To determine physical ends of the phage genome, we first predicted putative termini positions based on the analysis of the organization of similar phages (Bacillus virus BM15) and inspection of read mapping to the assembled genome. Then, we confirmed that these positions are actually ends of the DNA molecule by primer walking (Sanger sequencing) through them. The sequencing procedure was conducted on the 3130xl Genetic Analyzer (Hitachi, Ltd.) in Molecular Biology Techniques Laboratory (AMU, Poznań) using the following primers:
Thurquoise_end_101 5’-GCCTATGGGGAAAGCTACCAATC-3′,
Thurquoise_end_121 5’-ATATAGCAAAAGGATCGGCGGC-3′;
Thurquoise_end_-103 5’-TTACTTGACACCTCCAAGCTGC-3′;
Thurquoise_end_-140 5’-TGTCTAAATTAGGATCAAGATTTCTGTCAAG-3’.
The reaction was set up using BigDye™ Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems™) that utilizes AmpliTaq Polymerases there is known to add additional adenines at the end of the DNA molecule (Samuelson et al., 2004 (link)).
Thurquoise_end_101 5’-GCCTATGGGGAAAGCTACCAATC-3′,
Thurquoise_end_121 5’-ATATAGCAAAAGGATCGGCGGC-3′;
Thurquoise_end_-103 5’-TTACTTGACACCTCCAAGCTGC-3′;
Thurquoise_end_-140 5’-TGTCTAAATTAGGATCAAGATTTCTGTCAAG-3’.
The reaction was set up using BigDye™ Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems™) that utilizes AmpliTaq Polymerases there is known to add additional adenines at the end of the DNA molecule (Samuelson et al., 2004 (link)).