Two mycobacteriophage genomes were newly sequenced and annotated. Duggie and Hocus were discovered, enriched, and isolated from soil using Mycobacterium smegmatis mc2155. The bacteriophages are lytic Siphoviridae and belong to the B1 subcluster. The Hocus and Duggie genomes are highly similar to one another in both nucleotide sequence and gene content.
ABSTRACT
Two mycobacteriophage genomes were newly sequenced and annotated. Duggie and Hocus were discovered, enriched, and isolated from soil using Mycobacterium smegmatis mc2155. The bacteriophages are lytic Siphoviridae and belong to the B1 subcluster. The Hocus and Duggie genomes are highly similar to one another in both nucleotide sequence and gene content.
ANNOUNCEMENT
Bacteriophage genomes were among the first complete genomes sequenced due to their relatively small size and simplicity (1). Studying bacteriophage genomes helps to identify bacteriophages with therapeutic potential, especially in the treatment of antibiotic-resistant bacterial infections (2, 3). In this study, we annotated the genomes of the mycobacteriophages Duggie and Hocus. Duggie and Hocus were found at Doane University in Crete, Nebraska. Soil samples were collected from campus, phages were isolated by enrichment culture and purified by repeated serial dilution, and samples were amplified by generating a lysate using Mycobacterium smegmatis mc2155. The host bacteria were grown in 7H9 broth and incubated at 37°C with agitation. Transmission electron microscopy (TEM) imaging revealed that the phages are siphoviruses. Sequencing confirmed that the phages are lytic and belong in the B1 subcluster.
Bacteriophage genomic DNA was isolated using the Wizard DNA extraction kit (Promega) and sequenced at The Pittsburgh Bacteriophage Institute. Sequencing libraries were prepared from genomic DNA using a New England BioLabs (NEB) Ultra II kit with dual-indexed barcoding. Libraries were pooled and run on an Illumina MiSeq instrument, yielding at least 100,000 single-end 150-base pair reads and a 1,057-fold coverage (504,224 raw reads) for Hocus and 1,353-fold coverage (657,250 raw reads) for Duggie. No further quality control or adapter trimming was performed on the reads provided by the sequencer. These raw reads were assembled using Newbler v.2.9 (default settings). Each genome yielded a single contig, which was checked for completeness, accuracy, and phage genomic termini using Consed v.29 as previously described (4).
Autoannotation in DNA Master v.5.23.3 (http://cobamide2.bio.pitt.edu) running GeneMark v.2.8 (5) and Glimmer v.3.02 (6) predicted gene locations. Gene start sites were identified using Starterator (https://seaphages.org/software/), ribosomal binding site (RBS) scores within DNA Master, and BLASTp comparisons to similar bacteriophage genes. Gene functions were assigned using Phamerator (7), HHpred (8), PECAAN (9), and NCBI BLAST (10). ARAGORN v.1.1 (11) was used to confirm the absence of tRNA genes, which are not commonly found in subcluster B1 phages. Characteristics of the two genomes are shown in Table 1. All tools were run using default parameters, unless otherwise stated.
TABLE 1.
Characteristics of two bacteriophage genomes
| Phage name | GenBank accession no. | SRA accession no. | GC content (%) | Sequence length (bp) | No. of genes | No. of genes with assigned functions | Closest relative | Identity with closest relative (%) |
|---|---|---|---|---|---|---|---|---|
| Duggie | MN585965 | SRX7344740 | 66.4 | 68,885 | 102 | 31 | JakeO | 99.59 |
| Hocus | MN369738 | SRX7344741 | 66.4 | 68,083 | 100 | 27 | Mutante | 98.47 |
The Duggie and Hocus genomes display nucleotide similarity to each other and to other subcluster B1 phages, as determined by global nucleotide BLAST. Specifically, the genomes of Duggie and Hocus are 98.95% identical to one another. The genome of another B1 phage, Kloppinator, is 95% identical to both the Hocus and Duggie genomes. Notably, Duggie and Hocus differ from Kloppinator in a region between bp 55976 and 57353, where the Kloppinator genome contains a different gene, Kloppinator_71, that is not present in Duggie or Hocus. The Hocus genome includes two genes not found in Duggie or Kloppinator; Hocus_67 is an orpham with no known function, and Hocus_90 belongs to a pham of only six members, all of which are cluster B phages (12).
Data availability.
Genome sequences and raw sequence data are available in GenBank and the Sequence Read Archive. Accession numbers are shown in Table 1.
ACKNOWLEDGMENTS
We acknowledge the Pittsburgh Bacteriophage Institute for sequencing the Hocus and Duggie genomes and the Virginia Commonwealth University Nucleic Acids Research Facilities for sequencing Kloppinator. Additionally, we acknowledge the University of Nebraska—Lincoln Microscopy Core for taking TEM images of Hocus and Duggie. We thank Salvador Delgadillo, Trevin Alberts, Ayden Benavides, Brenna Mulvey, Nick Crespo, and Serenity Kinswoman for assistance with annotation of the three genomes. We also thank Kylie Crnkovich, Makenna Weddle, and Anna Korte for the discovery of Duggie and Hocus and assistance with annotation. Finally, we thank Uriel Sanchez and Madison Shoemaker for discovering Kloppinator and Duggie, respectively.
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Associated Data
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Data Availability Statement
Genome sequences and raw sequence data are available in GenBank and the Sequence Read Archive. Accession numbers are shown in Table 1.