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.
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UCE Project Team
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