Computational protocol: Genome sequences of two novel phages infecting marine roseobacters

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Protocol publication

[…] To prepare DNA template for genome sequencing, purified phage DNA was amplified using Genomiphi V2 kit (GE Healthcare, Piscataway, NJ, USA) according to the manufacturer's protocol. The initial sequence segments were obtained by random PCR amplification of phage DNA using degenerate primer RP-1 (5′-ATHGAYGGNGAYATHCAY-3′) and RP-2 (5′-YTCRTCRTGNACCCANGC-3′). The PCR was performed in 50 μl volume containing 1× reaction buffer (Genescript, Scotch Plains, NJ, USA) with 1.5 mM MgCl2, 100 μM of dNTPs, 50 pmol of each primer, 1 U Taq DNA polymerase (Genescript) and 10 ng phage DNA as templates. PCR program consists of an initial denaturing at 94°C for 2 min, followed by 30 cycles of denaturing at 94°C for 30 s, annealing at 48°C for 1 min and extension at 72°C for 1 min and a final extension at 72°C for 10 min. Multiple PCR amplicons could be obtained for both phages. The most dominant bands for each phage were excised and the DNAs were purified using gel purification kit (Qiagen, Valencia, CA, USA) and sequenced bi-directionally using the same primer set. The three fragments with unambiguous sequences are used as starting templates for primer walking. All the subsequent primer walking was done by using an automated sequencer ABI 310 (PE Applied Biosystems) in the Biological and Analytical Laboratory at the Center of Marine Biotechnology, UMBI. From each primer walking, unambiguous sequences were assembled together using AssemblyLIGN program (GCG, Madison). Open reading frames were predicted by using ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) and GeneMarkS (). Translated ORFs were compared with known protein sequences using blastp (). tRNA sequences were searched by using tRNAscan-SE (). The countcodon program was used to determine codon usage (http://www.kazusa.or.jp/codon/countcodon.html).Sequences alignment and phylogenetic analysis were performed using MacVector 7.2 program (GCG, Madison, WI). Jukes–Cantor distance matrix analysis was used to calculate the distances from the aligned sequences, and the neighbour-joining method was used to construct the phylogenetic tree. […]

Pipeline specifications

Software tools Open Reading Frame Finder, GeneMarkS, BLASTP, tRNAscan-SE, MacVector
Applications Genome annotation, Phylogenetics, Metagenomic sequencing analysis
Organisms Ruegeria pomeroyi DSS-3, Escherichia virus N4, Escherichia coli K-12