Exploitation of phage Carin-5’s own DNA polymerase to sequence its T-hypermodified genome

The bacteriophage Carin-5, which infects Cobetia marina, presented significant challenges for genome sequencing due to DNA modifications that hindered efficient amplification of its DNA. Initial attempts using an optimized Illumina library preparation protocol yielded only a partial genome. Among the obtained contigs, the gene encoding the replicative DNA polymerase was identified. This gene was subsequently cloned into an expression vector and the encoded DNA polymerase protein was purified. The purified DNA polymerase was used to replicate the Carin-5 genomic DNA in vitro, effectively bypassing the modification-induced amplification barriers. This approach enabled the amplification of the entire Carin-5 genomic DNA and the use of a standard NGS library preparation protocol and allowed to obtain the complete sequence of the Carin-5 genome. The DNA modifications were characterized by mass spectrometry, and they showed a clear analogy with the oligosaccharide-hypermodified thymidines of bacteriophage SP-15. The annotated genome of Carin-5 carried a gene block encoding enzymes for nucleotide sugar modifications and glycosyltransferases.