MO Genome complexity and metagenomics 11/10 Flashcards
Amplicon
piece of DNA or RNA that is the product of amplification events, usually produced by techniques like polymerase chain reaction (PCR).
Amplicons are defined by the specific region of the genome that is targeted by primers during the amplification process. Short fragments of genetic material that contain the sequence of interest for further analysis.
Uses targeted sequencing of specific marker genes, such as the 16S rRNA gene for bacteria and archaea or 18S rRNA for eukaryotes, to assess the composition and diversity of microbial communities.
It is particularly useful for taxonomic profiling because these genes contain highly conserved regions (for universal primer binding) and variable regions (for species differentiation).
Why 16S/18S rRNA is a Useful Marker and used for making the (new) tree of life
- Highly Conserved Yet Variable:
The 16S (for prokaryotes) and 18S (for eukaryotes) rRNA genes are universally present in all bacteria and archaea (or eukaryotes) and evolve slowly, making them ideal for studying long-term evolutionary relationships.
They contain both highly conserved regions (suitable for universal primer binding) and hypervariable regions that vary between species, enabling researchers to distinguish closely related taxa. - Single Copy in Most Genomes: The 16S/18S rRNA gene typically exists in a single copy, reducing complications from gene duplications and making it a reliable marker for estimating species abundance and diversity.
- Application in Metagenomics: 16S/18S rRNA sequencing is commonly used in amplicon-based metagenomics to profile microbial communities. Its resolution is sufficient to identify taxa at the genus and species level, making it a cornerstone for microbial ecology and environmental studies.
Co-occurrence networks
are graphical representations used to illustrate relationships between entities (e.g., genes, species, or microbial taxa) based on their simultaneous presence or abundance patterns across multiple samples.
Key Concepts:
1. Nodes:
In co-occurrence networks, nodes represent the individual entities being analyzed, such as microbial species, genes, or metabolites.
2. Edges:
Edges (lines connecting nodes) indicate a significant co-occurrence or co-exclusion pattern between the entities. For example, if two microbial species frequently appear together across different samples, they would be connected by an edge, suggesting a potential associations
The strength of the association can be quantified using various statistical measures, such as correlation coefficients (Pearson, Spearman) or more complex ecological similarity indices.
3. Positive vs. Negative Associations:
Positive Co-Occurrence: When two entities are found together more often than expected by chance, indicating potential interactions such as mutualism or shared environmental preferences.
Negative Co-Occurrence: When two entities are rarely found together, suggesting competition, antagonism, or different ecological niches.
4. Inferring Interactions:
Co-occurrence networks do not directly show causal relationships but provide hypotheses for potential biological interactions (e.g., symbiosis, competition, or predator-prey dynamics).
Further experiments or integrative modeling approaches are needed to validate and interpret these associations.
(look it up on google for image)
Metagenome-Assembled Genomes (MAGs)
are partial or complete genomes reconstructed directly from environmental metagenomic sequencing data. MAGs are used to characterize the genomes of uncultured microorganisms,
How MAGs are Constructed
- A metagenomic sample is sequenced, generating millions of short reads from multiple organisms present in the environment.
- These reads are assembled into longer contigs and binned into separate genomes using bioinformatics tools based on coverage, GC content, and tetranucleotide frequency.
- MAGs enable researchers to identify and characterize previously unknown microorganisms and their functional roles within a community.
Problematic is how to deal with conserved regions, because they are lost in the MAG approach
Advantage/Disadvantage MAGs
Advantages:
* Enables genomic study of organisms that are difficult or impossible to culture.
* Provides a comprehensive view of the functional potential of microbial communities.
Disadvantage:
* MAGs, as population consensus genomes, often aggregate heterogeneity among species and strains, thereby obfuscating the precise relationships between microbial hosts and mobile genetic elements (MGEs*).
Single Amplified Genome Catalogs (SAGs)
are genomes reconstructed from individual microbial cells. This method captures genomic information at the single-cell level, providing high-resolution insights into the diversity and functional potential of microorganisms within a community.
How SAGs are Constructed:
* Individual cells are isolated using techniques like flow cytometry or micromanipulation.
* The genome of each cell is then amplified using multiple displacement amplification (MDA) before sequencing.
* Unlike MAGs, which are reconstructed from a mixed population, SAGs represent the genome of a single cell, reducing contamination from other organisms.
Advantages:
* Captures intra-species diversity and enables the study of rare or low-abundance taxa that may be missed in bulk metagenomics.
* Allows exploration of functional variation and genome plasticity within a single population.
Applications:
* Useful in studies of microbial dark matter (previously uncultured or unknown taxa).
* Facilitates the study of functional differences between closely related strains within a community.
To conclude
- The difference in taxonomic composition between SAGs and MAGs indicates that combining both methods would be effective in expanding the genome catalog.
- By connecting mobilomes and resistomes in individual samples, SAGs could meticulously chart a dynamic network of ARGs on MGEs, pinpointing potential ARG reservoirs and their spreading patterns in the microbial community. The mobilome includes elements such as plasmids, transposons, integrons, insertion sequences, and bacteriophages. These elements are key drivers of genetic diversity and evolution in microbial populations.
A genome catalog is a comprehensive collection of genomes representing the genetic diversity of a specific group of organisms, environment, or ecosystem. Genome catalogs serve as organized references that include assembled and annotated genomes.
The mobilome refers to the collection of all mobile genetic elements (MGEs) within a genome or a microbial community
MGEs are DNA sequences that can move within a genome or between genomes, playing a crucial role in horizontal gene transfer (HGT) and genetic recombination.
The resistome encompasses the total collection of all antibiotic resistance genes present within a microorganism, microbial community, or environment. This includes both the resistance genes currently expressed by bacteria and the silent or cryptic resistance genes that may be activated under selective pressure.
Stable Isotope Metagenomics
is a method that combines stable isotope probing (SIP) with metagenomics to link metabolic activity to specific microbial taxa in complex communities.
How It Works:
* A substrate labeled with a stable isotope (e.g., 13C or 15N) is introduced into a microbial community.
* Microbes that metabolize the labeled substrate incorporate the isotope into their DNA.
* DNA is then extracted, and isotopically enriched (heavy) DNA is separated from unlabeled (light) DNA using density gradient centrifugation.
* The heavy DNA is sequenced to identify the active microbes and link metabolic processes to specific taxa.
Advantages:
* Directly links microbial activity to specific metabolic pathways and organisms in situ.
* Provides insights into functional roles within microbial communities that cannot be obtained through metagenomics alone.
Applications:
* Used in environmental studies to track the degradation of pollutants and understand nutrient cycling.
* Helps identify key players in microbial processes such as methanogenesis, sulfur cycling, and nitrogen fixation.
Species Taxonomy and Classification
- Genomic methods such as Average Nucleotide Identity (ANI) and 16S/18S rRNA gene sequencing have become standard for defining bacterial and archaeal species.
- ANI provides a quantitative measure of genome similarity, while 16S/18S rRNA sequences serve as reliable markers for taxonomic classification.
ANI (Average Nucleotide Identity) is a metric used to compare the genetic similarity between two genomes. It measures the average identity of nucleotide sequences shared between two microbial genomes and is typically expressed as a percentage.
