What follows is an email response to some questions regarding our protocols and methods for working with old fungal specimens. The core information of the email is replicated here, primarily so I do not have to write the bulk of it again for other inquiries.
Originally sent 02/01/2025. Specific details, methods, and opinions expressed are likely to change with more time and more experience.
What follows is a ChatGPT revision of the original email. The original email is at the bottom of this page.
Revised Letter for Clarity/Brevity
Subject: Working with Older Mycological Specimens: Protocols and Considerations
Hello [Name],
Thank you for reaching out about processing older mycological specimens. We do have considerable experience with this topic—most of our specimens are around 80 years old, though not usually as old as 100–200 years. Below are the most important points we’ve learned regarding DNA extraction, PCR, and sequencing from older collections.
1. Age vs. Storage Conditions
- Age Matters, But Not Most: While older specimens can yield DNA, how the material was dried and stored is often more critical than its age.
- Drying & Storage: Rapid, thorough drying immediately after harvest, followed by storage in a consistently dry environment, provides the best chance for successful DNA recovery.
- Sampling Strategy: Because success can be unpredictable, we recommend collecting multiple tissue samples (preferably from different parts of the specimen). This ensures you have backups in case a particular sample fails.
2. DNA Extraction
- Kit Choice: We’ve tested various standard kits (Promega, Zymo, Qiagen, Omega, etc.) and found no single product that consistently outperforms the others in terms of total DNA yield for older specimens.
- Hit-or-Miss Results: Regardless of the kit, success can vary depending on the specific piece of tissue or its contamination level.
- Our Preferred Protocol: We currently use a modified Promega Wizard extraction method. It strikes a good balance between cost, ease of use, and yield. Our detailed protocol is available here: [protocol link].
3. PCR Amplification
- Unpredictable Outcomes: PCR on older specimens can be temperamental. Identical conditions sometimes yield different results on different attempts.
- Brute-Force Strategy: We run four PCRs per specimen:
- Full-length ITS (undiluted template)
- Full-length ITS (1:50 diluted template)
- ITS2 region (undiluted)
- ITS2 region (diluted)
- Why Four Reactions?
- Increases the chance of at least one successful amplification.
- Replicates help validate results: if two reactions (with different primer sets or dilutions) give the same sequence, confidence in the outcome is higher.
- Dilution Effects: Sometimes dilution overcomes PCR inhibitors in older tissue. However, it can also reduce already low DNA levels, so trying both approaches is key.
4. Sequencing Technology
- Contamination Challenges: Over decades of storage, specimens often accumulate contaminants (e.g., yeasts, Aspergillus spp.). This can thwart Sanger sequencing.
- Nanopore vs. Illumina: We prefer nanopore for its longer read lengths, relatively simple library prep, and lower cost. Illumina can work but typically has more complex requirements.
- Throughput Example: For 960 specimens, we run four separate nanopore Flongle cells (one for each PCR approach mentioned above). This is scalable depending on how many specimens you have.
5. Sample Size & Ongoing Development
- Standard Amount: We typically use a piece of tissue about the size of a grain of rice and keep a backup tube with similar material in case the first extraction fails.
- Tiny Samples: Especially for precious or historic specimens (e.g., holotypes), the tissue available may be extremely limited. We are currently validating a rapid Extract-n-Amp/X-Amp approach with bead-beating and minimal tissue, which can be followed by Promega Wizard if the first attempt fails.
- Future Work: We are finishing a project on 6,000 University of Michigan herbarium specimens and plan to sequence ~3,000 holotypes in 2025. A larger 15,000-specimen project is also in the pipeline, so we anticipate continued improvements in protocols for older specimens.
6. Next Steps
Please feel free to reach out if you have additional questions or if you’d like to schedule a Zoom call. We’re happy to discuss any of these methods in more detail and share updates on our evolving protocols.
Thank you again for your interest. We look forward to any future collaboration and hope our experiences can help guide your work with historic fungal collections.
Best regards,
Steve Russell
Original email:
Hello and thanks for writing,
We do have extensive experience working with older specimens. However, most of our specimens have a median age of 80 years, not 100-200 years old. A couple quick notes/qualifiers. While age does matter, it is not the primary concern when getting successful DNA. From our perspective, how they were initially dried and how they were stored matters much more than age. In particular, specimens that were dried quickly and thoroughly after harvest will have the best chance of success. Next to that, as long as they were stored in a dry environment, they should work well.
Next consideration is DNA extraction. We have tried many of the standard kits for DNA extraction. They all work roughly the same. None of the kits from Promega, Zymo, Qiagen, Omega, etc. work fundamentally better than the other in terms of total DNA. Some of the kits may produce cleaner DNA than the others, but overall that is not a driving concern for us when working with small amounts of tissue. What does seem to be the case is that successful DNA extraction from any kit is hit-or-miss on really old specimens. It is possible to run the process for a small piece of tissue and have it not work, then to try it again from a different part of the same specimen and have it work. So the sampling from the actual tissue can play a role. Wherever possible, take multiple tissue samples from multiple sites of the mushroom, that way there is backup tissue that can have a chance of success if there is an initial failure.
Next, the sequencing technology will play a fundamental role in success rates. Many older specimens will have contaminants either originally from the time of collection or from the storage process itself. Many older specimens were not dried soon after harvest nor dried quickly (ex - sun drying). Both of these aspects increase the potential for yeasts or other fungi to contaminate the collections. Secondarily, fungi such as Aspergillus halophilicus are very common contaminants on collections that occur after decades in the dry museum environment. As a large percentage of these specimens have some form of contamination - either at the time of collection or during museum storage - Sanger sequencing will never be a good option for systematically sequencing old specimens. Next to that, Illumina or nanopore would be the next best option. We prefer nanopore for many reasons, among them the longer read length, single PCR library prep, and cost. Illumina could surely work, but with many limitations that are not present with nanopore.
Overall we have selected the following Promega Wizard protocol for working with older specimens. It has the right balance for our needs of success rates vs. cost vs. ease of protocol. The ability to scale the process is very important to our lab. Our standard protocol is available here: https://www.protocols.io/view/modified-promega-wizard-extraction-for-barcoding-m-rm7vzb3p4vx1/v3.
The next consideration is amplification. Much like DNA extraction for old specimens, PCR itself is fickle/temperamental. It can fail the first time you attempt it, and then work the second time with exactly the same template, PCR cocktail, and thermocycler parameters. Secondarily, with our extraction protocol, diluting the template often washes out PCR inhibitors. Inversely, diluting the template can sometimes dilute out low quantities of DNA from not working during PCR at all. So for older herbarium specimens we take what we would call a "brute force" approach at PCR. For every specimen, we perform four PCR reactions before we look at the results. Two attempts with full length ITS and two attempts with different primers for ITS2. Within each of these, one attempt is at standard template dilution and the other attempt is with a 1:50 dilution of the template. So the permutations are full ITS no dilution, full ITS diluted, ITS2 no dilution, and ITS2 diluted. We perform no DNA quantifications on individual specimens nor run gels for any individual specimens before sequencing. PCR success is validated solely by whether the specimen was successfully sequenced.
For sequencing, we do 960 specimens at a time on a nanopore Flongle cell x these four permutations. So for full length ITS no dilution, we do the 960 specimens on a single Flongle. For the full length ITS diluted we do the same 960 samples on a second Flongle cell. So in total for each 960 specimens, we build four libraries and sequence them on four independent cells. It would surely be possible to combine all of these permutations onto a single Flongle cell if there are less specimens to work with.
Doing these four PCR reactions for each specimens has many advantages. The first is that you get all of the data for the first attempt, right up front, without the need to resort/reorder specimens and spreadsheets for a second PCR attempt. Secondarily, and perhaps most importantly, this process typically yields more than one sequence for the same specimen. This helps to self-validate the results you are getting, as the same sequence should result from different, independent PCR reactions, libraries, and primer combinations. Having this replicated data right of front significantly helps confidence of results during analysis.
The final consideration is replicate material. All of the protocols above have an assumption of starting material about the size of a grain of rice. When we are initially sampling at the herbarium, we take a replicate tube of tissue for each specimen (where possible). We put a piece of tissue the size of a grain of rice in the primary tube for the initial extraction, then we put additional tissue into a backup secondary tube in case the first extraction attempt fails. The amount of tissue that goes into the secondary tube varies depending on the size of the specimen in the box and the requirements of the herbarium.
Our current area of protocol development involves validating a protocol for older older specimens with far less tissue. Tissue the size of a grain of rice is too large for some older specimens, especially with a replicate, and herbarium managers typically want to release as little tissue as possible, especially for holotypes. Our current methodological developments involve the following. A basic Extract-n-Amp/X-Amp extraction can work well for very old specimens. We have successfully sequenced the Crepidotus cinnabarinus type from the 1890's this way with full-length ITS. The process we are currently validating for tiny tissue would be 1.) Tissue in PCR tube with X-amp and 1mm bead. 2.) Bead-beat & heat. 3.) Use this as the template. 4.) For anything that does not work, transfer this material to a standard Promega Wizard extraction above. 5) If no success with that tissue then move to replicate tissue. The X-amp extraction can work fine for older specimens with tiny tissue amounts (with tissue disruption - beads, grinding, etc). It is also much more scalable than a kit based extraction, especially for a first PCR attempt round. We are currently still working to validate this protocol.
Aside from this, I would be happy to answer any additional questions you may have or meet for a Zoom discussion. We are currently finishing up 6,000 specimens at the University of Michigan herbarium. We hope to sequence all of the holotype specimens there in 2025 (~3,000 specimens). We also hope to start work on another large herbarium project of 15,000 specimens in 2025. So we will continue protocol development within this realm long into the future.
Best,
Steve Russell
Mycota Lab