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[…] Short fragment DNA libraries were generated using the Illumina NexteraXT DNA preparation kit and fragment sequencing was undertaken with the Illumina NextSeq 500 platform using 2 × 150 bp chemistry. Highly intact and high quality genomic DNA was extracted from Ef_aus0233 and subjected to Pacific Biosciences SMRT sequencing according to the manufacturer’s instructions and sequenced with two SMRT cells on the RS II platform (Pacific Biosciences) using P5-C3 chemistry. Genome assembly was performed using the SMRT Analysis System v2.3.0.140936 (Pacific Biosciences). Raw sequence data were de novo assembled using the HGAP v3 protocol with a genome size of 3 Mb. Polished contigs were error corrected using Quiver v1. The resulting assembly was then checked using BridgeMapper v1 in the SMRT Analysis System, and the consensus sequence corrected with short-read Illumina data, using the program Snippy ( The final chromosome assembly was validated by reference to a high-resolution NcoI optical map using MapSolver (version 3.10; OpGen, Gaithersburg, MD, USA). Common bacterial DNA base modifications and methyltransferase motifs were assessed using the protocol, RS_Modification_and_Motif_Analysis in the SMRT Analysis System v2.3.0.140936 (Pacific Biosciences). [...] Artemis Comparison Tool () was used to align the chromosomes of four fully assembled E. faecium genomes. The Ef_aus0233 chromosome was compared against other fully assembled E. faecium chromosomes using BLASTn DNA:DNA comparisons that were undertaken and visualized using Blast Ring Image Generator (). [...] Illumina sequence reads were de novo assembled into contigs using Spades v3.6.1 (). The closed Ef_aus0233 genome and Spades contigs were annotated with Prokka (v1.12b) () using the Enterococcus database ( as well as manually annotated protein files derived from two fully assembled E. faecium genomes (; ). Multilocus sequence types (STs) were determined using an in silico tool ( CRISPR databases were used to search for CRISPR sequences ( and (accessed 19th of May 2016). Sequence files were uploaded to the web based ISsaga ()(accessed 11th of February 2016) to detect both the abundance and diversity of insertion elements. Phage discovery was undertaken using the web based resource PHAST (accessed 15th of February 2016) (). [...] Snippy was used to map short read data against the full-assembled Ef_aus0233 genome to call core genome single nucleotide polymorphism (SNP) differences. Hierarchical Bayesian clustering was performed upon a core SNP alignment to assign genomes into discrete populations using hierBAPS with BAPS6 (a prior of 10 depth levels and a maximum of 20 clusters were specified)(). Nested clustering analyses were undertaken upon subsets of the original SNP alignment to a total depth of three levels or until no further clustering could be achieved. [...] Recombination within the core genome was inferred using ClonalFrameML v1.7 () using the whole genome alignment generated by Snippy. The ML tree generated with FastTree v2.1.8 was used as a guide tree for ClonalFrameML. Positions in the reference genome that were not present in at least one genome (non-core) were omitted from the analysis using the “ignore_incomplete_sites true” option and providing ClonalFrameML with a list of all non-core positions. Maximum likelihood trees with bootstrap support were constructed using a recombination free SNP alignment with the program FastTree (). Bootstrap support was derived from comparisons between the original tree against 1,000 trees that were built upon pseudo-alignments (sampled from the original alignment with replacement). [...] Orthologous proteins were identified through reciprocal blast using Proteinortho5 v5.11 (). A blast cutoff of 95% identity and alignment coverage of 30% were used. The resulting matrix of ortholog presence and absence was visualized using Fripan ( (downloaded on the 28th of April 2016). The General Feature Format files have been deposited in Figshare ( [...] The alignment of homologous sequences was undertaken using Mauve (). Alignments were performed using MUSCLE (). Sequences and alignments were visualized using Geneious Pro (version 8.1.8, Biomatters Ltd. ( […]

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