Complete Genome Sequences of Mycobacteriophages OKaNui and DroogsArmy Kayla M. Fast, a Kadaisha G. Johnson, a Kaitlyn N. Mayfield, a Leah A. Stephens, a T. Hunter Reid, a Emma D. Ryan, a Tracy W. Keener, a Michael W. Sandel a a Department of Biological and Environmental Sciences, University of West Alabama, Livingston, Alabama, USA ABSTRACT Mycobacteriophages OKaNui and DroogsArmy were isolated from soil using the bacterial host Mycobacterium smegmatis mc 2 155, which belongs to the phylum Actinobacteria. OKaNui was discovered in east Mississippi and DroogsArmy in west Alabama in the United States. The genomes of OKaNui and DroogsArmy were 51,424 bp and 53,254 bp long, respectively. A mong biological agents, bacteriophages (phages) are the most populous and ubiquitous in the environment (1). The phages named OKaNui and DroogsArmy were isolated from the bacterial host Mycobacterium smegmatis mc 2 155. Most Myco- bacteria species are saprophytic and reside in the soil or water or on plants (2). Although M. smegmatis is a nonpathogenic bacterium, an understanding of phage infection in this strain may contribute to advances in phage therapy for other Myco- bacteria species. M. smegmatis may be used as a delivery system for phages intended for infection of M. avium and M. tuberculosis (3). Phages isolated from M. smegmatis have be used in the experimental treatment of closely related hosts, including M. ulcerans (4). OKaNui was discovered in Meridian, Mississippi, in moist soil in a shaded area, and DroogsArmy was found in Lisman, Alabama, in a dark-colored, moist soil (Table 1). The soil samples were inoculated with the host bacterium and incubated at 37°C with shaking in 7H9 liquid medium (5). A filtrate was then plated on a lawn of M. smegmatis mc 2 155 along with a negative control (5). The formation of plaques follow- ing incubation at 37°C were indicative of phage presence. Isolates were purified using serial dilutions and a filtered high-titer lysate (HTL) collected from webbed plates (5). Phage genomic DNA was extracted using the Wizard DNA cleanup system with modified protocols (Promega, Madison, WI) (5). DNA libraries were built and pooled for sequencing using the NEBNext Ultra II FS kit with dual-indexed barcoding (New England BioLabs, Ipswich, MA). Sequencing was performed using the Illumina MiSeq platform at the Pittsburgh Bacteriophage Institute. The genome lengths, GC content, and coverage depths are listed in Table 1. OKaNui yielded 316,000 single-end 150-base reads, and DroogsArmy yielded 410,000 reads. Both genomes displayed defined ends with 10-bp overhangs (CGGCCGGTAA). Assembly was performed using Newbler 2.9 with default settings (6). A single contig for each genome was produced and used to determine the genome ends; the beginning of each genome was chosen based on similar genomes. These were then checked for completeness and accuracy using Consed 2.0 (7). Genome annotation was performed using the Phage Evidence Collection and Annota- tion Network (PECAAN; https://discover.kbrinsgd.org/) and DNA Master 5.23.3 (cobamide2.bio.pitt.edu/computer.htm). We used the following programs to determine gene presence, functions, and start sites: NCBI BLAST, Phamerator (https://phamerator .org/), PhagesDB BLAST, HHPred 3.0, Starterator (https://github.com/SEA-PHAGES/ Citation Fast KM, Johnson KG, Mayfield KN, Stephens LA, Reid TH, Ryan ED, Keener TW, Sandel MW. 2020. Complete genome sequences of mycobacteriophages OKaNui and DroogsArmy. Microbiol Resour Announc 9:e00791-20. https://doi.org/10.1128/MRA .00791-20. Editor Simon Roux, DOE Joint Genome Institute Copyright © 2020 Fast et al. This is an open- access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Kayla M. Fast, kfast@uwa.edu. Received 8 July 2020 Accepted 21 July 2020 Published 13 August 2020 GENOME SEQUENCES crossm Volume 9 Issue 33 e00791-20 mra.asm.org 1 Downloaded from https://journals.asm.org/journal/mra on 03 September 2023 by 54.211.6.52.