15. THE METAGENOME Flashcards
What is Metagenomics?
- Metagenomics is the study of genetic material taken directly from environmental or biological samples/compartments
What’s the difference between microbiome & the microbiota?
- Microbiome is also known as the metagenome, it includes micro-organisms such as bacteria, prokaryotes as well as the genes they contain & the environmental factors influencing them
- The microbiota refers to the combination of micro-organisms within an environment such as protists, fungi, bacteria etc.
What are some examples of human & environmental microbiota?
- ENVIRONMENTAL - soul microbiome, deep sea microbiome
- HUMAN - gut microbiome, skin microbiome, oral microbiome
What can differences in the human microbiome tell us?
- The human microbiome varies between individuals and is unique for each individual
- Significant differences in the microbiome have been associated with various diseases such as depression, cancer, IBS
- The gut microbiome can also be used to classfify individuals as obese or lean with greater than 90% accuracy
What’s the effect of Clostridum Difficil on the stool microbiome?
- A clostridum difficil infecion can change the stool microbiome & make it vary significantly from the healthy microbiome
- A faecal transplant can help restore the microbiome
What are the two sub-units of prokaryotes & eukaryotes & what do they consist of?
EUKARYOTES: - 60S = 5S & 28S rRNA - 40S = 18S rRNA PROKARYOTES: - 50S = 5S & 23 rRNA - 30S = 16S rRNA
What is 16S rRNA PCR targeted amplification?
- 16S rRNA is a component of the 30S small subunit of prokaryotic ribosomes, found in all bacteria
- Targeted 16S rRNA amplification assesses taxonomic diversity in a sample
- However, it’s biased as it only looks at bacteria
*What are the steps involved in Targeted PCR 16S rNA amplification?
- Sample collection
- DNA extraction
- 16S rRNA PCR amplification
- Sequencing
- Analysis
Name 3 analysis softwares for 16S rRNA targeted PCR amplification
- MOTHUR
- QIIMEZ
- DADA2
What are the two regions of the 16S rRNA?
- Conserved region
- Variable region (e.g V1)
- The variable region can be sequenced & can tell us about differences
What two factors need to be considered when choosing the variable region of the 16S rRNA?
- Phylogenetic signal
- Amplicon length
- If two sequences overlap, the errors in the overlap will be corrected
- Two short sequences won’t overlap as much, so not all the errors will be corrected
- Two longer sequences will overlap completely & the errors will be corrected
How can the sequencing machine affect the read length?
- There are two types of sequencing machine which can be used in 16SrRNA targeted PCR amplification:
1. Short read - Roche 54 (700bp), Illumina Miseq (2 x 300bp), Illumina Next seq & Hiseq (2 x150bp)
2. Long read - PacBio (10Kb), ONT MINON (15Kb)
What is the kitome?
- The kitome refers to the microbiome which has been contaminated
How can we avoid potential contamination of samples in 16S rRNA targeted PCR amplification?
- Randomise samples to even the samples out or so that a particular sample has less of an overall effect
- Note batch numbers to identify a bad batch
- Sequence the negative control so that any contamination can be accounted for
**What is Whole genome shotgun sequencing?
- Whole genome shotgun sequencing can assess taxonomic diversity & composite gene functions in a sample
- WGS is unbiased as it looks at all microrganisms
What are the two outcomes of WGS sequencing?
- Produce an ASSEMBLY - to show taxonomic diversity
2. BINNING - sequences can be compared to taxonomic sequences & can be grouped into taxonomic groups
Name 3 softwares used by WGS sequencing
- MG-RAST
- METAPHLAN
- KRAKEN2
Why is there no amplifications step in WGS sequencing?
- Amplification isn’t needed to enrich the bacterial DNA because the host cells are often in excess in the sample
- However, this is sample dependent
- Saliva, nasal & skin samples have a low biomass but produce greater than 90% reads
- Whereas faecal samples have a high biomass but produce less than 10% reads
*Give two ways in which we can enrich the sample without amplification in PCR?
- PRE-EXTRACTION - Differential lysis of mammalian cells to enrich microbial cells
- Possible bias for gram bacteria - POST-EXTRACTION - Enzymatic degradation of methylated nucleotides targets mammalian cell
- Possible bias for AT rich bacteria
Give three applications of WGS sequencing
- Animal microbiome - Rumen (chambers of cows gut) can be sequenced & used to reassemble whole genome
- Environmental - can sequence an area to identify unknown phylotypes & genes
- Clinical - Potential to develop diagnostic based on differences in the microbiome
