Week 5 High-throughput Screening And Sequencing Methods Flashcards
What is transcriptomics?
The study of overall gene expression - without targeting specific genes of interest as the choice of genes can sometimes limit the outcomes of any study
What are microarrays?
The hybridisation of probe and target sequences
What are the targets for microarrays?
DNA or cDNA
Roughly how do microarrays work (4 steps)
- Oligonucleotide robe is a known sequence and is immobilised onto a solid support
- Target molecules are labelled with a fluorescent marker
- Knowledge of the probe sequence allows gene identification
- Intensity of fluorescent signal indicates relative abundance of transcript in the sample
Name 3 benefits of microarrays?
- Can spot known spot unknowns form genes sequences in whole-genome
- Commercially available gene chips for commonly assayed organisms
- 100s and 1000s sequences targeted at once
- Multiple dyes possible
Name 2 disadvantages of microarrays
- Limit set by optical resolution and fluorescent carry over
- Issues with cross-hybridisation
- Semi-quantities at best - needs validation for qPCR
- All data needs to be independently tested - prone to false positives and false negatives
- Cost
- Archiving large volumes of data
A study by Biscontin et al. 2019 used a microarray to show what about Antarctic Krill?
That energy storage pathways appear to regulated by the endogenous clock in accordance with their ecological relevance in daily energy managing
Do sequencing technologies occur before a microarray?
Yes
Name 3 NGS technologies?
- Illumina sequencing
- Hydrogen Ion sequencing
- Single molecular real-time sequencing
- Nanopore sequencing
What is the study of metagenomics?
The study of a genome from a community not from pure culture
What is DNA sequencing and what does it allow us to do?
The acquisition of the sequence of nucleotides, allowing us to locate regulatory and gene sequences, making comparisons and identifying mutations
In 1970, two methods of sequencing were developed, what were they called?
- Chemical cleavage methods
2. Chain termination method (Sanger sequencing)
What signature thing does Sanger sequencing use, and how does it work?
It uses dideoxynucleotides which contain a hydrogen on carbon 3’ instead of OH. This prevents the addition of further nucleotides as a phosphodiester bond cannot form
What type of DNA does Sanger sequencing require?
Pure DNA
How does Illumina sequencing work?
- Genomic DNA is prepared as random fragments of genomic DA and ligate adapters are added o both ends of the fragments
- DNA is attached to the surface
- Bridge amplification - unlabelled nucleotides are added
- The double stranded molecules are denatured
What sort of sequencing does Illumina use?
Cyclic reversible chain termination method - detects single bases as they are incorporated into growing DNA strands
What technology does hydrogen Ion sequencing use and what does it have that differs to other technologies?
It uses a semiconductor technology and uses ion sensing (H+) instead of fluorescent tags
How does hydrogen ion sequencing work?
Everytime a new base is added it releases a H+ which changes the pH and the proton signal is detected
What are the third generation sequencing techniques?
- Single molecule real time
2. Nanopore sequencing
How does Single Molecule Real-Time (SMRT) sequencing work?
- Occurs in wells which allow detection of fluorescent light flashes only far enough to see a single molecule of DNA polymerase adding nucleotides onto a single DNA chain
- As the nucleotide is added onto the growing DNA chain, the flash of light is detected through the bottom of the zero-mode waveguide
What are the benefits of SMRT sequencing?
No PCR is required and it can read long sequence lengths
How does Nanopore sequencing work?
- Nanopore are small openings in a membrane that only allow one molecules through at a time
- There is a charge separation between compartments
- A detector measures how much the current is reduced (DNA is - charged). As each base alters the current by different amount (G>C>T>A) so the detector can sequence
How can you improve long read sequencing in metagenome assemblies?
While Illumina metagenome assembly yields good coverage, contiguity was greatly improved by Illumina+ONT and Illumina+PacBio hybrid assemblies
