14 - Metagenomics/transcriptomics Flashcards
How can you multiplex samples to sequence many samples in one run?
- Locate organisms from a complex sample
- Add indexing tag (eg. short 6 nt code) to differentiate the samples
- Pool and sequence in the normal way (eg for illumina, synthesis sequencing)
This approach can be used to address the great plate count anomaly problem. We can use this method to see how many species there are in a sample, what metabolisms are at work and what compounds are being degraded or produced etc..
What are the two common methods that employ DNA sequencing in microbial ecology?
Environmental PCR: Typically used to determine what and how many species are there in a wide sample.
16S amplicon sequencing: Small subunit (16S) rRNA genes are universal genetic markers and their sequence is used as molecular barcodes to classify microbial species and build phylogenetic trees.
Amplicons from PCR on 16S can be subjected to massively-parallel DNA sequencing (eg. 454 or illumina), giving a dataset that contains the 16S sequences of the organisms present in the sample (16S amplicon sequencing)
Shotgun metagenomic approach:
- Using blast and other to identify sequences and comparing with ref genomes to get relative abundance of gene pathways in community
When would you use RNA over DNA in a metagenomics study?
When the interest is in expressed genes.
Give some applications of metagenomics
- Determine which genes are in a sample
- Determine what the genes do
- Study microbial diversity in wild environments
- Find organisms which can feed off a compound in an environment (eg. oil spill) to eliminate it
- Finding potential pathways that may turn a toxic compound into something non-toxic
- Looking at complex microbial ecosystems (eg. the human microbiomes)
- Correlating species with health conditions (eg. obesity)
- Evolutionary history of microbiomes in humans (eg. different flora in different populations of world)
