Copy Number Detection (Cytogenetics) Flashcards
What are the main cytogenetics methods for detecting copy number in DNA samples?
- G-banding
- FISH
- BAC-arrays
- Oligo-arrays
- SNP-arrays
Briefly describe how G-banding is used for copy number detection.
Manipulation of cell cycle to obtain metaphase cells enabling comparison of banding pattern between homologues.
Advantage of using G-banding for copy number detection.
- Whole genome screen
- Detects balanced/unbalanced rearrangements
- Provides positional information
- Detects mosaicism
- Relatively robust and inexpensive
- Determine structural
- rearrangements e.g. ring chr 20
Disadvantage of using G-banding for copy number detection.
- Low resolution (>5Mb)
- Labour intensive
- Slow turnaround time
- Unable to detect UPD
- Requires dividing cells and manipulation of the cell cycle
- Risk of cultural artefacts e.g. prenatal cases
- Some abnormalities (usually mosaic aneuploidies) not detected in cultured cells
Examples of using G-banding for copy number detection.
- To detect pre/postnatal aneuploidy
- To detect unbalanced rearrangements in dysmorphic child
- To detect balanced rearrangements in recurrent miscarriage couple
- Detection of abnormal clones in cancer
- Detect clinically significant structural rearrangementse.g. t(15;17) in APL
Briefly describe how FISH is used for copy number detection.
- Fluorescently labelled ssDNA probes are hybridised to specific denatured DNA sequences e.g. metaphase spreads or interphase nuclei.
- Probes typically target specific loci e.g. centromere, telomere, locus-specific, whole chromosome paint or BACs
Advantage of using FISH for copy number detection.
- Positional information if metaphases analysed
- Detects mosaicism
- Detects ploidy
- Aids interpretation of G-banding
- Fast turnaround time (able to perform shorter hybridisation in urgent cases e.g. ?T18)
- Higher resolution than G-banding
- Large number of individual cells can be examined
- Can be used to analyse single cells
- Probes available for almost any genomic region
Disadvantage of using FISH for copy number detection.
- Targeted test
- Probes can be expensive
- Cannot detect MCC in certain cases
- Cannot detect UPD
- Limited number of probes can be used at one time, only 2 or 3 colours possible
- Interphase FISH provides no positional info
- May require metaphases
- Atypical rearrangements may be normal by FISH e.g. some t(15;17) arrangements
- Microdupsmay be undetected due to limited resolution on metaphase spread
- Co-localisation can occur: two signals overlap and appear as one
- Cross-hybridisation can occur: probe binds to regions with repetitive sequences
Examples of using FISH for copy number detection.
- Aneuploidy screen
- Microdeletion/duplication detection
- Aid G-banding e.g.origin of marker chr
- Detection of cryptic rearrangements e.g.t(12;21) in cancer
- Used if G-banding fails e.g. AML screen using disease specific probes
- Detection of mosaicism
- Detection of abnormal clones/clonal evolution in cancer
- Gene amplification e.g. HER2 in br. Ca. or N-MYC in neuroblastoma
- Gene deletions e.g. TP53 or ATM
- PGD
- Variant gene fusionse.g. BCR-ABL1 in CML
- Post-transplant chimaerism monitoring
What is Array Comparative Genomic Hybridisation (arrCGH) ?
Array Comparative Genomic Hybridisation (CGH) is a molecular technique based upon c_ompetitive hybridisation_ of a test sample and a control sample to identify any possible gains or losses at particular sites of interest.
Within a genetic context, arrays can be used on either DNA or RNA samples
Give an overview of the array CGH protocol
- Patient and control DNA is labelled with fluorescent dyes and applied to the microarray (Cy3 vs Cy5)
- Labelled DNA is cleaned, combined and precipitated.
- Pre-hybridisation to Cot-1 DNA: hybridises to repetitive sequences in genome and prevents cross-hybridisation when on the array platform.
- Sample then hybed to array: Patient and control DNA compete to hybridise to the array probes. Automated and highly controlled in incubator or oven (rotates slide for even hyb)
- Dual wavelength laser scanner is used to measure fluorescent signals on array
- Software analyses the data and generates plots.
What are the expected Cy3 vs Cy5 ratios for various copy number changes detected by arrCGH?
- Equal hybridisation gives 1:1 ratio of red to green (the colours read from the fluorescence; after normalising for variation already mentioned).
- Deletion gives excess of control DNA sample colour.
- Duplication gives excess of patient DNA sample colour
How are the Cy3 vs Cy5 ratios interpreted to gain information about copy number value?
- In ratio world the raw values are 2.0 for a doubling of the test sample and 0.5 for half the test sample
- i.e. reduction ratios are squished between 1 and 0 whereas duplication ratios will be plotted further and further away from 1.
- This means that the plot is asymmetrical and exaggerates the difference between equal magnitude gains and losses
- Log transformation turns the equal hybridisation (ratio 1.0) value to 0.
- Means that the plot of deletions and duplications becomes symmetrical around 0.
- Doubling of test DNA = log(2)2 = 1
- Halving of test DNA = log(2)0.5 = -1
What are the log 2 values for heterozygous deletions and duplications detected by arrCGH?
Why are duplications ‘harder’ to detect by arrayCGH than deletions?
- To do with the log2 values generated for dels vs dups
- Deletion of test DNA = log(2)0.5 = -1
- Duplication of test DNA = log(2)1.5 = 0.58
- Thus, the magnitude of movement away from normal copy number is not as great for duplications
What key quality criteria are assessed when determining the success of an arrCGH experiment?
- Inclusion rate of 95+%, ie. the percentage of DNA spots (features) on the array that show good hybridisation – this is a measure of technical reproducibility
- Standard deviation of autosome of <0.075 – a measure of the variation of replicate features across the array – this is a measure of biological reproducibility
Briefly describe how BAC-arrays are used for copy number detection.
- Patient and reference DNA labelled with different fluorescent dyes
- Then hybridised to different BAC clones (on a glass slide) spread throughout the genome
What types of probes are used on BAC arrays and how are they produced?
- Large PAC, cosmid and fosmid clones of approx. 150-200kb in size are used (derived from the mapping stages of the human genome project).
- BAC clones are propagated in vectors in bacteria, purified and amplified and then spotted onto a glass slide using ultra fine needles.
- Multiple copies of each BAC are spotted onto the array and distributed across the array.
What are the advantages of using the large probes found on BAC arrays?
Due to the large size, BACs are very stable and hybridisation is specific (high signal to noise ratio).
Advantage of using BAC-arrays for copy number detection.
- Whole genome screen
- Can detect unbalanced copy no changes to ~1Mb resolution
- Can concentrate probes to particular regions of interest e.g. gene rich (can design custom arrays to target specific areas/genes as well as providing a lower resolution coverage over the whole genome)
- Simultaneously process large numbers of samples
- Cheap and reliable - still widely used for PGS (24sure)
Disadvantage of using BAC-arrays for copy number detection.
- No positional/structural information
- Doesn’t detect balanced rearrangements/ ploidy
- Lower resolution than other platforms (0.5-1Mb detection levels)
- Expensive
- Labour intensive
- Difficulty with interpreting some results
- Resolution not even across the genome
- Requires relatively large amounts of good quality DNA
Advantages of using Oligo-arrays for copy number detection.
- High resolution imbalance detection (~50-200Kb depending on coverage)
- Can concentrate probes to particular regions e.g. gene rich (can design custom arrays to target specific areas/genes as well as providing a lower resolution coverage over the whole genome)
- Simultaneously process large numbers of samples
- Analysis of single cells possible (with whole genome amplification)
- Uses DNA from uncultured or cultured material
Disadvantages of using oligo-arrays for copy number detection.
- No positional information obtained
- Doesn’t detect balanced rearrangements/ploidy
- Difficulty with interpreting some results-CNVs of unclear significance
- Expensive
- Labour intensive
- Requires relatively large amounts of good quality DNA
- Low-level mosaicism not detected.
Describe how the Illumina SNP array platform works.
- Bead based oligo probe ~50bp in length designed to be immediatley adjacent to a SNP site.
- A single base extension of a fluorescently labelled nucleotide that is complementary to the allele carried by the test DNA is then incorporated
- The result is the appropriately coloured signal.
- Beads are randomly distributed onto a slide so their genomic location has to be ‘decoded’ during the analysis
- Beads are very small in size enabelling millions of beads, and thus probes to be distributed onto a single slide giveng very high resolution arrays.
Describe how the Affymetrix SNP array platform works.
- Oligo probes are synthesised in situ on a solid slide
- The genomic location of the probe and it’s coordinates on the array are recorded during manufacturing
- The SNP site is positioned to be in the centre of the oligo
- There are probes for the major ad minor allele of each SNP
- The test DNA will bind with the highest affinity where there is a 100% with the probe.
- Thus, if hom then most binding will be on the major allele probe with only a small amount of test DNA binding to the mismatched minor allele
- If het then ~50:50 binding of the major and minor alleles is seen
What SNP array profile is characteristic of a normal copy number state i.e. diploid?
In a normal diploid individual, you get a balance of AA, BB and AB i.e. a normal allelic balance
The heterozygous line (really A-B) is also known as the allelic balance line.
What SNP array profile is characteristic of a heterozygous duplication?
- In this case there is a heterozygous gain of material we lose the allelic balance line as we never see the A and B alleles in equal proportions
- i.e. either have a gain of A or a gain of B = pushed out the tracks
- N.B. When we have a gain (i.e. 3 alleles choices , you get 4 lines)
What SNP array profile is characteristic of a heterozygous deletion?
- In this case there is a heterozygous loss of material.
- Here we loose the allelic balance line as either have a loss of A or a loss of B
- i.e. cannot have an AB heterozygous line and the tracks are pushed inwards.
- N.B. When we have a loss (i.e. 2 allele choices, you get 2 lines)
What SNP array profile is characteristic of a copy neutral Loss of Heterozygosity (LOH)?
Terminology, ‘absence of heterozygosity’ important as it does not imply any mechanism, see only AA, or BB patern, nothing in the AB heterozygosity track indicating heterozygosity.
