Lecture 14_ Genomics

Page 1: Introduction to Genomics and Proteomics

• Genomics: Whole genome sequencing

  • “Shotgun” cloning: Randomly fragmenting and cloning DNA.

  • Metagenome sequencing: Sequencing DNA from environmental samples.

  • RNA expression analysis: Study of the quantity and type of RNA in a sample.

    • DNA microarray: A high-throughput technique for analyzing gene expression.

  • Proteomics: The study of proteins' structures, functions, interactions, and abundances in biological samples. Which proteins are expressed under various conditions within a single cell type or to compare protein expression patterns between different organisms.

  • The most prominent disease being studied with proteomic approaches is cancer, but this area of study is also being applied to infectious disease. Examine the feasibility of using proteomic approaches to diagnose various types of hepatitis, tuberculosis, and HIV infection, which are rather difficult to diagnose using currently unavailable techniques.

    • 2D gel electrophoresis: Separation of proteins based on isoelectric point (pI) and molecular weight.

    • Biomarkers- a recent and developing proteomic analysis relies on identifying proteins.

Learning Objectives

• Understand genomics analysis-Genomics analysis refers to the study of an organism's entire genome using bioinformatics, computational biology, and statistical techniques.

• Infer function and physiology from genomic data.

  • Apply machine learning to predict functions from sequence motifs and domains.

  • Identify genes associated with metabolic pathways, stress responses, and adaptations.

• Identify signatures of horizontal gene transfer

  • HGT is the movement of genetic material between organisms, bypassing traditional inheritance.

Genomics Analysis

• Creation of a library that represents the entire genome or significant portions.

• Increasing affordability of genomic analyses.

• Analyze genetic diversity and functional potential of microbial communities.

• Understanding gene expression patterns and regulatory mechanisms by measuring simultaneous gene expression using hybridization techniques.

• Employing 2D electrophoresis to separate proteins based on isoelectric point and size using SDS-PAGE.

Practical Consequences of Genomics

• Capability to provide extensive data.

• Microarrays and proteomics provide useful snapshots of genomic data.

• Importance of data processing, analysis, and presentation skills.

Page 2: Genomics Research

• Cluster diagram: Algorithm that clusters genes based on response patterns.

• Pie chart: Displays which gene is most upregulated.

• Snf2 superfamily proteins: DNA-binding proteins that are secreted.

• Hypothesis-generating tool: Observing patterns and examining upregulated genes.

  • Most upregulated gene identified: Death box helicase gene.

Dr. Orwin's Work at CSUSB

• Genomics Research: Study on V. paradoxus.

  • Comparing EPS strain biofilms vs. broth vs. stationary phase.

  • Observations of growth stage changes in biofilms.

• Differential gene expression: Notable gene alterations related to biofilms formation on leaves, roots, and dirt.

• Example of data analysis:

  • Venn diagram: Illustrates subsets of upregulated genes in specific growth types.

    • Identification of most upregulated and downregulated genes in biofilms.

• Unexpected findings in the lacI TF group, highlighting a gene with 200,000-fold increased expression in biofilms with unknown function, hypothesized to stabilize biofilm structure.

Page 3: RNA-Seq Results and Applications

• Approximately 1700 genes uniquely regulated in biofilms.

• Various classes of genes enriched in biofilms.

Purpose of Genomics

• Serve as a regulatory model and structural model.

• Genomics and taxonomics functions as hypothesis-generating tools.

Page 4: Advanced Regulatory and Structural Models

• Regulatory Model:

  • RNA degradosome: Complex responsible for breaking down mRNAs into individual nucleotides.

• New Regulatory Model Hypothesis:

  • Proposed faster degradation of specific RNA types.

  • Hypothesis: Changing proteins in the degradosome may alter transcript distribution.

• Observations: Most upregulated genes are specific to biofilms, leading to potential new models for gene expression control.

• Recent findings present challenges to established theories.

Discovery of Functional Proteins

• Small secreted proteins with predicted DNA-binding motifs identified as highly expressed in biofilms.

  • Proposal: DNA-binding proteins neutralize the charge of DNA in the biofilm matrix.

  • Suggested role: Facilitation of denser packing in biofilms, similar to the function of histones in eukaryotes.

Page 5: Innovations in Research Methods

• Importance of research in generating hypotheses.

• Acknowledgement of scientific methods that go beyond intuition and observational skills.

• Capability of long-read sequencing and hybrid read sequencing for comprehensive genomic mapping.

  • Comparative Genomics: Efficiently identify genomic positions.

• Challenges in sequencing completion, particularly in identifying the last 2% of sequence missing.

Phylogenetic Studies

• Phylogenetic analysis revealed similarities among Variovorax species.

• Discovery of diverse genomic variations through ANI cluster maps.

Page 6: Understanding the PanGenome

• PanGenome diagram: Visual representation of gene regions exclusive to specific strains, common genes across species, and unique genes.

• Volcano plot: Display of significance versus magnitude of gene expression changes, with emphasis on p-values as measures of data reliability.

Implications of the PanGenome

• Exploration of core genes and their impacts on growth variations.

• Analysis of significant vs non-significant data, focusing on p-value interpretations to assess data quality.

Page 7: Biofilms and Gene Transfer

• Biofilm Types:

  • Colony biofilms: Formations visible on plates.

  • Submerged biofilms: Structures formed in liquid cultures on surfaces.

• Horizontal Gene Transfer: Mechanisms of genetic material exchange.

  • Conjugation: Direct DNA transfer between cells.

  • Transformation: Uptake of environmental DNA.

    • Generalized: Phage-mediated transfer of any genomic part.

    • Specialized: Phage transfer of specific DNA segments.

• Transposition: Integration and excision of DNA within microbial genomes.

Key Questions in Research

• Investigating common genes in all isolates, treatment of genes on second replicons, and comparing colony with submerged biofilms.

• The role and importance of horizontal gene transfer in microbial genetics and evolution, as well as practical implications in addressing infectious diseases and environmental challenges.

What genes are shared in all isolates?

• Helps identify essential genes and antibiotic resistance patterns.

2⃣ How do genes on second replicons (like plasmids) behave?

• Important for drug resistance and survival traits in bacteria.

3⃣ How do genes differ in colony vs. submerged biofilms?

• Biofilms resist antibiotics—understanding gene changes can help develop better treatments.

4⃣ How does horizontal gene transfer (HGT) shape bacteria?

• Bacteria swap genes to gain resistance or adapt to new environments.

5⃣ Why does HGT matter for disease and the environment?

• Helps track superbugs, improve treatments, and use bacteria for pollution cleanup.

Page 8: Evolutionary Implications of Gene Transfer

• Differentiating between vertical and horizontal gene transfer.

• Implications of Horizontal Gene Transfer:

  • Evolution through vertical and horizontal mechanisms.

  • Major physiological changes can occur without cell division, fundamentally altering microbial genetics and evolution theories.

  • Concept of selfish genes in the context of microbial genetics, suggesting rapid evolution through gene movement.

• Considerations for plasmids carrying beneficial genes, particularly those conferring antibiotic resistance, enhancing survival chances of bacteria.

Thought-Provoking Questions

• Exploring the impact of horizontal gene transfer on phylogeny, levels of interspecies gene transfer, and considerations for microbial evolution.

• Advocating for a re-evaluation of perspectives on microbial genetics in light of gene transfer mechanisms.