microarrays and next generation sequencing Flashcards
What is a microarray?
- collection of microscopic DNA spots attached to a solid surface (glass -like microscope slide).
Why do scientists use microarrays?
- used to measure the expression levels of large number of genes or to genotype multiple regions of a gene.
- to investigate which genes are activated and which genes are repressed when two populations of cells are compared, every gene is measured simultaneniously.
- each DNA spot contains a picamole of specific DNA sequence known as a probe.
What is next gen sequencing (sanger sequencing)
technology used to work out the order of nucleotides in entire genomes.
- cycle sequencing method
- one reaction per sequence
- highly accurate
- slow
- used until 2007s
- cost 3 billion to map human genome
- each base is a different colour
How does next gen sequencing work?
- DNA library construction : DNA is fragmented chemically, enzymatically, or physically (sonication).
=> library is a collection of random fragments. - Cluster generation : hybridise library to flow cell as a random process.
compare sanger sequence an next generation sequence.
- sanger sequence is analogue NGS produces digital readouts.
- Sanger sequence is one sequence read and NGS is consensus of multiple short read sequences.
What are applications of NGS?
-more efficient and cost effective to only sequence areas of interest, 80% of mutations in exon so it would make sense to only sequence exon - saves alot of money.
What is third generation sequencing?
- single molecule sequencing
- DNA passes through nanopore and base sequence is converted into an electrical current.
- very expensive
What are advantages and disadvantage of third gen sequencing?
advantage :
- no expensive machine required, the flowcell is machine itself and scaleable
disadvantage: - very expensive, high error rates and teach is still developing.
What are expression levels of all genes in my samples?
transcriptome
- discover the biology of your samples
- classify samples
- predict which class a sample belongs to
how do gene expression microarrays work?
- lots of copies of the same probe in a spot
- each spot gives the relative expression for one transcript
- detects all known transcripts in one sample.
Describe the process of expression profiling workflow.
two colour array:
- we isolate RNA from messenger RNA
- we label one in red, called Si5 and the other control in green called Si3
- we recreate cDNA using reverse transcriptase.
- we hybridize the cDNA of red dye and gree dye
- then we scan it and get different colour results.
- we look at relative colours so if both are roughly the same it will be yellow.
- if test sample is stronger it will be red and if control sample is much stronger it will be green
what are the steps in data analysis workflow (cell file)?
- feature extraction = getting the raw data from the array (called cell file)
- quality control = try adjust any issues about conc or quality of the sample
- normalisation = attempt to make the data look like everyone else.
- analysis
=> differential expression analysis between our test and control sample.
=> biological interpretation
=> submit data to public repository
How does a microarray work?
- you have 6 and a half million locations on each array and on each location we have millions of DNA strands, each strand is made up of 25 base pairs.
- we then add DNA fragment that has been tagged with fluorescence.
- anything that is complimentary to the DNA will hybridise
- when you shine a laser light on them the fluorescence will glow and be visible through the microscope .
Why do we microarray genes?
- look for expression of all genes on a cell or tissue
- look at SNPs genotyping and structural variations
What is hierarchal clustering?
- looking to see if there is a pattern in the data
- organises data with similar pattern into classes
- objects within the same class are more similar
What is quantitative PCR used for?
- to measure the amount of PCR product
- check microarray results
What must be done first with the microarray results to do qPCR?
- we need to amplify RNA in some way so we can see the relative quantity in our sample.
- we must amplify the RNA by converting it to DNA making it more robust so we can carry out PCR.
How do we convert RNA to cDNA so we can carry out PCR?
- using reverse transcriptase enzyme
- then we carry out PCR
Why must we also PCR our house keeping gene along with our gene of interest?
- house keeping genes are maintenance genes
- widely expressed in all cells in all tissues
- so act as internal controls for gene expression
What is a common house keeping gene?
beta actin.
What does DNA amplification plot for qPCR show?
-y axis fluorescence signal
-x axis shows cycle number
shows the inversely proportional relationship between sample copies and threshold value
- sample with highest copies of transcript = lowest threshold value (CT).
Why do we do qPCR?
- to independently confirm differences and levels between samples.
- probe binding is noisy and differences, detected that are not real <2 folds.
What can we use qPCR in clinics for?
- > predicting breast cancer
- > At low EPclin score, endocrine therapy (ET) alone is sufficient.
- > at higher scores ET and chemotherapy beneficial
- > Give only those who need chemotherapy the chemo treatment bc it has many unpleasant side effects, eg: hair loss.
What are SNP microarrays?
- GWAS are possible bc we can genotype large numbers of SNPs in large numbers of subjects.
- this is possible by using microarrays that hybridise with genomic DNA adajacent
- the SNP is then extended by one base that is fluorescently labelled and detected using high definition scanner.
