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.
