fbpx
search 

Collect Tissue for Sequencing

Gather your materials

Label your tubes

Collect tissue

Instructions for specific fungi


Before you start:  To have your specimens sequenced by FunDiS, your project must be registered; you must either have a sequencing grant or have purchased sequencing from FunDiS in our Shop; all specimens must be properly documented (you have taken pictures in the field; collected the mushroom properly; and uploaded photos and descriptive data to iNaturalist or Mushroom Observer for each specimen; and your specimens must be dried (with exceptions noted below).


Gather your materials

  • You will need cutting implements (tweezers, razors or scalpel), rubbing alcohol & tissue; and special tubes..
  • Eppi tubes: We use 1.5 milliliter (mL) Eppendorf tubes (Eppi tubes), which can be purchased in our Shop under Eppi Tubes for Paid orders. (Grant awardees receive Eppi tubes as part of the grant. )
    • If you have old tubes with liquid in them, do NOT use them. Our current sequencing partner, BOLD, only accepts dried tissue. There is no charge for replacing old tubes with buffer solution for grant awardees. Go to the Grant Package section of the Shop and order as many as you need to complete your old grant and email coordinator@fundis.org to let us know what you are doing.
    • Note that specimens to be sequenced under old (pre-9/2020) grants must be received by FunDiS by 12/31/2020. 

Label your tubesCollection Tubes

  • Tubes must be labeled with the iNaturalist or Mushroom Observer observation number that corresponds to each specimen. 
  • Labels must be printed: No handwritten numbers (they can be illegible at this small scale). Print observation numbers from your exported spreadsheets (see Submit Tissue for Sequencing). The observation numbers need to be small enough to fit vertically on the side of the tubes. 
    • A simple way to do this is copy the ID number column from your exported spreadsheet, place it in several columns of a new spreadsheet, resize (try 8 point), print and cut out the numbers.
  • Use clear tape to attach the numbers to the tubes.

Collect tissue

  • Clean all surfaces. Be as clean as possible during tissue collection to reduce DNA contamination. Do these cleaning steps between every specimen or your DNA sample may be contaminated. 
    • Clean the surface where you will be working with rubbing alcohol (or a solution of 10% liquid bleach).
    • Wipe your fingertips with an alcohol wipe.
    • Clean the tools you will use (tweezers, razor blade or scalpel) with alcohol wipes and use immediately. Remember to wipe the inside surface of the tweezers that touches the sample, in addition to the outside. If still not clean, or to be certain, swirl the tweezers/scalpels in some alcohol and then dry with the wipe.

  • Which part to sample? While it is possible to take dried tissue from any part of the mushroom to obtain DNA, you’ll do best selecting parts with high DNA concentrations (usually the spore producing surface: lamellae, tubes or gleba) and low levels of contamination.
    • Avoid cap surfaces to reduce contamination by soil and spores deposited by other fungi. 
    • Avoid fuzzy areas as these may be parasitized by other fungi. 

  • Tweeze or cut off and collect a piece of dry fungal tissue about the size of a grain of rice (1-3 mm) using sterilized tweezers or blade, and place in an Eppi tube labeled with its iNat or MO number.
    • For collections with multiple look-alike specimens, wrap the sampled specimen in a small piece of paper and write, DNA source.

Instructions for specific fungi


Mushrooms 

Preferably tear a dried mushroom apart, exposing the middle of the stipe/stalk (a cut on the margin may be needed to start the tear) to expose uncontaminated surfaces to collect samples from. Tweeze a piece of flesh from the top of the stalk where it joins the cap plus (interior flesh if it is a larger mushroom), place it in the vial, then tweeze a piece of gill or tubes and add it to the vial using the clean technique above. This will yield the highest concentrations of DNA and least contamination. Don’t destroy the entire upper stalk as there are important diagnostic structures on the surface that are needed for identification.


Polypores

These fungi are typically tough, so you will need a razor blade or scalpel. Healthy looking pore surface should be cut from the underside of the polypore to about 1 mm deep and placed in the vial.


Crust fungi

There is only a 50%-60% success rate. Success is higher with recently collected specimens than old ones. For thin crusts, take fungal tissues together with some underlying wood. Some use a dissecting needle to pry out clean bits of tissue.


Stinkhorns

Take pieces of the spore-bearing surface (gleba) for obtaining the highest concentration of DNA.


Puffballs

Pieces of the spore-bearing interior can be tweezed off and placed in a vial for sequencing.


Fleshy Ascomycetes (Cup fungi, Morels, etc.) 

To remove a piece of the spore producing surface from cup fungi (fresh or dry), ensure the inside of the cup is clean (gently wipe dirt out with a brush or tissue). Try to tear or break the cup in half, and then tweeze a clean piece of tissue from the cup’s inner (spore-producing) surface (if hard and dry, you can soak a piece in water first and then redry). For morels, spore-bearing tissue can be used from the indentations on the ‘cap’ or from the upper stalk, but ensure the tissue is clean. False morels (Helvella) are often contaminated by soil, so extra careful cleaning is required when removing a healthy piece of the ‘flap (fresh or dry)’. Use a fine paint brush or pick it clean with tweezers. Success rate may be low.


We are not currently sequencing…

These species require special primers and techniques and we are not equipped to sequence them at this time. You may store them dried (bag, tag and hold) in the event we are able to sequence them in the future -- but we cannot guarantee it.

  • Hard Ascomycetes (“carbonaceous”, e.g., Hypoxylon and Dead Man’s Fingers)
  • Chanterelles (Cantharellus & Craterellus)
  • Lichens 
  •  Slime molds

About Fungal Diversity Survey

FunDiS is dedicated to a world in which the fungal kingdom is fully documented, understood, appreciated and protected.