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Background   Enterococcus faecalis  and  Enterococcus faecium  are typical enterococcal bacterial pathogens. Antibiotic resistance means that the identification of novel  E. faecalis  and  E. faecium  phages against antibiotic-resistant Enterococcus have an important impact on public health. In this study, the  E. faecalis  phage IME-EF4,  E. faecium  phage IME-EFm1, and both their hosts were antibiotic resistant. To characterize the genome termini of these two phages, a termini analysis theory was developed to provide a wealth of terminal sequence information directly, using only high-throughput sequencing (HTS) read frequency statistics.    Results  The complete genome sequences of phages IME-EF4 and IME-EFm1 were determined, and our termini analysis theory was used to determine the genome termini of these two phages. Results showed 9 bp 3′ protruding cohesive ends in both IME-EF4 and IME-EFm1 genomes by analyzing frequencies of HTS reads. For the positive strands of their genomes, the 9 nt 3′ protruding cohesive ends are 5′-TCATCACCG-3′ (IME-EF4) and 5′-GGGTCAGCG-3′ (IME-EFm1). Further experiments confirmed these results. These experiments included mega-primer polymerase chain reaction sequencing, terminal run-off sequencing, and adaptor ligation followed by run-off sequencing.    Conclusion  Using this termini analysis theory, the termini of two newly isolated antibiotic-resistant  Enterococcus  phages, IME-EF4 and IME-EFm1, were identified as the byproduct of HTS. Molecular biology experiments confirmed the identification. Because it does not require time-consuming wet lab termini analysis experiments, the termini analysis theory is a fast and easy means of identifying phage DNA genome termini using HTS read frequency statistics alone. It may aid understanding of phage DNA packaging.

A novel termini analysis theory using HTS data alone for the identification of Enterococcus phage EF4-like genome termini