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genomics notes > microarrays and next generation sequencing > Flashcards

microarrays and next generation sequencing Flashcards

1
Q

What is a microarray?

A
  • collection of microscopic DNA spots attached to a solid surface (glass -like microscope slide).
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2
Q

Why do scientists use microarrays?

A
  • 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.
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3
Q

What is next gen sequencing (sanger sequencing)

A

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
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4
Q

How does next gen sequencing work?

A
  1. DNA library construction : DNA is fragmented chemically, enzymatically, or physically (sonication).
    => library is a collection of random fragments.
  2. Cluster generation : hybridise library to flow cell as a random process.
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5
Q

compare sanger sequence an next generation sequence.

A
  • sanger sequence is analogue NGS produces digital readouts.
  • Sanger sequence is one sequence read and NGS is consensus of multiple short read sequences.
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6
Q

What are applications of NGS?

A

-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.

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7
Q

What is third generation sequencing?

A
  • single molecule sequencing
  • DNA passes through nanopore and base sequence is converted into an electrical current.
  • very expensive
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8
Q

What are advantages and disadvantage of third gen sequencing?

A

advantage :

  • no expensive machine required, the flowcell is machine itself and scaleable
    disadvantage:
  • very expensive, high error rates and teach is still developing.
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9
Q

What are expression levels of all genes in my samples?

A

transcriptome

  • discover the biology of your samples
  • classify samples
  • predict which class a sample belongs to
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10
Q

how do gene expression microarrays work?

A
  • 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.
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11
Q

Describe the process of expression profiling workflow.

A

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
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12
Q

what are the steps in data analysis workflow (cell file)?

A
  1. feature extraction = getting the raw data from the array (called cell file)
  2. quality control = try adjust any issues about conc or quality of the sample
  3. normalisation = attempt to make the data look like everyone else.
  4. analysis
    => differential expression analysis between our test and control sample.
    => biological interpretation
    => submit data to public repository
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13
Q

How does a microarray work?

A
  • 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 .
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14
Q

Why do we microarray genes?

A
  • look for expression of all genes on a cell or tissue

- look at SNPs genotyping and structural variations

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15
Q

What is hierarchal clustering?

A
  • 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
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16
Q

What is quantitative PCR used for?

A
  • to measure the amount of PCR product

- check microarray results

17
Q

What must be done first with the microarray results to do qPCR?

A
  • 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.
18
Q

How do we convert RNA to cDNA so we can carry out PCR?

A
  • using reverse transcriptase enzyme

- then we carry out PCR

19
Q

Why must we also PCR our house keeping gene along with our gene of interest?

A
  • house keeping genes are maintenance genes
  • widely expressed in all cells in all tissues
  • so act as internal controls for gene expression
20
Q

What is a common house keeping gene?

A

beta actin.

21
Q

What does DNA amplification plot for qPCR show?

A

-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).

22
Q

Why do we do qPCR?

A
  • to independently confirm differences and levels between samples.
  • probe binding is noisy and differences, detected that are not real <2 folds.
23
Q

What can we use qPCR in clinics for?

A
  • > 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.
24
Q

What are SNP microarrays?

A
  • 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.
25
Q

What in the SNP microarrays spots?

A
  • lots of copies of the same single - stranded oligonucleotide - a ‘probe’
  • each probe is for genotyping one SNP.
26
Q

How does SNP microarrays work?

A
  • the two probes when they hybridise with complementary bases they give off a colour.
    => probes to detect rs852641073 = yellow
    => probe to detect rs121908424 = red
    => we have millions of probes across array.
    => each spot is a genotype for one SNP.
27
Q

Why do we do array CGH (comparative genome hybridisation) ?

A
  • looking for structural abnormalities (human karyotypes)
28
Q

What are we looking for?

A
  • structural variants (SV)
  • copy number variants (CNV)

Examples of these include:
-deletions, insertion, inversion, tandem duplication, copy number variant.

29
Q

what is the method of array CGH?

A
  1. you have a patient(green) sample and a control (red) sample which you mix.
  2. then you hybridise this to array of DNA fragment
  3. then you laser scan, the colours show SNV/CNVs if it is yellow = no change.
    if it is green there has been duplication and if it stays red there has been deletion.
30
Q

What is an example of a disease causing SNP?

A
  • not all SNP are disease causing but an example of one that is :
    micro deletion on 16p13.3, 120kb deletion causing Rubinstein - Taybi syndrome.
31
Q

What are advantages of array CGH?

A

used for pre-natal testing

=> array CGH is able to examine all 46 chromosomes in greater detail than other techniques
=> so we can identify small pieces of additional or missing chromosome.

32
Q

What are disadvantages of array CGH?

A

=> some genetic conditions are not caused by chromosome imbalance but by mistake in individual genes.
=> array CGH cannot detect the tiny changes in individual genes.

33
Q

What are expression levels of all genes in my sample?

A

The transcriptome

genomics notes (18 decks)

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