Cathy and Andrea recently participated in the MASS program through Scripps Academy (http://www.scrippscollege.edu/academy/math-and-science-scholars). This program is designed to connect high school girls with faculty at the Claremont Colleges. Faculty work closely with students on a research project, with a final culmination in a research presentation.
Students travelled from high schools in Los Angeles to Claremont on four Saturdays during the fall. They were trained in DNA extraction, PCR amplification, Gel Electrophoresis, DNA alignment and Phylogenetic Analysis. The students also learned a bit about deep-sea corals! It was a fun experience, and we look forward to participating in the program again next year~
Two of our undergraduate summer researchers presented their research at the annual Summer Research Poster Celebration at Harvey Mudd College this month. Thanks again to Aaron Friend (senior), who spent the summer at the AMNH, and MIke Adams (junior) who spent the summer at HMC working on species delimitation of corals.
We have successfully designed 16,449 probes to target 1,795 loci across the anthozoa. We used several existing genomic and transcriptomic resources as well as our newly assembled Renilla muelleri genome (hybrid assembly). Brant Faircloth's amazing package Phyluce was used primarily to design these probes. Probes are being synthesized by MycroArray and we plan to test them on a subset of taxa before finalizing the probe set.. We will make the probe sequences publicly available.
And, we thank all of our collaborators who have provided essential data for use in probe design.
My name is Craig Dawes and I am an NSF-REU Scholar at the American Museum of Natural History, in New York City, working under the supervision of Dr. Estefania Rodriguez (Associate Curator of Marine Invertebrates) and Mercer R. Brugler (Assistant Professor at NYC College of Technology [CUNY]). I am also a full-time student in the Biomedical Informatics program at NYC College of Technology in Brooklyn, NY. I have been working in Dr. Brugler’s deep-sea molecular lab since January 2015, initially as a part of the Emerging Scholars program and then as an LSAMP (Louis Stokes Alliance for Minority Participation) Scholar. Based on my experience in the lab, Dr. Brugler recently placed me in a mentoring role; i.e., I am teaching new students how to extract and quantify DNA, set up PCR, visualize PCR on an agarose gel, set up a cycle sequencing reaction, and obtain DNA sequence data using a traditional ABI-3730xL Sanger sequencer. I am originally from Jamaica and moved to NYC about six years ago to pursue a degree in Nursing. After taking a Biology course with Dr. Brugler I was inspired to explore research as a career option.
My NSF-REU summer internship includes three projects:
1. I participated in a NOAA-funded ocean-going research expedition during Summer 2015 to the Flower Garden Banks National Marine Sanctuary in the Gulf of Mexico to collect mesophotic black corals. Mesophotic corals are defined as those organisms living in the middle of the photic zone, i.e. areas of low light penetration. We collected a total of 25 black corals representing three families and six genera across a depth range of 64 - 157 meters. Using three mitochondrial intergenic regions and three nuclear genes, I am obtaining a molecular barcode for these corals, in an effort to elucidate any undescribed species and/or extend the range of known species. We also surveyed banks within the sanctuary for Acanthopathes thyoides and Elatopathes abietina.
2. Based on morphology, Acanthopathes and Elatopathes are currently classified in the same family; however, they do not group together in a molecular phylogeny. These species are considered ‘wandering taxa’ as they change position depending on the gene (mitochondrial v. nuclear) or algorithm (Parsimony v. Likelihood v. Bayesian) used to build the phylogeny. We successfully collected two A. cf. thyoides and six E. cf. abietina. Elucidating 1) intraspecific variability within A. thyoides and E. abietina or 2) closely related cryptic species could potentially stabilize their phylogenetic position.
3. We recently obtained tissue samples from ten black corals that were collected during the 2015 Hohonu Moana Expedition (aboard the NOAA ship Okeanos Explorer) that explored deep waters surrounding the Hawaiian Archipelago. Dr. Dennis Opresko (Smithsonian NMNH), the world’s foremost expert on black coral taxonomy and systematics, noted that several individuals might be new to science based on a rough morphological examination. Thus, I am also barcoding these samples using mitochondrial and nuclear DNA in hopes of elucidating potentially new species.
Other projects - Molecular characterization of Deep-Sea “Sea Anemones” from the Arctic Ocean
We also obtained three specimens from Beaufort Sea, outlying the Arctic Ocean, at a depth of 1000m. Two of these specimens were tentatively identified as Kadosactis rosea, Allantactis parasitica and an unknown species presumed to be a member of the order Actiniaria. We amplified three mitochondrial, genetic markers to confirm the morphological identification of the first two specimens and reveal the identity of the unknown specimen. Our DNA analysis of the unknown suggests that we may have found a representative of a new genus. Currently I am analyzing the morphology of the animal via histological and microscopic examination. Future work will place these three specimens in a phylogenetic context.
For the past several weeks we have been busy preparing deep-sea octocorals for multi-locus DNA Barcoding from our recent RV Celtic Explorer voyage to Whittard Canyon. Initial identification of samples, based on morphology, indicates that the octocorals contributing to the diversity within Whittard Canyon are: Isididae, Plexauridae, Primnoidae, Acanthogorgiidae, Alcyoniidae, Chrysogorgiidae, Paragorgiidae, Clavulariidae, and Pennatulacea. So far, we have extracted high quality DNA from each sample and have amplified both the mtMutS and COI markers. We have recently begun amplifying a third marker, nuclear 28s rDNA. Additionally, we are amplifying the IGR4 region of the Isididae samples in order to further delineate taxonomic relationships. Once amplified, our samples will be sent off for sequencing; these sequences will then be edited and aligned in order to construct preliminary phylogenetic trees. Our ultimate goal with these markers is to better understand the species richness of Octocorallia in Whittard Canyon. Furthermore, we will be calculating genetic distances for between our samples and compare these to previously studied specimens from around the world in order to aid in our species identification and estimates of species richness. We hope to have our analyses completed in the coming few weeks!
Thomas Byrne, Pomona College '18
Over the past three weeks we have been working hard in the McFadden lab to prepare 47 Paramuricea samples for Restriction Site Associated DNA Sequencing (RADSeq). These samples come from Canada, the Gulf of Mexico, and Whittard Canyon off the coast of Ireland. Ultimately we wish to compare the phylogenetic tree we build from the RADSeq data to a tree built using genes that have been conventionally used for octocoral systematics to understand species boundaries in this genus. In order to do this we have successfully extracted high quality DNA from Paramuricea samples collected during the RV Celtic Explorer cruise that took place last month. We have also been working to amplify mtMutS and COI, and n28S from all Paramuricea samples collected off Ireland; those from the Gulf of Mexico and Canada are completed (see Doughty et al. 2014). Because the DNA from some of the older samples collected off Canada and in the Gulf are degraded, we will be re-extracting the DNA from original tissue samples over the next week or so. Plus, we just obtained tissue samples from Paramuricea in the Mediterranean. We hope to submit samples for RADSeq by the end of the month. We will update you on the results!
Mike Adams (Harvey Mudd '18)
Excited to say that we have draft genome assemblies for nine anthozoans! I used bbmerge and bbduk (https://sourceforge.net/projects/bbmap/) to clean and trim sequences. DiscovarDenovo (https://www.broadinstitute.org/software/discovar/blog/) was used for the draft assemblies. Computation time on nine species was ~1 week (on a 512 GB RAM 64 processor). That is all!
We are currently comparing the draft assemblies to other assemblers (SOAPdenovo, SPAdES) and I am also blasting the contigs (and also the trimmed reads) for environmental contaminants. This latter step is the tricky part. If you are reading this and have any advice, please send it my way.
Finally, we have sequenced Renilla on PacBio as well. I will be coupling these data with the Illumina data to generate a hyrbid assembly. Exciting! Stay tuned for more~
Happy Summer Solstice! Its been a great start to summer 2016. I was fortunate to spend 3 weeks off Ireland surveying deep-sea coral communities in Whittard Canyon. Check out http://scientistsatsea.blogspot.com/ for more information about the cruise (led by Louise Allcock at NUIG).. Beautiful weather and a lot of hard work by many (including several students) equalled a very successful cruise!
Stay tuned for more blogs this summer! For now, a beautiful sunrise~
Recently, Co-PI Estefania Rodriguez shared her knowledge of anemones in the SciCafe lecture series at the American Museum of Natural History. If you have a moment and want to learn more about the anemones-check out the video below!
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This past month we have been working diligently to obtain high quality, high molecular weight (HMW) DNA for genome sequencing. We have made substantial progress and have currently submitted four anthozoans for sequencing on an Illumina HiSeq2500. We have chosen a sea pansy (Renilla sp.) that is also part of the aquarium trade, a possible new species of bamboo coral (Keratoisidinae) collected in Whittard Canyon, a corallimorpharian (Corallimorphus profundus), and a colonial anemone or zoanthid (Mesozoanthus fossii). We are sending additional octocorals this week, including Alcyonium digitatum, Parasphaerasclera valdiviae, and Cornularia pabloi.
We will also be sequencing two of these species (Renilla and a Ceriantharia) and an antipatharian black coral on the PacBio platform, and are currently amassing 10 ug of DNA per sample for this sequencing effort. PacBio is useful for obtaining long reads (10 kb average read lengths), but can be prone to high error rates. Combining both PacBio and Illumina reads will give us high coverage and long reads, and thus high quality assemblies. We will then use these two reference genomes to aid in assemblies of other species.
Our DNA extractions have been most successful when we have used a few mg of recently preserved (95-100% EtOH) or frozen material; however, we have also obtained high quality (260/280 ratios 1.8-2.0, 260/230 ratios ~2.0) and HMW DNA from samples that were frozen and stored in liquid nitrogen for 20 years as well as samples that have been preserved in 95% EtOH for ~8 years. Mainly, we have extracted DNA using a CTAB protocol or a Gentra Puregene protocol (Qiagen) with a few modifications. Firstly, we are not macerating or homogenizing the tissue before submerging it into the CTAB or cell lysis solution. Secondly, we are adding 5ul of ProK twice—at the start of lysis and then again after 4 hours; samples then sit overnight. We then add RNase (1.5 or 6.0 ul) and incubate this for 30 min to an hour at 37degC before finishing the rest of the extraction protocol. Our protocol has worked well, resulting (for the most part) in little degraded DNA.
I wanted to mention that additional genomic resources have become recently available. For cnidarians, four myxozoan genomes are now available (Chang et al. 2015): Polypodium hydriforme, Enteromyxum leei, Sphaeromyxa zaharoni, and Kuda iwatai. All of these will be useful as outgroups to Anthozoa. Also, the dinoflagellate Symbiodinium genome is now available (Lin et al. 2015). This genome not only is critical for studying the evolution of coral-algal symbioses, it will be extremely useful in transcriptomic and genomic pipelines by enabling coral sequences to be separated from those of symbionts.
UCE Project Team
All things Anthozoa, Evolution and Ecology
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