Diagnostic Use of 16S rRNA Gene Sequencing & Metagenomics in Medical Microbiology Flashcards
What can you do when you dont know which bacteria you are looking for?
1) PCR of 16s rRNA
2) Sanger sequencing
3) Compare sequence to known bacterial sequences in database (ex BLAST)
4) Bacterial identification
Why is 16S rRNA (ribosomal RNA) a good barcode molecule?
1) Present in all bacteria
2) Contains sufficient phylogenetic information. The mutation rate is even and predictable
3) Easily amplified by PCR - the multiple conserved regions are used as primer sites (universal PCR)
4) Variable regions between priming sites used for identification
5) Comprehensive databases on 16S rDNA sequences used for comparison
What is BLAST?
The Basic Local Alignment Search Tool (BLAST) is a program for comparing biological sequence information (eg nucleotides of DNA/RNA sequences or amino acids of proteins).
The program compares the nucleotide sequence (query) to a library or database of sequences and calculates the statistical significance of matches.
BLAST can be used to inter functional and evolutionary relationships between sequences as well as help identify members of gene families.
What approaches can be used for minimising bias?
1) Sampling: use only sterile materials/sterile methods
2) DNA extraction: limit contamination, use clean facilities/reagents, include controls
3) PCR amplification: always include negative and positive controls
4) Interpretation: manual inspection of sequences and filtering of contaminants, use databases that you trust, consider the clinical relevance of the pathogen identified
How is broad-range 16S rDNA PCR performed?
The primers that are used target the highly conserved regions of the 16S ribosomal subunit, present in all bacterial ribosomal genes (rDNA). These regions also consist of variable regions that differ between bacterial genera and species.
What is taxonomy?
Characterisation, classification, naming (nomenclature) of biologic groups.
Important for understanding of diversity in the biosphere.
Used for communication.
What is the microbiome?
The microbiome is the community of microorganisms that exists in a particular environment. In humans, the term is often used to describe the microorganisms that live in or on a particular part of the body, such as the skin or gastrointestinal tract. These groups of microorganisms are dynamic and change in response to a host of environmental factors, such as exercise, diet, medication, and other exposures.
What is the metagenome?
All genetic material (DNA/RNA) present in an environmental or clinical sample - which originate from all the individual organisms present.
What is metagenomics?
The study of the structure and function of entire nucleotide sequences isolated ad analyzed from all the organisms (typically microbes) in a bulk sample.
Diagnostic metagenomics is detection and identification of (all) pathogenic microbes directly from clinical samples by sequencing.
How is metagenomics performed?
1) Sequencing of 16S rRNA genes
2) Shotgun sequencing
What is amplicon sequence variant (ASV)?
An amplicon sequence variant (ASV) is any one of the inferred single DNA sequences recovered from a high-throughput analysis of marker genes.
Makes it possible to distinguish sequence variation by a single nucleotide change.
The uses of ASVs include classifying groups of species based on DNA sequences, finding biological and environmental variation, and determining ecological patterns.
