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Microbiology

Sequencing

This section focuses on sequencing, specifically 16S rRNA gene sequencing, as a powerful method for bacterial identification. It covers the theory, interpretation, and application of this widely used molecular technique

Theory: Unlocking Bacterial Identity Through the Ribosome’s Code

  • What is 16S rRNA Gene Sequencing?
    • 16S rRNA gene sequencing is a molecular technique used for bacterial identification and phylogenetic analysis
    • It involves sequencing a highly conserved (present in all bacteria) and highly variable (different between species) region of the 16S ribosomal RNA (rRNA) gene
    • The unique sequence of the 16S rRNA gene is used to identify the bacterium to the species level
  • Why Use 16S rRNA Gene Sequencing?
    • Universal: Present in all bacteria, allowing for identification of a broad range of organisms
    • Highly Informative: The 16S rRNA gene contains conserved regions for primer design and variable regions for species-level differentiation
    • Culture-Independent: Can identify bacteria without the need for prior culture (e.g., in complex environments)
    • High Accuracy: Provides accurate and reliable species identification
    • Phylogenetic Analysis: Allows for the determination of evolutionary relationships between bacteria
    • Identification of Non-Culturable Bacteria: Useful for identifying bacteria that are difficult or impossible to culture
  • The 16S rRNA Gene
    • The 16S rRNA gene is a highly conserved gene found in all bacteria
    • It encodes for the 16S ribosomal RNA, a structural component of the bacterial ribosome
    • The gene contains conserved regions (highly similar across bacterial species) and variable regions (different between species)
    • The conserved regions are used for designing universal primers for PCR amplification
    • The variable regions are used for species-level identification
  • General Principle of 16S rRNA Gene Sequencing
    1. DNA Extraction: Extract DNA from the bacterial sample
    2. PCR Amplification: Amplify the 16S rRNA gene using universal primers that bind to the conserved regions
    3. Sequencing: Determine the nucleotide sequence of the amplified 16S rRNA gene
    4. Sequence Analysis: Compare the sequence to a database of known 16S rRNA gene sequences
    5. Identification: Identify the bacterium based on the sequence match

Interpretation: Decoding the Genetic Fingerprint

  • Sequencing Results
    • Sequence Data: The result of the sequencing reaction is a DNA sequence representing the 16S rRNA gene
    • Sequence Alignment: The sequence is aligned with a database of known 16S rRNA gene sequences
    • Percentage Identity: The percentage identity between the unknown sequence and the closest match in the database is calculated. This is the most important result
    • Closest Match: The database provides the closest matching species based on the sequence alignment
    • E-value: The E-value is a statistical value that estimates the number of hits one can expect to see by chance when searching a database of a particular size. A lower E-value indicates a more significant match
    • Confidence Level: The identification is often reported with a confidence level (e.g., high, medium, low)
    • Phylogenetic Tree: The results can be used to construct a phylogenetic tree showing the evolutionary relationships between the unknown bacterium and other bacteria
  • Interpreting the Percentage Identity
    • Species-Level Identification: Generally, a percentage identity of >98-99% is considered sufficient for species-level identification
    • Genus-Level Identification: A percentage identity of 95-98% may indicate genus-level identification
    • Unidentified: If the percentage identity is low (e.g., <95%), the bacterium may be a novel species or may not be present in the database
  • Factors Affecting Accuracy
    • Database Quality: The accuracy and completeness of the database are critical
    • Sequence Quality: The quality of the sequencing data affects the accuracy of the identification
    • Primer Specificity: The primers used for PCR amplification must be specific to the 16S rRNA gene
    • Multiple Organisms: If the sample contains multiple organisms, the results may be ambiguous
    • Strain Variation: Strain variation can affect the sequence, but the method is usually robust enough to identify down to the species level

Application: Putting Knowledge into Practice

  • Quality Control (QC)
    • Control Strains: Use known positive and negative control organisms for each run
    • Sequencing of the Control Strains: Sequence known species to check if the sequencing process and database are working correctly
    • Sequence Quality Checks: Assess the quality of the sequencing data (e.g., read length, Phred scores)
    • Documentation: Record QC results in a logbook or LIS
    • Repeat if Necessary: If QC fails, repeat the sequencing run
  • Procedure
    1. Sample Preparation: Prepare the bacterial sample (e.g., from a pure culture, from a clinical sample)
    2. DNA Extraction: Extract DNA from the sample using a suitable extraction method
    3. PCR Amplification: Amplify the 16S rRNA gene using universal primers
    4. PCR Product Purification: Purify the PCR product to remove excess primers and other contaminants
    5. Sequencing
      • Prepare the sequencing reaction
      • Load the sample into the sequencing instrument
      • Run the sequencing reaction
    6. Sequence Analysis
      • Obtain the sequence data
      • Trim the sequence to remove low-quality regions
      • Align the sequence with a database
      • Determine the percentage identity and closest match
      • Generate a phylogenetic tree (optional)
    7. Result Interpretation: Interpret the results based on the percentage identity and the closest match
    8. Documentation: Record the results in the LIS
    9. Correlation: Correlate the results with Gram stain, colony morphology, and other clinical information
    10. Reporting: Report the identification to the clinician
  • Examples of Applications
    • Identification of Bacteria from Culture
    • Identification of Bacteria from Clinical Samples
    • Identification of Bacteria that are difficult to culture
    • Identification of Bacteria from environmental samples (e.g., water, soil)
    • Outbreak Investigations
    • Phylogenetic Studies
  • Troubleshooting
    • Poor Sequencing Results
      • Low DNA Concentration: Ensure sufficient DNA for sequencing
      • Poor DNA Quality: Ensure good DNA quality (e.g., no degradation)
      • Contamination: Use clean reagents and maintain aseptic technique
      • Primer Issues: Optimize primer design and reaction conditions
    • Ambiguous Identification
      • Low Percentage Identity: The bacterium may be a novel species
      • Multiple Closest Matches: The sample may contain a mixed population of bacteria
      • Database Limitations: The bacterium may not be present in the database
    • Technical Issues
      • Instrument Malfunction: Contact the sequencing instrument manufacturer for technical support
      • Reagent Problems: Use fresh reagents and follow the manufacturer’s instructions
      • Poor Sample Preparation: Ensure proper sample preparation techniques are followed

Key Terms

  • 16S rRNA Gene Sequencing: A molecular technique for bacterial identification based on sequencing the 16S rRNA gene
  • 16S rRNA Gene: A gene that encodes for the 16S ribosomal RNA
  • Ribosome: A cellular structure responsible for protein synthesis
  • Conserved Region: A region of a gene or protein that is highly similar across different species
  • Variable Region: A region of a gene or protein that is different between different species
  • DNA Extraction: The process of isolating DNA from a sample
  • PCR (Polymerase Chain Reaction): A technique for amplifying specific DNA sequences
  • Primer: A short DNA sequence that initiates DNA synthesis
  • Sequencing: Determining the order of nucleotides in a DNA sequence
  • Nucleotide: The basic building block of DNA and RNA
  • Sequence Alignment: Comparing two or more DNA sequences to identify similarities and differences
  • Percentage Identity: The percentage of identical nucleotides between two DNA sequences
  • Closest Match: The bacterial species that has the highest percentage identity with the unknown sequence
  • E-value: A statistical value that estimates the number of hits one can expect to see by chance
  • Phylogenetic Tree: A diagram that shows the evolutionary relationships between organisms
  • Quality Control (QC): Procedures used to monitor and ensure the reliability of laboratory testing
  • Control Strains: Known organisms used as positive and negative controls
  • Gram Stain: A differential staining technique used to classify bacteria
  • Colony Morphology: The visual characteristics of bacterial colonies on solid media
  • LIS (Laboratory Information System): A computer system used to manage laboratory data
  • Aseptic Technique: Procedures used to prevent contamination
  • Amplification: The process of making multiple copies of a DNA sequence
  • Taxonomy: The science of classifying organisms
  • Phylogeny: The evolutionary history of a species or group of species
  • Annotation: The process of identifying and describing the features of a genome
  • Strain: A genetic variant or subtype of a species
  • Genome: The complete set of genetic material in an organism
  • Read Length: The number of nucleotides in a DNA sequence read
  • Phred Score: A measure of the quality of a nucleotide base call in a DNA sequence
  • Trim: To remove low-quality regions from a DNA sequence
  • Consensus Sequence: A single sequence that represents the most likely sequence for a given region of DNA