The Metagenome Flashcards
What is metagenomics?
The study of genetic material recovered directly from environmental or biological systems/compartments.
Define the term “Microbiota”
Ecological community of commensal and pathogenic microorganisms - Includes bacteria archaea, protists, fungi and viruses
Define the term “Microbiome”
Collective genomes of the microorganisms in these communities
What does the variation in the human microbiome between individuals mean for microbiome studies?
- Varuation makes it very hard to conduct microbiome studies of patients with a particular condition
- Means you will need 100’s of people involved in any particualr study just to observe differences in the microbiome that might be associated with the particualr disease you’re studying
Give some examples of diseases/conditions associated with changes in the microbiome?
- Irritable Bowel Syndrome
- Depression
- Cancer
- Asthma
What is the theory behind chnages in the microbiome leading to emotional changes in a person?
- Idea of the gut-brain axis in which bacteria in the gut produce neurochemicals via neurotransmitters which travel up the gastrointestinal tract and can have an effect on the brain
- These effects can include changes in emotion/mood
Is the relative abundance of bacteria or the genetic content of those bacteria thought of as more important on the effect of the human microbiome on health?
- Genetic content of the bacteria within the microbiome more important
- Certain bacteria will have certian metabolic pathways which will contribute to the health of that person
What are the 2 technological methods used to study metagenomics?
- Targeted PCR amplification
- Whole genome shotgun sequencing
What are some of the characteristics of the 16S rRNA gene?
- Is around 1500 bases long
- It’s split into about 9 variable regions interspaced with conserved regions
- You can determine the different species present within a bacterial sample by looking at what variable regions are present within the sample
Explain the process of 16S PCR amplification?
- You first collect your bacterial sample
- Then you extract the DNA from the 16S rRNA gene of all the bacteria present within the sample
- Then you conduct a 16S PCR amplification on the DNA extracted - Each amplified gene sequence will be represented by a different colour
- You then place the products of the PCR reaction into a sequencing machine which will generate sequences that are complementary to the amplified products of the PCR reaction
* These generated sequences should also match the bacterial content of the original sample.
How can data from 16S PCR amplification be used to identify the quantity of each species of bacteria within a particular sample?
- The generated short-read sequences are compared to a 16S database - this is a database containing 16S genes from all types of bacterial strains
- By matching the generated short-read sequences with the sequences from the database you can determine how many of any particular bacteria was present within the sample
What do the read lengths of the sequencing technologies used for 16S PCR amplification mean for process of sequencing the 16S rRNA gene.
The read lengths mean that these technologies aren’t able to sequence the entire length of the 16S rRNA gene which means only certain parts of the gene can be amplified and sequenced
What are some of the common regions of the 16S rRNA gene that are sequenced during PCR amplification?
Commonly sequenced regions include:
- V1-V2
- V1-V3
- V3-V4
- V3-V5
- V4
What are some of the factors that decide which variable regions of the 16S rRNA gene are sequenced during 16S PCR amplification?
- Phylogenetic signal
- Amplicon length
How can phylogenetic signal be used to decide which variable regions are sequenced during 16S PCR amplification?
- Phylogenetic trees can be created based on different variable regions of the 16S rRNA gene within different species of bacteria
- They can be used to decide which variable regions you sequence based on how distinct the different variable regions are for a set of bacteria.
- If you’re looking for a particular type of bacteria within a microbiome smaple you would sequence the variable regions in which that type of bacteria has a distinct difference in phylogenetic tree compared to other bacteria so it’ll be easier to identify
How can amplicon length (read length) be used to decide which variable regions are sequenced during 16S PCR amplification?
- Different sequencing technologies produce different read lengths which may lead to errors being present within the final PCR product depending on the length of the variable regions sequenced.
- E.g. Illumina MiSeq technology produces 2 x 300bp reads from each end of the sequence
- If you sequenced V1-V3 regions of rRNA gene because of its length you would end up with a small section of that V1-V3 sequence will be amplified twice during PCR
- During PCR amplification DNA polymerase will produce errors in the amplified DNA.
- Only in the part of V1-V3 sequence that’s amplified twice will those errors be corrected because the polymerase has a template (section that’s been amplified twice) that can be used to correct the errors so they don’t end up in final PCR product
- If you amplified V4 using Illumina MiSeq because it’s a shorter sequence than V1-V3 the whole sequence is amplified twice during PCR
- This means any errors throughout the whole sequence can be corrected by DNA polymerase so no errors will be present within PCR product.
Explain the process of whole genome shotgun sequencing?
- Process is very similar to that of 16S rRNA sequencing with the differences being that instead of extracting DNA from the 16S rRNA gene you extract DNA from the entire genome
- Also once you’ve extracted the DNA instead of doing 16S PCR amplification you just sequence the entire genome
How can data from whole genome shotgun sequencing be analysed?
- You can assemble the short sequence reads produced to figure out how the genomes of each of the bacteria sequenced look like
- You can then use the sequence reads to create phylogenetic trees which look at taxonomic diversity between species (how genetically different 2 species of bacteria are)
- You can also look at which genes are present within the sample as you are sequencing the entire genome - this can then be used to look at metabolic pathways
