To Split or Not to Split: Division of Mycobacteriophage Subcluster A3 Brittany Grandaw, Daphne Hussey, Warren Taylor Abstract The purpose of this experiment was to determine whether or not sufficient proof is available to determine whether or not the A3 subcluster should be divided into separate subclusters. In order to test this hypothesis, average nucleotide identity, ANI, was used to compare the genomes of every member of the A3 subcluster for similarity. Additionally, a Case It analysis of the Pham 266 gene (present only in A3 phages) and the protein product was used to further compare the members of the A3 subcluster for similarity and difference. The results proved that the identified two A3 subcluster groups exist and they share a marked similarity with members within their group and not with members of the other group. These results supported our hypothesis and it is the opinion of the group, based on these results, that there is sufficient proof available to determine that the A3 subcluster should be divided into separate subclusters. Introduction The assignment of clusters is dependent on many factors, looking especially for similarity between genomes of different phages to determine if, in fact, they belong to the same cluster. As time goes on, phages may be reassigned a cluster or subcluster based on their similarity to other phages. In the case of the A3 subcluster, based on the results of the Phamerator map of every single A3 phage, there does appear to be a definite split, where a new subcluster could be created. It was the hypothesis of the group that the A3 subcluster could be split into two distinct groups and a new Cluster A subcluster be created (1) (2) (3) (4). Data References 1. Lawrence, Dr. Jefferey, and Adam Retchless. DNA Master. Computer software. PhagesDB. Vers N.p., 16 Jan Web. 29 Apr Bergland, Mark, Karen Klyczek, and Chi-Cheng Lin. Case It. Computer software. Vers University of Wisconsin-River Falls, n.d. Web. 25 Apr Hatfull, Graham F. "Mycobacteriophage Database." Mycobacteriophage Database. HHMI, Mar.-Apr Web. 25 Apr Cresawn, Steve. Phamerator. Computer software. PhagesDB. N.p., Web. 25 Apr Acknowledgements Biology Department- UWRF Howard Hughes Medical Institute Science Education Alliance Conclusions/Discussion The results ultimately showed that there is a distinct split between the A3 phages such that two definite groups are distinguishable. This fully supports the groups hypothesis concerning the A3 phages and the fact that they could split into two distinct groups. The tests to prove this hypothesis went smoothly, save that two phages were unavailable for the Case It analysis, but we feel confident, that regardless of this, the results would be the same. The experiments seemed to prove effective at determining differences and similarities in the distinct groups, so no changes appear necessary. In order to make an accurate decision on the split of A3 phages, other comparison methods need to be used, however we feel that, based on our results, the A3 subcluster should be split. Summary of results The results of the Case It analysis indicated that four phages possessing the Pham 266 gene and its protein product are markedly different in terms of composition than the other A3 phages. The ANI results proved similar in that a small group of phages were much more similar to one another than to the other phages tested (Compare the percent of similarity values between the blue and red phages) Methods In order to compare A3 genomes as well as Pham 266 gene and protein sequences, two computer programs were used: the CaseIt program (2) and the DNA Master ANI Comparison tool (1). The first program digested the genome and protein and then compared and grouped the digests based on similarity. DNA Master annotates each genome of the target phages and then gives a percentage amount for how much each genome is similar to every other genome. Table 1. DNA Analysis of Pham 266 of A3 Phages with Case It Table 2. Protein Analysis of Pham 266 of A3 Phages with SplitsTree Table 3. ANI Results for all A3 subcluster phages ( Blue indicates group 1, red indicates group 2)