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.
Felipe Zapata (postdoctoral researcher in the Dunn Lab) and colleagues used a transcriptomic approach to address uncertainties in evolutionary relationships within the Phylum Cnidaria. Their work was recently (Oct 14, 2015) published in PlosOne. This paper sets a milestone towards furthering our understanding of the evolution of a key group of basal metazoans with a broad range of life history traits, morphological characters, and ecological niches.
Resolving evolutionary relationships among cnidarians (i.e., jellyfish, corals, anemones, hydroids) has proved challenging in part due to the ancient divergence (~500 million years) of the clade and the lack of single copy nuclear markers available for robust species tree analyses. Although the Medusozoa and Anthozoa have consistently been recovered as two monophyletic clades, alternative hypotheses have been suggested for the major lineages within each group. For example, long-standing views have recognized octocorals (sea fans, soft corals, sea pens) and hexacorals (stony corals, anemones) as sister taxa in the Anthozoa. This relationship was based on morphological characters, life cycle traits, and primarily on nuclear rDNA data. Recent studies, however, using mitochondrial genomes suggested that Anthozoa is paraphyletic, with octocorals sister to the medusozoans (Park et al. 2012; Kayal et al. 2013). Furthermore, Stampar et al. (2014) recovered the Order Ceriantharia (tube anemones) as sister to all remaining anthozoans, and proposed Ceriantharia as a new sub-class. With additional inconsistencies within the Medusozoa (Collins et al. 2006, Marques and Collins 2004), it has become clear that multiple, single-copy nuclear markers are sorely needed to resolve evolutionary relationships among cnidarians.
Zapata and co-authors set out to address these uncertainties by using a phylogenomic approach to resolve relationships among major cnidarian lineages. They first sequenced transcriptomes of 15 new cnidarians, adding considerably to the existing genomic resources of cnidarians. Let me stress that again--Fifteen transcriptomes!! They then combined these newly sequenced data with existing transcriptomes for a dataset that included 31 cnidarians and 7 outgroups. [Three cnidarians had to be excluded from analysis due to poor (<5%) gene occupancy]. In total, they recovered 1,262 orthologous genes (365,159 aa), and generated a matrix with 38 taxa and 54% gene occupancy. Some people may argue that this is overall poor gene occupancy, but the majority of the nodes are in fact well supported. From my experience in working with a group (the Octocorallia) that has used only mitochondrial data and nuclear rDNA to construct phylogenies, I would say this work represents outstanding progress in Cnidaria Evolution.
And, so what did Zapata et al. find? Their results strongly support several traditional views of Cnidaria, including monophyletic Anthozoa (octocorals and hexacorals) and Medusozoa clades. Also, these results further support the Marques and Collins (2004) cladistics work on Medusozoa, based on life history traits and morphology. Interestingly, this study also demonstrated the instability of the enigmatic Ceriantharia. This may be due to the low number of genes recovered for this group (16.6%) compared with others. It is clear that more data and more taxa are needed to resolve the relationship of Ceriantharia to other anthozoans.
Zapata et al. (2015) has provided the scientific community with a wealth of new data to build upon and tools to use to further explore evolutionary hypotheses within the Cnidaria. I have to say these are exciting times for those working on the phylogenetics of any cnidarian group!
Collins et al. 2005 http://dx.doi.org/10.1080/10635150500433615
Kayal et al. 2013. http://www.biomedcentral.com/1471-2148/13
Marques and Collins 2004. http://onlinelibrary.wiley.com/doi/10.1111/j.1744-7410.2004.tb00139.x/abstract
Park et al. 2012. http://www.sciencedirect.com/science/article/pii/S1055790311004374
Stampar et al. 2014 http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0086612
Have you heard of Iplant Collaborative? If not, you should click here and check out the project and the team asap. Iplant is an innovative platform that aims to connect scientists to applications, tools, datasets, storage, and an atmosphere (aka-a ‘cloud’). In fact, their tagline is, “it is where scientists in all domains of life sciences can connect to public datasets, manage and store their own data and experiments, access high-performance computing, and share results with colleagues.” Most importantly-it is open-source. In other words, it is free for academic scientists to use –of course with appropriate credits.
I attended a workshop at UC Davis last week to find out more about Iplant and what it has to offer in genomic resources. Prior to this workshop, I did not know too much about the program. In fact, I only knew that we were going to store our datasets from our Anthozoan UCE project on their servers. In all honesty, and most likely because of the name, I thought it was a program focused on sequencing genomes of plants, and providing resources to botanists. Of course, I could not have been more wrong. Although I should mention that there is a genomic bias to Iplant, you do not necessarily have to work with genomic data to utilize Iplant Collaborative.
There are many benefits to signing up for an Iplant account. Firstly, you can upload and store large datasets. This system provides a pretty nice backup (100 GB or more if requested) for any data that you may store locally (i.e., on your own laptop or desktop). Secondly, you can invite your colleagues to sign up for their own accounts. And guess what? You can share your data with your colleagues and vice versa, or with the broader scientific community. Thirdly, you can explore and use applications that have been added to the website by the team. Have you ever been frustrated because you cannot correctly compile a program on your computer? Is BLAST too slow on your laptop? Is RAxML too slow on your local computer? Do you have multiple copies of the same program in 10 places on your computer? Iplant likely has your application of interest and you can run it on their server, in what is known as the Discovery Environment (DE). And if they do not have a particular application of your interest, you can contact them to add it. This means if you want to use a certain program, such as RAxML, it may be much easier to do so on their DE. Also, they have decent processors (16CPU, 128 GB RAM) and you can even make your own workflows that link multiple programs (i.e., MAFFT to RAxML). This means that you can run a workflow in one click. Also, it seems like a great teaching tool for both undergraduate and graduate courses.
Iplant is pretty outstanding. Here is some advice from me that I gathered from this past week’s workshop: If you work with genomic data or in fact ANY large datasets, you should create an account, log in, and explore the applications. If you are ever frustrated about sharing large files with your colleagues, you should create an account, log in, store your data, invite your colleagues, and share those data with them. If you are intimidated by using bash scripts, python, R, or even the command line in general-you should create an Iplant account and check out what they have to offer. But in the end, I personally think that while familiarizing yourself with Iplant and their resources, you may want to take a course and start learning how to use the command line. (Check out datacarpentry.org). In fact, learn how to program in any language (codecademy has great resources for beginners).
The workshop last week was very useful. I am thankful to know that if I hit a roadblock, I can jump on Iplant to see if their resources can improve my workflow. Additionally, I will definitely use it to store data and for teaching purposes in the future. Iplant Collaborative is a great resource, run by a great team, and they are sharing it with others…for free.
We are excited to announce our new project that will inform our understanding of the evolution of one of the most important groups of metazoans on earth: the anthozoan cnidarians! Stay tuned for updates and blog posts.
UCE Project Team
All things Anthozoa, Evolution and Ecology
Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation