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
