Genomic technologies Flashcards
What technologies do we use to analyse genomes, trandscriptomes or methylomes?
Microarrays
Sequencing techniques
Why are microarrays important for?
Identifying disease mechanisms
Locating which genes are switched on or off
Expression of genes following therapy
What are methods to carry out analysis?
Amplification based methods - amplify DNA or RNA (Q-PCR)
Hybridisation based methods - DNA microarrays
Sequencing based methods - next generation sequencing
What are the advantages of amplification based methods?
Extremely sensitive
What are the disadvantages of amplification based methods?
Error prone
Specific primers needed
Can be expensive
What are the advantages of hybridisation based methods?
Sensitive
Economical
Easy to analyse
High throughput
What are the disadvantages of hybridisation methods?
Limited to array content
What are the advantages of sequencing based methods?
Unlimited content
Highly parallel analyss
Base level sensitivity
What are the disadvantages of sequencing based methods?
Costly
Difficult to analyse
What is essential to determine before using a microarray?
The quality and integrity of the sample
What information do techniques of microarrays provide?
Genotyping
Methylation
Expression of genes
What samples can micrarrays be used on?
Gene
Exon
Transcriptome
Describe how a microarray is carried out
- Microarray chips contain mRNA sequences complementary for certain genes
- Scientists extract normal and abnormal cells and label them with different colours
- The mRNA transcribed from the genes from both cells will bind to their complementary mRNA sequences
How do we interpret a microarray chip?
If a certain gene is upregulated in the abnormal cell, the colour of the abnormal cell will predominate in that well
If a certain gene is downregulated in the abnormal cell, the colour of the normal cell will predominate in that well
If a certain gene is expressed equally in both cells, the colour of that well will be an even mixture of the two colours
What is microarrays used for currently?
Expression of genes - Studying gene expression patterns in tumours over time
Genotyping - detecting SNPs and structural variations
Methylation - detection of aberrant methylation patterns and biomarkers
Which cancer have been identified through microarrays?
Lymphoblastic leukaemia
Breast cancer
Prostate cancer
Lung cancer
Colon cancer
Microassay principles
- Synthesised or spotted oligonulceotide/cDNA arrays on glass 50 bases in length
- Hybridization of RNA samples
- Labelling
- Washing
- Signal amplification
- Detection
What is the output of expression microarrays?
Relative expression of genes
What is the output of genotyping microarrays?
Genotype for a given probe
What is the output for methylation microarrays?
Relative levels of methylation - yes or no
Important factors to consider when designing a microarray experiment
Sample requirements
Quality control
Array types
Costs
Time-frames
What are affymetrix microarrays?
Special microarrays designed to bind to genes, exons and transcriptomes
Contain up to 1 million different oligonulceotides, which are repeated continuously and represent known locations on the genome
What do affymetrix microarrays allow us to do?
Observe which genes are upregulated or downregulated in certain conditions
How are agilent microarrays different from affymetrix arrays?
You can look at the whole genome through agilent microarrays
Can only look at the exome, genome or transcriptome on affymetrix microarrays
What is the normal processing time for microarrays?
Around 5 days
What is normalisation?
Process of adjusting individual hybridisation intensities
Why must data be normalised?
Procedures have different starting quantities of RNA
Dyes may be different at labelling
Unequal probe hybridization
What are the 3 aims of microarray normalisation?
Preserving biological variation between samples
Minimising experimental variation
Allowing comparison of different experiments
What percentage of DNA is coding?
0.1%
What are GWAS?
Looks at many common genetic variants in different individuals to see if any variant is associated with a trait
Typically focus on the associations between SNPs and major diseases
What is loss of heterozygosity?
Loss of the entire genes and the surrounding chromosomal region
Common in cancer = absence of a functional tumour suppressor gene in the lost region
What is copy number variation?
A form of structural variation
Alterations of the DNA of a genome that results in the cell having an abnormal variation in the number of copies of one or more sections of the DNA
What type of assay is illumina?
Genotyping assay
What are the 4 steps to carrying out an illumina assay?
- Sample prep - adaptors added to allow the strand to bind to the complementary sequences on the lanes on the Illumina
- Cluster generation - sample is amplified through hybridisation and bridge synthesis
- Sequencing by synthesis - single fluorescent nucleotides bind to the formed sequences, releasing a fluorescent signal
- Data analysis
What does the intensity of fluorescence in Illumina reveal?
Information about the allelic ratio
Why are microarrays commonly used over illumina?
Cheap
Fast
High throughput
What is the main disadvantage of microarray technology?
Cannot sequence the whole DNA sample
Disadvantages of Illumina
More expensive
Low throughput
Needs bioinformatician to analyse data
