1. PowerPoint ® Lecture Presentations prepared by John Zamora Middle Tennessee State University C H A P T E R © 2015 Pearson Education, Inc. Microbial Genomics 6 6.2 Sequencing Genomes

2. © 2015 Pearson Education, Inc. Entering the era of Science Fiction Artificial Genomes Sequencing the Oceans The Human Ecosystem Personalized Genomics

3. © 2015 Pearson Education, Inc. 6.1 Introduction to Genomics Genome Entire complement of genetic information Includes genes, regulatory sequences, and noncoding DNA Genomics Discipline of mapping, sequencing, analyzing, and comparing genomes Sequencing: determining the precise order of nucleotides in a DNA or RNA molecule

4. © 2015 Pearson Education, Inc. 6.2 Sequencing Genomes Sanger method Dideoxy analogs of dNTPs used in conjunction with dNTPs Analog prevents further extension of DNA chain Bases are labeled with radioactivity Gel electrophoresis is then performed on products Figure 6.1 Fredrick Sanger

5. © 2015 Pearson Education, Inc. Figure 6.2a Fredrick Sanger 6.2 Sequencing Genomes

6. © 2015 Pearson Education, Inc. Figure 6.2b Fredrick Sanger GA T C 6.2 Sequencing Genomes Sequence from bottom: 5’ – A G C T A A G – 3’ Sequence of unknown strand: 3’ –T C G A T T C – 5’

7. © 2015 Pearson Education, Inc. Figure 6.2c Fredrick Sanger 6.2 Sequencing Genomes Radioactivity replaced by fluorescent dye Sequence from bottom: 5’ – A G C T A A G – 3’ Sequence of unknown strand: 3’ –T C G A T T C – 5’

8. © 2015 Pearson Education, Inc. 6.2 Sequencing Genomes Virtually all genomic sequencing projects use shotgun sequencing Entire genome is cloned, and resultant clones are sequenced Much of the sequencing is redundant (7-10 fold = DEPTH)

9. © 2015 Pearson Education, Inc. Second-generation DNA sequencing Generates data 100x faster than Sanger method Massively parallel methods = DEPTH Large number of amplified samples sequenced side by side Uses increased computer power and miniaturization 454 and Illumina: Enzymes generate light, which is quantified 6.2 Sequencing Genomes – 2 nd GEN

10. © 2015 Pearson Education, Inc. Illumina 6.2 Sequencing Genomes – 2 nd GEN

11. © 2015 Pearson Education, Inc. Illumina 6.2 Sequencing Genomes – 2 nd GEN

12. © 2015 Pearson Education, Inc. Pacific Biosciences SMRT (PacBio) Single-stranded DNA fragments attached Complementary strand synthesized Fluorescent tags monitored 6.2 Sequencing Genomes – 3 rd GEN “Nanocontainers”

13. © 2015 Pearson Education, Inc. Figure 6.4a Ion torrent semiconductor sequencing 6.2 Sequencing Genomes – 4 th GEN

14. © 2015 Pearson Education, Inc. Nanopore sequencing “Pocket Sequencing” Figure 6.4b 6.2 Sequencing Genomes – 4 th GEN

15. © 2015 Pearson Education, Inc. Figure 6.5 6.3 Bioinformatics and Annotating Genomes

16. © 2015 Pearson Education, Inc. 6.3 Bioinformatics and Annotating Genomes Functional ORF: an open reading frame that encodes a peptide or protein Computer algorithms used to search for ORFs Look for start/stop codons and Shine–Dalgarno sequences ORFs can be compared to ORFs in other genomes = comparative genomics

17. © 2015 Pearson Education, Inc. 6.3 Bioinformatics and Annotating Genomes

18. © 2015 Pearson Education, Inc. Figure 6.6 6.3 Bioinformatics and Annotating Genomes

19. © 2015 Pearson Education, Inc. 6.3 Bioinformatics and Annotating Genomes Number of genes with role that can be clearly identified in a given genome is 70% or less of total ORFs detected Hypothetical proteins: uncharacterized ORFs; proteins that likely exist but whose function is currently unknown BIOPROSPECTING Enzyme Discovery & Engineering

20. © 2015 Pearson Education, Inc. 6.4 Genome Size and Content Correlation between genome size and ORFs On average, a prokaryotic gene is 1,000 bp long ~1,000 genes per megabase Figure 6.7

21. © 2015 Pearson Education, Inc. 6.4 Genome Size and Content NCBI Genomes Jan 25 = 94,992 Plus incomplete, unassembled, and unannotated…

22. © 2015 Pearson Education, Inc. 6.4 Genome Size and Content Comparative analyses allow for predictions of metabolic pathways and transport systems Figure 6.9

23. © 2015 Pearson Education, Inc. Figure 6.10 6.4 Genome Size and Content Percentage of an organism's genes devoted to a specific cell function is to some degree a function of genome size Replication and translational indispensable; Regulation increases in complexity with genome size

24. © 2015 Pearson Education, Inc. Figure 6.11 6.5 Genomes of Organelles Encode proteins required for photosynthetic reactions and CO 2 fixation Contains rRNA used in chloroplast ribosomes, tRNA for translation and several proteins used in transcription and translation. Some chloroplast proteins are encoded in the nucleus

25. © 2015 Pearson Education, Inc. Figure 6.12 6.5 Genomes of Organelles Primarily encode proteins for oxidative phosphorylation Use simplified genetic codes rather than "universal" code Some contain small plasmids Mammalian mitochondria encode 13 proteins

26. © 2015 Pearson Education, Inc. Figure 6.12 6.5 Genomes of Organelles Many insects and some other invertebrates contain symbiotic bacteria Symbionts are not capable of independent life Restricted genomes Host receives essential amino acids and other nutrients

27. © 2015 Pearson Education, Inc. 6.6 Eukaryotic Microbial Genomes The haploid yeast genome Entire genome is ~13,400 kbp Encodes ~6,000 ORFs; ~4,000 encode proteins with known functions About 900 ORFs are essential (single deletions) Contains a large amount of repetitive DNA Genes contain introns

28. © 2015 Pearson Education, Inc. Figure 6.16 6.6 Eukaryotic Microbial Genomes Avg introns = 10 per gene

29. © 2015 Pearson Education, Inc. Explore 6.1 III. Functional Genomics “From the growth of the Internet through to the mapping of the human genome and our understanding of the human brain, the more we understand, the more there seems to be for us to explore.” -Martin Rees

30. © 2015 Pearson Education, Inc. 6.7 Microarrays and the Transcriptome Transcriptome The entire complement of RNA produced under a given set of conditions Microarrays Small solid-state supports to which genes or portions of genes are fixed and arrayed spatially in a known pattern

31. © 2015 Pearson Education, Inc. Figure 6.17 DNA segments on arrays are hybridized with mRNA from cells grown under specific conditions and analyzed to determine patterns of gene expression 6.7 Microarrays and the Transcriptome

32. © 2015 Pearson Education, Inc. 6.7 Microarrays and the Transcriptome What can be learned from microarray experiments? Global gene expression Expression of specific groups of genes under different conditions Expression of genes with unknown function; can yield clues to possible roles Identification of specific organisms

33. © 2015 Pearson Education, Inc. 6.7 Microarrays and the Transcriptome RNA-SEQ: all RNA molecules from a cell are sequenced Don’t need a genome or other template Quantitative: can be used to determine induction Figure 6.19 Exponential Phase: 4.5 hours Stationary Phase: 14 hours

34. © 2015 Pearson Education, Inc. 6.8 Proteomics and the Interactome Proteomics study of the structure, function, and regulation of an organism's proteins Proteome The entire set of proteins expressed by a genome, cell, tissue or organism at a certain time Figure 6.20 2-D Gel Electrophoresis

35. © 2015 Pearson Education, Inc. 6.8 Proteomics and the Interactome Proteins with >50% sequence similarity typically have similar functions Proteins with >70% sequence similarity almost certainly have similar functions Protein domains Distinct structural modules within proteins Have characteristic functions that can reveal much about a protein's role, even in the absence of complete sequence homology

36. © 2015 Pearson Education, Inc. Figure 6.22 6.8 Proteomics and the Interactome Interactome Complete set of interactions among molecules

37. © 2015 Pearson Education, Inc. Interactions are a Social Network

38. © 2015 Pearson Education, Inc. 6.9 Metabolomics and Systems Biology Metabolome The complete set of metabolic intermediates and other small molecules produced in an organism Mass spectrometry is one of the primary techniques for monitoring metabolites Figure 6.23

39. © 2015 Pearson Education, Inc. Figure 6.24 6.9 Metabolomics and Systems Biology Me

40. © 2015 Pearson Education, Inc. 6.10 Metagenomics Metagenome The total gene content of the organisms present in an environment Several environments have been surveyed by large- scale metagenome projects Examples: human body, marine ecosystems, fertile soil HMP: Human Microbiome Project

41. © 2015 Pearson Education, Inc. 6.10 Metagenomics The Human Microbiome Project The NIH Common Fund Human Microbiome Project (HMP) was established in 2008, with the mission of generating resources that would enable the comprehensive characterization of the human microbiome and analysis of its role in human health and disease. The HMP has characterized the microbial communities found at several different sites on the human body: nasal passages, oral cavity, skin, gastrointestinal tract, and urogenital tract. The project has examined the role of these microbes in human health and disease. The HMP is an interdisciplinary effort involving four sequencing centers: The Broad Institute, the Baylor College of Medicine, Washington University School of Medicine, and the J. Craig Venter Institute. Jeff Gordon

42. © 2015 Pearson Education, Inc. 6.10 Metagenomics The Human Microbiome Project The 5 stated aims of the project include: 1. Development of a reference set of 3,000 isolate microbial genomes 2. Initial 16S & mWGS metagenomic studies at each of the 5 target sites (i.e. "core" microbiomes) 3. Demonstration projects to determine the relationship between disease and changes in the human microbiome 4. Development of new tools and technologies for computational analysis, establishment of a data analysis and resource repositories 5. Examination of the ethical, legal and social implications (ELSI)

43. © 2015 Pearson Education, Inc. Table 6.6 “Omics” – Summary

44. © 2015 Pearson Education, Inc. 6.11 Gene Families, Duplications, and Deletions Some Terms: Homologous: related sequence that implies common genetic ancestry Gene families: groups of gene homologs Paralogs: genes within an organism whose similarity to one or more genes in the same organism is the result of gene duplication Orthologs: genes found in one organism that are similar to those in another organism but differ because of speciation

45. © 2015 Pearson Education, Inc. Figure 6.27 6.11 Gene Families, Duplications, and Deletions

46. © 2015 Pearson Education, Inc. 6.11 Gene Families, Duplications, and Deletions Gene duplications thought to be mechanism for evolution of most new genes Figure 6.28 “neofunctionalization”

47. © 2015 Pearson Education, Inc. 6.11 Gene Families, Duplications, and Deletions Gene duplications thought to be mechanism for evolution of most new genes http://www.personal.psu.edu/rua15/Stage3.jpg XXX

48. © 2015 Pearson Education, Inc. 6.11 Gene Families, Duplications, and Deletions Deletions can eliminate gene no longer needed Gene analysis in the three domains of life suggests that many genes present in all organisms have common evolutionary roots

49. © 2015 Pearson Education, Inc. 6.12 Horizontal Gene Transfer and Genome Stability The transfer of genetic information between organisms, as opposed to vertical inheritance from parental organism(s) May cross phylogenetic domain boundaries Figure 6.29

50. © 2015 Pearson Education, Inc. 6.12 Horizontal Gene Transfer and Genome Stability Detecting horizontal gene flow: Presence of genes typically found only in distantly related species Presence of a DNA with GC content or codon bias that differs significantly from remainder of genome Horizontally transferred genes typically do not encode core metabolic functions

51. © 2015 Pearson Education, Inc. 6.12 Horizontal Gene Transfer and Genome Stability Transposons—pieces of DNA that can move between chromosome, plasmids, and viruses Figure 6.30

52. © 2015 Pearson Education, Inc. 6.12 Horizontal Gene Transfer and Genome Stability Transposons may transfer DNA between different organisms Transposons may also mediate large-scale chromosomal changes within a single organism Presence of multiple insertion sequences (IS) Recombination among identical IS can result in chromosomal rearrangements Examples: deletions, inversions, or translocations

53. © 2015 Pearson Education, Inc. 6.13 Core Genome versus Pan Genome The "pan"/"core" concept: genomes of bacterial species consist of two components Core genome: shared by all strains of the species Pan genome: includes all the optional extras present in some but not all strains of the species

54. © 2015 Pearson Education, Inc. Figure 6.31 6.13 Core Genome versus Pan Genome Core = Black Pan = Everything

55. © 2015 Pearson Education, Inc. Figure 6.32 6.13 Core Genome versus Pan Genome

56. © 2015 Pearson Education, Inc. 6.13 Core Genome versus Pan Genome Figure 1. Circular representation of the genome of Campylobacter jejuni NCTC 11168. Genome maps (in order of presentation from outside to inside) are: (A) NCTC- K12E5 from an infected human being; (B) NCTC 11168-GSv; and (C) NCTC 11168-V26. The scale on the outside of the outermost map represents genome location (x 10 4 bases). Red and orange bars represent mutations relative to the original annotated reference NCTC 11168-GS strain deposited in GenBank Comparative Variation within the Genome of Campylobacter jejuni NCTC 11168 in Human and Murine Hosts (2014) Thomas DT, Lone AG, Selinger LB, Taboada, EN, Abbott, DW, Inglis GD. PLOS One 9(2):e88229.

57. © 2015 Pearson Education, Inc. 6.13 Core Genome versus Pan Genome Chromosomal islands believed to have a "foreign" origin based on several observations Extra regions often flanked by inverted repeats Base composition and codon usage in chromosomal islands often differ from rest of genome Often found in some strains of a species but not others

58. © 2015 Pearson Education, Inc. 6.13 Core Genome versus Pan Genome Chromosomal islands: Region of bacterial chromosome of foreign origin that contains clustered genes for some extra property such as virulence or symbiosis Figure 6.33 PAI: Pathogenicity Island CI: Chromosomal Island GC Content Red = different Blue = average Gene comparison Red = virulence Green = conserved

59. © 2015 Pearson Education, Inc. 6.13 Core Genome versus Pan Genome Chromosomal islands contribute specialized functions not essential to growth Virulence Biodegradation of recalcitrant compounds Symbiosis