Computational protocol: Adaptation of Surface-Associated Bacteria to the Open Ocean: A Genomically Distinct Subpopulation of Phaeobacter gallaeciensis Colonizes Pacific Mesozooplankton

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[…] To sequence the complete 16S rRNA gene and the downstream internal transcribed spacer (ITS) region, isolates were grown in marine broth, harvested by centrifugation and DNA was extracted by the Qiagen Blood & Tissue Kit. Amplification products were generated with primers 27f (5′-AGA GTT TGA TCM TGG CTC AG-3′; ) and 23S-130r (5′-GGG TTB CCC CAT TCR G-3′; ) and sequenced by Sanger sequencing. ITS sequences were aligned using ClustalW implemented in MEGA6 () and manually curated. The phylogenetic tree was calculated employing the best fit option of the Maximum Likelihood method (Kimura 2-parameter model with gamma distribution, complete gap deletion) in MEGA6. [...] Based on the results of the phylogenetic analysis, two strains originating from distant sampling locations were chosen for genome analysis. Genomic DNA was extracted with the JETFLEX Genomic DNA Purification Kit (Genomed). SMRT sequencing was carried out on the PacBio RSII (Pacific Biosciences, Menlo Park, CA, United States) using the P6 chemistry (details see Supplementary Material). PacBio reads were assembled de novo in the SMRT Portal 2.3.0 and were corrected by paired-end Illumina reads, which were sequenced on the MiSeq (PE150), using the Burrows-Wheeler Aligner () and the CLC Genomics Workbench 7.0.1. The final assembly was circularized and adjusted to the replication system as start point. Genome sequences were automatically annotated using Prokka 1.8 () and were deposited in the NCBI GenBank (accession numbers: CP021040–CP021052). Information for all other Phaeobacter strains which were used for genomic comparisons are given in Supplementary Table . [...] Polymorphic genome sites in the Phaeobacter genus were extracted from a core genome alignment using Parsnp and Gingr (). A phylogenetic network was calculated from the resulting matrix which contained 68,858 characters with the NeighborNet algorithm in SplitsTree 4.13.1 (). Phylogenetic distances of P. gallaeciensis were inferred from pairwise comparisons of complete genome sequences via the GGDC 2.1 web service (formula 2; ). A BIONJ tree was calculated with the R package ape and rooted at midpoint (; ). Whole chromosome alignments of P. gallaeciensis were obtained with Mauve (), sequence similarity between genomes estimated by BLAST () and plotted together with the R package genoPlotR (). Nucleotide diversity between Pacific and the other strains was calculated for all aligned ortholog genes along the genome with the R package PopGenome (). Prophages were identified with PHASTER () and classified by Virfam (). Genomic islands were predicted with the IslandViewer web server (). Mobile elements of the other P. gallaeciensis had been identified previously (Freese et al., submitted) and were included in the analysis. Orthologs of P. gallaeciensis were inferred via proteinortho () and the core genome was defined as orthologs present in all strains. Subsequently, clade-specific orthologs were identified and their KEGG orthologies (ko identifiers) were determined using KAAS () using a threshold of 30%. Methylation motifs were detected using the SMRT Portal with the RS Modification and Motif Analysis and were identified using REBASE (). Methylase sequence homologs for the hits were identified using BLAST. […]

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