Lecture 4 Flashcards
What was an early sequence based approach to identifying bacteria
16S rRNA sequencing.
Why was 16s rRNA important
Because of its important function, most of it is very highly conserved, meaning that its possible to reliably amplify by PCR from most species.
Ribsomes tend to evolve slowly as they’re fundamental (they have catalytic properties)
V loops of rRNA
tend to be stuck on the outside, slightly less important so evolve more rapidly, contain more phylogenetic information
What did application of 16S rRNA sequencing to environmental samples reveal
That the vast majority of bacteria cannot be cultured in the lab.
The sequences from unculturable bacteria are found across the 16S rRNA tree of known bacterial phyla
How can we investigate dark matter organisms
16S rRNA profiling and metagenomics
What does 16S rRNA profiling involve
- Extract the DNA from sample from environment and PCR amplify it across a v loop. Can add adaptor sequences such as illuminia and get lots of sequencing reads and work out what they are
What does metagenomics involve
We sequence genomic DNA obtained from an environment rather than just targeting rRNA genes.
The sequence data doubles the number of protein sequences in GenPept overnight
Whats an issue with 16S rRNA sequencing and metagenomics
Because these involve PCR amplification, they’re highly sensitive and prone to contamination
What’s a contminant
DNA in the reagents used for DNA extraction. If there’s not much DNA in the actual sample, then the contaminant DNA from the reagents can be amplified and sequenced and wrongly interpreted as evidence
What does the observation of kit-ome mean
It’s essential to perform appropriate controls to ensure that the conclusions of a study are not influenced by contamination
Misinterpretation of metagenomes
The use of incorrect plasmid annotation meant that the genes were not in the correct place and the authors had incorrectly thought that sequence data had been obtained from Y.pestis murine toxin gene.
Single cell genomics
Another approach to sequencing dark matter - individual cells are isolated by laser microdissection, microfluidics or cell sorting and the genomic DNA is extracted
The single copy of the genome is PCR amplified using multiple displacement amplification - uses random primer sequences and a DNA polymerase which extends the random primers and displaces already synthesised strands
Amplified DNA is sequenced and assembled
Whats the IChip
Another alternative approach to characterising microbial dark matter - cultures organisms within their natural environment. It’s microfluidic device which can separate individual
Its microfluidic which can separate individual cells into seperate chambers - filled with molten agar and covered with a semi-permeable membrane and the device is placed back into the environment
What was the IChip used for
Was applied to discover a novel antibiotic teixobactin which it is claimed it can kill S. aureus or M. tuberculosis without any detectable resistance
How did we improve assemblies
SAG sequences was digitally normalized to reduce over represented regions caused by amplification bia.
What was the first instance of lateral gene transfer from eukaryote to arheon
Nanoarheaota genome encodes an oxidorereductase most cloesly related to the slime Dicyostelium discoideum and sits within the eukaryal evolutionary radiation for this gene
What does single cell genome data provide us with
An 11% greater coverage of known phylogenetic diversity than currently available genomes - represents 4.5 fold increase in phylogenetic diversity per genome
How was teixobactin identified
Using IChip, which simultaneously isolates and grows uncultured bacteria.
- Sample diluted so that one bacterial cell is delivered to a given channel
- Device is covered with two semi-permeable membrane and placed back in the soil
- Diffusion of nutrients and growth factors through chambers enables growth of uncultured bacteria in natural environment
- Once colony is produced, substantial number of uncultivated isolates are able to grow in vitro
How many isolates were obtained and screened for antimicrobial activity on plates overlaid with S.aureus and what was the new species discovered
10,000- B-proteobacteria named Eleftheria
What does Teixobactin contain
It’s an unusual dipsipeptide which contains enduracidine, methylpenylalanine and 4-d-amino acids
Whats teixobactins mechanism of actin
It inhibits synthesis of peptidoglycan- no effect on label incorporation into DNA
Teichoic acids anchor autolysins preventing uncontrolled hydrolysis of peptidoglycan - helps liberate autolysins contributing to excellent lytic and killing activity
Resistance has not developed to this compound
What are two widely used techniques
Deep sequence surveying of PCR amplified marker genes such as 16S rRNA
Whole genome shotgun metagenomics (entire complement of community DNA is sequenced en masse)
What are some caveats and limitations that can distort taxonomic distributions and frequencies observed in sequence dataset
-Choices relating to sample collection
-Sample storage and preservation
-DNA extraction
-Intro of contaminating microbial DNA during sample preparation
Examples of contaminants
Molecular biology grade water, PCR reagents and DNA extraction kits
Where are contaminating sequences greater
Contaminating sequences are greater in low biomass samples than in high biomass samples- critical tipping point where contaminating DNA becomes dominant in sequence libraries
