed with: Qiagen Buffer QBT: Water 750 mM NaCl = Moderate starting salt. Small molecules likely won’t bind under these conditions, but large molecules (e.g. HMW DNA) will. 50 mM MOPS, pH 7.0 = MOPS is another buffer molecule like Tris, but it works better around neutral pH (7). At pH 7, negatively charged DNA binds to positively charged anion exchange column. 15% isopropanol = Helps solubilize any hydrophobic things that might otherwise gum things up. I’m guessing? 0.15% Trion X-100 = Helps reduce viscoscity & allows the equilibration buffer to “wet” the whole column.
And so, the dripping of the anion exchange columns begins. There are a few distinct steps, all with a lot of dripping: 1) Application of initial lysate (that yellow liquid) 2) Washing column several times with buffer QC, (3) Elution with buffer QF. Each step requires the dripping of 10s of mL through the columns. How long does this take? A long time! By my watch over 24 hours. Luckily they can be left overnight… A tip from Qiagen: vortexing the DNA for 5s before application to the column shears the DNA enough that the viscosity is reduced and it drips faster. I didn't do that step, so there was much slow dripping to be had.
Can’t resist another explanation of anion exchange, on the wash and elution buffers:
Wash buffer, Qiagen Buffer QC: Water 1.0M NaCl = More salt than equilibration buffer. Helps remove things that might be stuck to the column with the DNA, like undegraded RNA. 50 MOPS, pH 7.0 = Same pH as the equilibration buffer. 15% isopropanol = Probably there to help wash off hydrophobic things.
Elution buffer, Qiagen Buffer QF: Water 1.25 M NaCl = Even higher salt, now the DNA is eluting 50 mM Tris-Cl pH 8.5 = Higher pH than past two buffers. This higher pH (more basic), means the anion exchange column itself is less positively charged, so the negatively charged DNA binds less strongly and returns to solution, where it elutes from the column! 15% isopropanol = Probably there to keep things consistent + setup for next step of isopropanol precipitation.
See here for the figure from Qiagen’s anion exchange kit manual that demonstrates how this all works:
So by washing at 1.0M NaCl, at pH 7, everything but DNA gets washed off the column. Then by increasing the salt, and increasing the pH, the DNA will come off. Got it? Okay good. Moving on...
The bound HMW DNA is eluted from the anion exchange column with QF buffer. Since HMW DNA is so long, it actually changes the properties of the solution. Water with a lot of HMW DNA in it is quite viscous/sticky. This property of stickness mainifests visibly during the elution of the DNA from the column: note how the elution isn’t going drop by drop, but actually makes a long “snake” of liquid down the side of the tube. Neat! Also means it drips extra extra slowly : )
Once eluted, isopropanol is added to the DNA solution, mixed slowly by inversion of the tube over several minutes, and lo and behold, precipitated DNA appears! (See the white blob floating around above) Tip: For me, adding glycogen was an essential step to get robust precipitation of HMW DNA. For 5mL of elution (QF buffer), I added 1 uL 10mg/mL RNA grade glycogen, & 3.5 mL isopropanol, then mixed at room temperature. Spinning down for 30 minutes @ 3,500g & 4˚C in a swinging bucket centrifuge effectively pelleted the DNA.
Finally, the precipitated DNA pellet is washed twice in 2mL 4˚C 70% ethanol, and redissolved in 80 uL of dissolution buffer. This gives the final purified high-molecular-weight DNA in soluble form (inside the plastic tubes shown above). We handed off these DNA samples to the sequencing provider, who will check the DNA size distribution by pulsed-field gel-electrophoresis (PFGE), and if everything looks good, start preparing the PacBio sequencing library (converting long intact HMW DNA -> into less long DNA with the proper adaptors for sequencing on the PacBio instrument). Au revoir DNA! Good luck!
Dissolution buffer, Qiagen QLE: Water 10mM Tris-Cl pH 8.5 = DNA is more stable at slightly basic pH like 8.5 0.1mM EDTA = Chelates metal ions, helps keep DNAses inactive if DNAses happen to get into the purified DNA sample.
Whew! I hope you enjoyed the update on the firefly genome project, and maybe even learned a bit of biochemistry along the way. Stay tuned for more information on how the PacBio library preparation and sequencing go.
Edit: See below for the QC results!
Fire Fly Aliquot # | ng/uL | Amount in Aliquot (~80 uL) |
1 | 214 | 17120 |
2 | 57.4 | 4592 |
3 | 111 | 8880 |
4 | 31.2 | 2496 |
5 | 284 | 22720 |
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