Computational protocol: Isolation, characterization, and complete genome analysis of P1312, a thermostable bacteriophage that infects Thermobifida fusca

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Protocol publication

[…] Genomic DNA was obtained from the purified phage P1312 after phenol/chloroform extraction and ethanol precipitation. Whole genome sequencing of P1312 was performed using Illumina Miseq (Tri-I Biotech, Inc). The count of reads was 115,622 with the average length of 175 bases per read. The sequence data could be assembled into a circular single contig of 60,284 base pairs using the de novo assembly algorithm of CLC Genomics Workbench (Qiagen). The nucleotide sequence of the genome of P1312 is deposited at GenBank under accession number KT021004. Identification of potential open reading frames (ORFs) within the phage genome was performed by using Bacterial Annotation System (van Domselaar et al., ) as well as Glimmer/RBSfinder program (Delcher et al., ), and the functions of proteins encode by the ORFs were predicted based on BLASTp program and conserved domain search (http://www.ncbi.nlm.nih.gov/). The probable replication origin of P1312 was predicted by the GenSkew program (http://genskew.csb.univie.ac.at/). [...] Protein sequences of terminase large subunit from a variety of bacteriophages were retrieved from the biological database, National Center for Biotechnology Information (NCBI). Multiple sequence alignments of those terminase sequences were performed by using the ClustalW program with default parameters in MEGA 6.0 version (Tamura et al., ). Phylogenetic tree was built by the neighbor-joining method and phylogenies were determined by bootstrap analysis of 10,000 replicates in MEGA 6.0 version. […]

Pipeline specifications

Software tools CLC Genomics Workbench, BASys, Glimmer, BLASTP, Clustal W, MEGA
Applications Phylogenetics, WGS analysis
Organisms Thermobifida fusca