Some very preliminary results

Early in the DNA extraction process--differently colored lobsters produce different hues of lobster juice

I've gotten some interest recently in what I've been able to accomplish with everyone's samples over the last nearly a year. I've received 31 donations of dried lobster mushroom or uninfected host samples from super generous people and was able to collect further samples of my own in Oregon. Through a combination of "things always take longer than you think" and activities for my thesis, I've only been able to fully process a few samples. My thesis is actually a project about fungus-farming ants, rather than lobster mushrooms, but the two study systems are united by the presence of a fungal parasite in each that are both in the same taxonomic order (so, fairly closely related, in the grand scheme of fungal diversity)--Hypomyces lactifluorum attacking lobster mushroom hosts, and Escovopsis attacking the fungal crop of ant farmers, both in the order Hypocreales. Over the winter, I spent three weeks in Costa Rica collecting fungus-farming ants and their colonies, and months before I left planning the trip and applying for permits. In March, I had my qualifying exam for my degree program, which consisted of a written proposal for my project and an oral defense. Both of these things took up a lot of my time, but here's what I was doing in the meantime with my lobster samples:

- Using different extraction techniques to try to obtain DNA without compounds from the fruiting body interfering with the extraction process

- Optimizing DNA amplification protocols (PCR) to prepare samples to be for both host and parasite (an unexpected roadblock arose when the DNA sequence I was targeting to try to identify the parasite came back from the host instead--I was sure that the parasite DNA was so much more plentiful that getting the host DNA would be a problem!)

- Sent three samples, including my only white sample, in for whole-genome sequencing (these were successfully sequenced and the genomes assembled)

Right now, I have whole genomes from three samples on our lab's server. These genomes were sequenced with high-throughput technology that indiscriminately "reads" all of the DNA in a sample. This normally means that any contaminants in a sample will also be sequenced, but can usually be filtered out relatively easily, especially if they're distantly related to the target organism. However, as it turns out, my lobster mushroom samples are diverse microbial communities, so what I have essentially sequenced are "metagenomes", a composite of genomic sequences from all sorts of organisms living in the lobster sporocarp. Here is a chart from the program Blobtools, assigning taxa to the sequences found in the metagenome from one of my samples:

The bars represent different taxa assigned by Blobtools by comparing DNA sequences in the metagenome to sequences in a database. As you can see, the highest proportion of reads after "no-hit" reads that were unable to be assigned to a specific organism is Pseudomonas, a very common bacterium. Because these lobster samples were dried and not stored in a nucleic acid preservation buffer, I can't know if these bacteria were growing on the living lobster mushroom sporocarp, or on the sample after it was dried. 

You may have noticed that there are no Hypomyces sequences represented in this figure--but don't worry! Trichoderma is a diverse genus that has been studied for its potential to target fungi that attack crop plants, but it is very closely related to Hypomyces. Hypomyces is understudied, and there aren't any sequences from it in the database Blobtools uses to assign sequences to taxa, so its DNA probably got identified as its close relative Trichoderma.

You might also have noticed that there are no Russula or Lactifluus in the sample. This took some more digging through the genome, during which I used a program that pulls out a specific part of genomes used for identification of fungi. For this particular sample, I was able to recover a region from a host in the family Russulaceae. Because of how complex taxonomy is for this group, I won't be able to do anything very useful with this host DNA until I can build a phylogeny (tree of genetic relationships) with DNA from other lobster mushroom hosts. For my other two lobster metagenomes, I actually didn't recover any host DNA at all. This is frustrating, but I think I may need to pull tissue from the interior of dried samples, where there is likely to be a higher amount of host DNA, and try sequencing it again.

When I'm able to get into the lab more frequently, I will continue preparing and sequencing my lobster samples, with the ultimate goal of building phylogenies of host and parasite and seeing if lineages of each correspond, suggesting adaptation of Hypomyces lactifluorum to specific evolutionary groups of hosts. In the meantime, I am separating out the so-called "Trichoderma" sequences from my metagenomes so I will have complete H. lactifluorum genomes, and I'm excited to see what surprises I'll find in the DNA of this unusual and apparently highly specialized fungal parasite.