Fun and Fights with Fungi, Part 2

Derek Jacoby and Vince Geisler

When last we left the stories of our fungal adventures, we had just sent the first two successful PCR-amplified mushroom samples off for sequencing. I was so sure that this was to be a triumphant presentation of dozens of barcoded samples. But we’re still in the midst of it. Today we’ll follow through on the bioinformatics of a successful barcoding sample, but most of our mushrooms are still unknown. We’ve just received the next level in the chemical arsenal to break usable DNA out of the mushroom, but we can report on our attempts so far. Outside of this last effort (which uses decidedly DIY-unfriendly chemicals such as chloroform) we’ve had interesting failures with sonicated samples (evenly sized DNA fragments [!] that are too short for our barcoding), with alkaline cell lysis buffers and detergent-based cell lysis buffers.

But let’s start with a description of the main problem. We have a mushroom sample we found at one of the mushroom shows, we may or may not know what mushroom it comes from, or more interestingly, we may think we know but are wrong. This mushroom has a genetic region that is not under evolutionary pressure, so it randomly accumulates mutations over evolutionary timescales. It turns out that due to this rate of genetic drift, different species of mushroom tend to have different DNA mutations in this region, causing it to be known as a barcoding region. There are a number of steps we have to follow ...

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