Whole Genome Sequencing Flashcards
Name 4 roles of WGS in Microbiology
Epidemiology
Detection of virulence markers
Detection of drug resistance
Vaccine development
What are the two most widely used NGS technologies?
Illumina sequencing
Thermofisher personal genome machine
Illumina sequencing uses a ‘reversible terminator chemistry’. Describe the three steps of this method.
- Library preparation: tagging each DNA fragment with adaptor and index sequences.
- Clonal amplification: primer sequences complimentary to the adaptor sequences are used to create clusters of NA on a solid phase. This is to create a signal strong enough for detection.
- Sequencing by synthesis.
How does the Thermofisher PGM identify sequences?
Detection is based on the change of pH of a solution on a semiconductor chip.
It is based on when a nucleotide is incorporated to a growing strand of nucleic acid a hydrogen ion is released. Due to the charge on the H+ ion a change in the pH of the solution will occur. The voltage generated from this is measured and allows recording of nucleotide in seconds.
How do you assemble a sequence from fragments, once you have generated them?
Compare it to a reference sequence or reference genome.
To do this small fragments of the genome (25 to 120bp) which overlap assembled to a larger part of the genome.
What software is available to help support genome assembly?
STAMPY or SAM are popular software for genome assembly.
They will map to a reference genome and any positions that differ. There may be gaps and work best of your sequence is closely related to the reference genome. For this to work well every species of microorganisms needs to be sequenced and available as a reference genome.
Can you assemble sequences without a reference strain?
Yes, this is de-novo assembly.
The DNA of the target organism is broken down into millions of small fragments which are then read on a DNA sequencer.
The gene sequences are then joined up or assembled through overlapping regions known as contigs (long continuous sequences).
This overcomes the issues with needing a reference strain, but the sequence needs to be aligned before comparisons can be made. Software such as Mauve can be used to align genomes.
What is the benefit of using a ‘gene by gene’ approach to NGS?
This is where example genes are retrieved from a genome using software such as BLAST. This is a useful technique when looking at generic markers which code for virulence or antimicrobial resistance. It can also be used for performing ID e.g. 16s rDNA.
What considerations of e-infrastucture are needed prior to implementing NGS.
Need for high performance computers and software and large scale data storage (e.g. network or cloud solutions).
A single illumnia run can generate 1.8TB of data, data assembly requires a significant amount of RAM. These exceed the capabilities and capacity of a standard NHS PC.
Most key manufacturers would audit the e-infrastucture and offer solutions.
What are the advantages of using NGS over Sanger for HIV resistance testing?
Sanger is 3 tests: protease RT, integrate and V3 loop of hiv envelope Vs 1 read of WGS which results in a lower cost of WGS.
NGS is highly sensitive and detects drug resistant variants in mixed populations.
What is the Influenza mutation conferring oseltamivir resistance
H275Y
