 Hello, my name is Jolene Ramsey. I'm a postdoc at the Center for Phage Technology at Texas A&M University. We'd like to acknowledge that we live and work on the unceded lands of the Tunkawa and Sana tribes. The project I'm going to tell you about today was completed by myself, Jason Snowden and Curtis Ross in Ray Young's lab, analyzing phage proteins in galaxy. Phage, or the viruses of bacteria, are incredibly abundant on this earth and are estimated to outnumber their hosts by about 10 to 1. When they infect an individual cell and begin to replicate, they must finally, at the end, escape by bursting or lysing the host cell in what is a dramatic cellular event. This mechanism is carefully orchestrated at the molecular level and allows the phage to specifically disrupt each of the barriers posed by a cell to escape from the inside to the outside. Phages encode specific proteins for each of those barriers. These are the three main classes, the holins, endolysins, and spannins used by tailed phages. We're going to focus today on the spannins, which disrupts the final barrier in a rather unique mechanism that we'd like to understand more biologically, which is where the bioinformatic part of this project comes in. We need more examples to look at. So, spannins, while having a somewhat unique mechanism of action, actually have some relatively generic protein features, including lipid modifications and transmembrane domains. And that's true of the three main types of spannins, the O-spannins, the I-spannins, and the U-spannins. Now, these spannin proteins were looked at in a bioinformatic study by our group several years ago, taking advantage of generic tools that looked at those features of spannin proteins, as well as the Perl script written by Helena Rash. These were used together in a manual process to curate over a thousand spannins that were compiled into a BLAST database now in use in a phage annotation pipeline at the CPT Galaxy. We wanted to improve upon and modify this particular tool kit by combining the tools into a Galaxy workflow and by writing several new custom scripts in Python that were tailored to detect each of the spannin types based on their known features. So, we did this and this is what the current iteration of the workflow looks like. It has 26 steps, takes about 30 minutes or less to complete, and is housed on our Galaxy instance at the Center for Phage Technology. It incorporates the BLAST database of curated spannins as well as the generic tools for looking at spannin protein features and the four Python scripts that are new for each of the different kinds of spannins as well as pairing I and O spannins. If you'd like additional details on how these work, I would encourage you to visit the poster that we have here at the GCC conference for further discussion. We applied this particular workflow to a subset of phages for validation and we chose to use a couple of different groups of phages that infect medically relevant escape pathogens of humans. In particular, we looked at the phages of acinetobacter, which is what the A stands for, and proteus, which falls under these entrobacteriaceae. So we found in the NCBI virus public database, records deposited through January, 105 phages for acinetobacter, and 36 phages for proteus. We got their genomic fast day files and then applied our workflow to them. In the case of the protease group of phages, the majority of them had detectable spannin proteins and spannins of all different types. For a total of 89% having detectable spannins, this is very much in line with what was previously seen in the 1100 set. In contrast for the acinetobacter phage group, we really only were able to find detectable spannins in 7.6% of the group. So the majority of them didn't have detectable spannins. This is something that we find to be interesting biologically and we'll be following up on, and we will continue to apply the workflow to different groups of phages to see how well this holds up, and if this is something that's really unique to acinetobacter phage spannins. All of the spannin annotations that we've made that are new, as well as the previous set, have been migrated to a new home at the debop or database of phage where the public can both browse and download this data for further use. We've also generated a set of training materials available for people to use who are interested in learning about and annotating spannin proteins, and we will continue to add to this material. I would like to acknowledge those who took part in this work, in particular Jason Snowded, who did most of the annotation, and Curtis Ross, who did our programming, all of our sources of funding at the Center for Phage Technology. If you have questions, I would encourage you to visit our poster or to connect with us on Twitter. Thank you for your attention.