Note: Front Cover -- New Approaches to Prokaryotic Systematics -- Copyright -- Dedication -- Contents -- Contributors -- Preface -- Chapter 1: The Need for Change: Embracing the Genome -- 1. A brief history of genomic sequencing of prokaryotes -- 2. Why Sequence the Genomes of Prokaryotes? -- 3. The State-of-the-Art -- 4. Where We Are Going -- Acknowledgement -- References -- Chapter 2: An Introduction to Phylogenetics and the Tree of Life -- 1. Introduction -- 2. Step 1: Posing a question -- 3. Step 2: Choosing Relevant Sequences -- 3.1. Obtaining 16S rRNA Sequences for Bacteria, Archaea and Eukarya -- 3.2. A note on the availability and use of data and methods -- 4. Step 3: Aligning Sequences and Editing the Alignment -- 5. Step 4: The theory of Fitting and Selecting a Phylogenetic Model -- 5.1. Markov nucleotide substitution models -- 5.2. Inferring phylogenies under Markov substitution models -- 5.3. Frequentist inference -- 5.4. Bayesian inference -- 5.5. Model comparison and assessment -- 5.6. Frequentist methods -- 5.7. Bayesian model choice -- 6. Step 5: Inferring Trees-Practical Guidelines for fitting and comparing Markov substitution models -- 6.1. Alignment formats for phylogeny programs -- 6.2. Inferring maximum likelihood phylogenies using RAxML -- 6.3. Bayesian analyses with PhyloBayes -- 6.4. Posterior predictive checks -- 7. Step 6: Interpreting the Phylogenetic Tree -- Conclusions -- Acknowledgements -- References -- Chapter 3: The All-Species Living Tree Project -- 1. Introduction -- 2. Sources of Information -- 2.1. Classification of microbial databases -- 2.1.1. Taxonomy (LPSN and Bergey's Manual) -- 2.1.2. Type-strain information (StrainInfo database) -- 2.1.3. Sequences and alignments (ARB and SILVA) -- 3. Database creation and updating -- 4. Features of the Database -- 4.1. Optimized SSU and LSU alignments. , 4.2. Curated hierarchical classification -- 4.3. Risk-group classification -- 4.4. Taxonomic thresholds -- 5. Phylogenetic trees -- 6. LTP as a Taxonomic Tool -- Acknowledgements -- References -- Chapter 4: 16S rRNA Gene-Based Identification of Bacteria and Archaea using the EzTaxon Server -- 1. Introduction -- 2. Use of 16S rRNA gene sequences in prokaryotic systematics -- 2.1. Sequencing of 16S rRNA genes -- 2.2. Calculation of nucleotide sequence similarity values of 16S rRNA gene sequences -- 3. Identification of Bacteria Using the EzTaxon Database -- 3.1. EzTaxon database -- 3.2. Algorithm for ``EzTaxon search´´ -- 3.3. Overall workflow from Sanger DNA sequence data -- 3.4. Assembly and trimming of sequences -- 3.5. Manual editing of sequences using the secondary structure information -- 3.5.1. Manual editing of sequences with EzEditor -- 3.6. Identification of strains using the EzTaxon server -- 3.7. Phylogenetic analysis -- Concluding remarks -- Acknowledgement -- References -- Chapter 5: Revolutionizing Prokaryotic Systematics Through Next-Generation Sequencing -- 1. Introduction -- 2. Sequencing Approaches -- 3. Bioinformatic Analyses -- 3.1. De novo assembly and mapping -- 3.2. Annotation -- 3.3. Comparative genomic analysis -- 3.4. SNP extraction and functional characteristics -- 3.5. Phylogenetic analyses -- 4. Applications of Next-Generation Sequencing Technology -- 4.1. Prokaryotic systematics -- 4.2. Pathogen evolution, transmission and adaptation -- 4.3. Genetic basis of phenotypic characteristics -- 4.4. Metagenomics -- 4.5. Target resequencing -- 4.6. RNA-Seq and transcriptomics -- Conclusions -- Acknowledgements -- References -- Chapter 6: Whole-Genome Analyses: Average Nucleotide Identity -- 1. Introduction -- 1.1. Calculation of average nucleotide identity -- 1.2. Theoretical background: BLAST/MUMmer software. , 2. Preparation and DNA Sequencing -- 2.1. Strain cultivation -- 2.2. DNA extraction and quantification -- 2.3. Whole-genome sequencing -- 3. ANI Calculations Using JSpecies -- 3.1. Installation -- 3.2. Operation -- 3.3. Calculations on-line -- 4. Interpretation and publication of results -- 5. Application to Prokaryotic Classification: Case Studies -- Concluding remarks -- Acknowledgements -- References -- Chapter 7: Whole-Genome Sequencing for Rapid and Accurate Identification of Bacterial Transmission Pathways -- 1. Introduction -- 2. The Sequencing Revolution -- 2.1. Second-generation sequencing technologies -- 2.1.1. 454 pyrosequencing -- 2.1.2. Illumina sequencing technology -- 2.1.3. Ion Torrent -- 3. Bacterial typing with next-generation sequencing -- 4. Identifying transmission pathways using whole-genome sequencing - The toolkit -- 4.1. Mapping and alignment of whole genomes -- 4.1.1. Indexing -- 4.1.1.1. Hash tables -- 4.1.1.2. The Burrows-Wheeler transform -- 4.1.2. Realigning indels -- 4.1.3. The SAM format -- 4.1.4. Identifying variation from mapped reads -- 4.2. De novo assembly and genome alignment -- 4.2.1. Read correction -- 4.2.2. Assemblers -- 4.2.2.1. Overlap-layout-consensus -- 4.2.2.2. de Bruijn graphs -- 4.2.2.3. Platform-specific assemblers -- 4.2.3. Scaffolding and gap filling -- 4.2.4. Identifying variation using co-assembly -- 4.3. Identifying variation from whole-genome assemblies -- 4.3.1. Whole-genome alignment -- 4.3.1.1. BLAT -- 4.3.1.2. MUMmer -- 4.3.1.3. Mauve -- 4.3.1.4. Mugsy -- 4.4. Identifying transmissions using whole-genome variation -- 4.4.1. SNP distances - Defining a cutoff -- 4.4.2. Phylogenetic evidence -- 4.4.3. Phylodynamics -- 5. Combining genomic and epidemiological evidence -- 6. Future Directions -- References. , Chapter 8: Identification of Conserved Indels that are Useful for Classification and Evolutionary Studies -- 1. Limitations of the phylogenetic trees for understanding microbial classification -- 2. Characteristics that are well-suited for classification -- 3. Conserved signature indels and Their usefulness for classification and evolutionary studies -- 4. Identification of Conserved Signature Indels in Protein Sequences -- 4.1. Creation of multiple sequence alignments -- 4.2. Identification of potential conserved indels in the sequence alignments -- 4.3. Blast searches on potential conserved indels to identify useful conserved indels -- 4.4. Formatting of the conserved indels -- 5. Interpreting the Significance of Conserved Indels -- 6. Correspondence of the Results Obtained from CSIs with rRNA and Other Phylogenetic Approaches -- 7. Importance of the discovered CSIs for understanding microbial classification and phylogeny -- Acknowledgements -- References -- Chapter 9: Reconciliation Approaches to Determining HGT, Duplications, and Losses in Gene Trees -- 1. Introduction -- 2. Bacterial species tree -- 3. Gene Family -- 4. Evolution of Genes in Bacterial Genomes -- 5. Gene Tree/Species Tree Reconciliation -- 5.1. Protocol for running AnGST -- 5.2. Interpreting the results of AnGST analyses -- 6. Analysis at the Genome Scale -- 6.1. Protocol for running COUNT -- Concluding Remarks -- Acknowledgements -- References -- Chapter 10: Multi-Locus Sequence Typing and the Gene-by-Gene Approach to Bacterial Classification and Analysis of Population -- 1. Introduction -- 1.1. Historical perspective -- 1.2. Multi-locus population analyses -- 2. Multi-locus sequence typing -- 3. Whole-Genome Data Analyses -- 3.1. Gene-by-gene analysis of WGS data -- 3.2. The Bacterial Isolate Genome Sequence Database -- 3.3. Isolate and sequence databases. , 3.4. Reference sequence and profile definitions database -- 3.5. Database Integrity -- 3.6. Gene nomenclature -- 3.7. Typing and analysis schemes -- 4. Examples of Gene-by-Gene Analysis: Neisseria -- 4.1. Ribosomal multi-locus sequence typing -- 4.2. Neisseria rplF assay -- 5. Examples of Gene-by-Gene Analysis: Campylobacter -- 5.1. Core genome multi-locus sequence typing -- 5.2. Whole-genome multi-locus sequence typing -- Conclusions -- References -- Chapter 11: Multi-locus Sequence Analysis: Taking Prokaryotic Systematics to the Next Level -- 1. Introduction -- 2. Multi-Locus Sequence Analysis -- 2.1. Underlying concepts -- 2.2. Selection of gene loci -- 2.3. Generating sequences -- 2.4. Data analysis -- 2.4.1. Properties of loci -- 2.4.1.1. Sequence alignments -- 2.4.1.2. Loci statistics -- 2.4.1.3. Establishing STs -- 2.4.2. Phylogenetic analysis -- 2.4.2.1. Models of evolution -- 2.4.2.2. Evaluating phylogenetic congruence -- 2.4.2.3. Construction of phylogenetic trees -- 2.5. Comparison with other taxonomic methods -- 2.6. MLSAs: Advantages and disadvantages -- 2.7. MLSA databases -- 3. Application of MLSAs in Prokaryotic Systematics -- 3.1. The genus Streptomyces -- 3.1.1. The Streptomyces MLSA scheme -- 3.1.2. DNA:DNA hybridization and MLSAs -- 3.2. Classification of the S. pratensis phylogroup -- 3.3. MLSA of phytopathogenic Streptomyces species -- 3.4. MLSA: Actinobacteria -- 4. Detection of Ecotypes Based on MLSAs -- 5. MLSA Based on Whole Genome Sequences -- References -- Chapter 12: Bacterial Typing and Identification By Genomic Analysis of 16S-23S rRNA Intergenic Transcribed Spacer (ITS) Seque -- 1. Introduction -- 2. Methods -- 2.1. Search and download bacterial whole-genome sequences -- 2.2. Annotation of rrn alleles. , 2.3. Extraction of the gene components (16S, 23S and 5S) and extra genic regions (ITS1, ITS2, pre-16S and post-5S) that make.