Abstract
The worrying rise of antibiotic resistance in pathogenic bacteria is leading to a renewed interest in bacteriophages as a treatment option. Novel sequencing technologies enable description of an increasing number of phage genomes, a critical piece of information to understand their life cycle, phage-host interactions, and evolution. In this work, we demonstrate how it is possible to recover more information from sequencing data than just the phage genome. We developed a theoretical and statistical framework to determine DNA termini and phage packaging mechanisms using NGS data. Our method relies on the detection of biases in the number of reads, which are observable at natural DNA termini compared with the rest of the phage genome. We implemented our method with the creation of the software PhageTerm and validated it using a set of phages with well-established packaging mechanisms representative of the termini diversity, i.e. 5′cos (Lambda), 3′cos (HK97), pac (P1), headful without a pac site (T4), DTR (T7) and host fragment (Mu). In addition, we determined the termini of nine Clostridium difficile phages and six phages whose sequences were retrieved from the Sequence Read Archive. PhageTerm is freely available (https://sourceforge.net/projects/phageterm), as a Galaxy ToolShed and on a Galaxy-based server (https://galaxy.pasteur.fr).
Highlights
Phages, the viruses of Bacteria, come in a diversity of shapes and sizes[1]
One can distinguish four main mechanisms used by phages to initiate and terminate packaging while recognizing their own DNA rather than the host DNA: (i) The terminase can recognize a specific site where it introduces a staggered cut, generating fixed DNA termini with cohesive ends that can either have 5’ or 3’ overhangs. (ii) A fixed position can be recognized on the phage DNA where direct terminal repeats (DTR) will be generated by extension synthesis at the 3’ ends of staggered nicks
In this study we investigate how the information gathered by high throughput sequencing technologies, and in particular Illumina technologies, can be used to determine the DNA termini and packaging mechanisms of double stranded DNA (dsDNA) phages
Summary
The viruses of Bacteria, come in a diversity of shapes and sizes[1] They all produce viron particles consisting of a protein shell, with in some cases the addition of a membrane. (ii) A fixed position can be recognized on the phage DNA where direct terminal repeats (DTR) will be generated by extension synthesis at the 3’ ends of staggered nicks The size of these DTRs can range from just over a hundred bases (e.g. T36, T77) to several thousand bases (e.g. T58). (iii) The terminase can initiate packaging on the phage concatemer at a specific packaging site (pac site), but following cuts are made when the phage head is full at variable positions (e.g. P110, P2211) This leads to capsids containing circularly permutated genomes with redundant ends used to circularize the phage genome through recombination after injection in the host cell. These phages will usually degrade the host DNA, ensuring that only their own DNA is packaged
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