Microbial Genomics

BIOL 457 – Topic 9: Microbial Genomics and the Other -omics

Introduction to Genomics

  • Genome: Entire complement of genetic information, including genes, regulatory sequences, and noncoding segments.

  • Genomics: Discipline of mapping, sequencing, analyzing, and comparing genomes. Over 240,000 prokaryotic genomes sequenced or in progress.

Steps to Sequence a Genome

  1. Sequence the DNA: Determining the precise order of nucleotides.

  2. Assemble the sequences: Create chromosomes or large fragments (contigs).

  3. Annotation: Predict genes and identify their functions.

DNA Sequencing Methods

  • Sanger Sequencing:

    • Dideoxy analogs of dNTPs prevent DNA extension.

    • Enables identification of DNA sequence by termination.

  • Next Generation Sequencing (NGS):

    • More parallel sequences and shorter reads than Sanger.

    • Various technologies include:

      • 454 Pyrosequencing: Uses massively parallel liquid handling and can generate data 90 times faster than Sanger.

      • Illumina: Similar principles, capable of generating billions of clusters, very accurate (99.9%), but more expensive ($2000/sample for large samples).

      • PacBio: Long reads (up to 10,000 bases), suitable for genome sequencing but has a higher error rate (1-15%).

      • Nanopore Sequencing: Measures electrical current through a protein pore, allowing very long reads (>9 kbp) but with a high error rate (10-20%).

Limitations of Sanger Sequencing

  • Major limitations include time and cost for large-scale genome sequencing, and the reliance on radioactive methods.

Metagenomics and Its Applications

  • Metagenome: Total gene content from organisms in a sample.

  • Can sequence entire complement of DNA in mixed samples using NGS or PCR with universal primers.

  • Can assess functional genes like nitrogen fixation and community diversity through techniques like 16S metagenomics.

Transcriptomics

  • Transcriptome: Entire RNA produced under specific conditions.

  • Measures global gene expression and specific groups of genes

  • Methods include qRT-PCR, microarrays, and RNA-seq, allowing for detailed analysis of gene expression.

Proteomics vs Transcriptomics

  • Transcriptomics: Focuses on all transcripts, provides insights on rRNAs, tRNAs, and potential expression but misses post-transcriptional effects.

  • Proteomics: All proteins present, more informative but complex to execute; methods include 2D-gels and mass spectrometry.

Interactomes

  • Interactome: Complete set of interactions between biological molecules; represented by network diagrams showing interactions such as protein-DNA or protein-protein networks.

  • Example: Human-SARS-CoV-2 interactome studied for viral interaction mechanisms.

Metabolomics

  • Metabolome: Set of metabolic intermediates produced in an organism; includes primary metabolites essential for growth and secondary metabolites relevant in specific environments.

  • Techniques involve mass spectrometry for monitoring metabolites.

Horizontal Gene Transfer (HGT)

  • Mechanisms of HGT can involve transformation, conjugation, and transduction, often facilitating genetic exchanges across domains.

  • Detecting HGT involves assessing gene presence in diverse organisms and examining GC content or codon bias discrepancies.

Gene Families, Duplications, and Deletions

  • Gene duplications facilitate the evolution of new genes, while deletions eliminate unnecessary genes.

  • Core genome includes shared genes across species whereas accessory genomes contain unique genes.

  • Pan-genome: Combination of core and accessory genes, helping define genetic diversity within a species.