Phage Annotation Guide: Guidelines for Assembly and High-Quality Annotation

Abstract

All sequencing projects of bacteriophages (phages) should seek to report an accurate and comprehensive annotation of their genomes. This article defines 14 questions for those new to phage genomics that should be addressed before submitting a genome sequence to the International Nucleotide Sequence Database Collaboration or writing a publication.

Keywords: annotation guide; bacteriophages; genome annotation; genomics; phage taxonomy; phages.

FIG. 1.

A recommended workflow for the annotation of structural, functional, and other features in assembled bacteriophage genome sequences. Examples of recommended tools for processes are detailed in yellow, but they do not represent an exhaustive list. CDD, Conserved Domains Database; HMMs, Hidden Markov Models; ncRNAs, noncoding RNAs; ORFs, open reading frames; PHROGs, Prokaryotic virus Remote HOmologous Groups; tRNAs, transfer RNAs.

FIG. 2.

Genome CDS comparison between Pseudomonas phages phiKMV (AJ505558) and vB_PaeP_ASP23, short ASP23 (MN602045) using clinker. Homologous CDSs are in the same color and linked through gray bars with the percentage amino acid identity, as indicated in the legend. (A) Direct comparison and visualization of GenBank records (accessed October 25, 2021). (B) Manual rearrangement of the ASP23 genome in TextEdit on Mac and reverse complementation with the Sequence Manipulation Suite, followed by reannotation with Prokka. CDS, coding sequence.

FIG. 3.

An example of an embedded gene—Rz1 within R of phage lambda, adapted from Rajaure et al.

FIG. 4.

(A) BLASTX analysis of the Lactobacillus phage LL-H large subunit terminase gene (NC_009554.1, 1647..3785) in which it appears that this region contains three ORFs, two of which on translation show homology to TerL proteins and one similarity to a homing endonuclease. (B) BLASTX analysis of Bacillus phage CampHawk (NC_022761.1, 118288..121224), which encodes a UvrD-like helicase with an intein. A nearly identical sequence is found in Bacillus phage vB_BsuM-Goe2, but not in Bacillus phage SP8. The CampHawk helicase contains 978 amino acids, whereas its homolog in SP8 possesses only 703 residues.

FIG. 5.

An example of an over-annotated genome. (A) Bacteriophage Felix 01 GenBank record (AF320576), graphical view of the region 3500..6100. (B) RefSeq curated record for Enterobacteria phage Felix 01 (NC_005282.1) graphical view of the region 3500..6100. Annotated coding sequences are depicted as red rectangles, with arrows denoting presence on the forward or reverse strand.

FIG. 6.

Sequence logo of a statistically over-represented motif (Shine–Dalgarno sequence or ribosome-binding site) identified by using MEME from 103 bp sequences encompassing the predicted start codon and upstream region.