20.02.05 Copy number detection techniques

Question 1

What is G-banding?

Answer 1

Manipulation of cell cycle to obtain metaphase cells enabling comparison of banding pattern between homologues

Question 2

Advantages of G-banding

Answer 2

1) Whole genome screen
2) Detects balanced/unbalanced rearrangements
3) Provides positional information
4) Detects mosaicism
5) Relatively robust and inexpensive
6) Determine structural rearrangements e.g. ring chr 20

Question 3

Disadvantages of G-banding

Answer 3

1) Low resolution (>5Mb)
2) Labour intensive
3) Slow turnaround time
4) Unable to detect UPD
5) Requires dividing cells and manipulation of the cell cycle
6) Risk of cultural artefacts e.g. prenatal cases
7) Some abnormalities (usually mosaic aneuploidies) not detected in cultured cells

Question 4

What do we use G-banding for?

Answer 4

1) To detect pre/postnatal aneuploidy
2) To detect unbalanced rearrangements in dysmorphic child

3) To detect balanced rearrangements in recurrent miscarriage couple
4) Detection of abnormal clones in cancer
5) Detect clinically significant structural rearrangementse.g. t(15;17) in APL

Question 5

What is FISH?

Answer 5

• Fluorescently labelled ssDNA probes are hybridised to specific denatured DNA sequences (metaphase spreads or interphase nuclei)
• Probes can be centromere, telomere, locus-specific, whole chromosome paint or BACs

Question 6

Advantages of FISH

Answer 6

1) Positional information if metaphases analysed
2) Detects mosaicism
3) Detects ploidy
4) Aids interpretation of G-banding
5) Fast turnaround time (able to perform shorter hybridisation in urgent cases e.g. ?T18)
6) Higher resolution than G-banding
7) Large number of individual cells can be examined
8) Can be used to analyse single cells
9) Probes available for almost any genomic region

Question 7

Dsiadvantages of FISH

Answer 7

1) Targeted test
2) Probes can be expensive
3) Cannot detect MCC in certain cases
4) Cannot detect UPD
5) Limited number of probes can be used at one time, only 2 or 3 colours possible
6) Interphase FISH provides no positional info
7) May require metaphases
8) Atypical rearrangements may be normal by FISH e.g. some t(15;17) arrangements
9) Microdups may be undetected due to limited resolution on metaphase spread
10) Co-localisation can occur: two signals overlap and appear as one
11) Cross-hybridisation can occur: probe binds to regions with repetitive sequences

Question 8

What do we use FISH for?

Answer 8

1) Aneuploidy screen
2) Microdeletion/duplication detection
3) Aid G-banding e.g.origin of marker chr
4) Detection of cryptic rearrangements e.g.t(12;21) in cancer
5) Used if G-banding fails e.g. AML screen using disease specific probes
6) Detection of mosaicism
7) Detection of abnormal clones/clonal evolution in cancer
8) Gene amplification e.g. HER2 in br. Ca. or N-MYC in neuroblastoma
9) Gene deletions e.g. TP53 or ATM
10) PGD
11) Variant gene fusionse.g. BCR-ABL1 in CML
12) Post-transplant chimaerism monitoring

Question 9

What is QF-PCR?

Answer 9

• Quantification of polymorphic repeat sequences to determine copy number
• Uses fluorescently labelled primers

Question 10

Advantages of QF-PCR

Answer 10

1) Detects mosaicism (above 15%), triploidy and aneuploidy
2) Requires little starting material
3) Fast turnaround times
4) Relatively inexpensive
5) High resolution/High throughput

Question 11

Disadvantages of QF-PCR

Answer 11

1) No positional information obtained
2) Targeted test
3) MCC may prevent interpretation of results
4) May not detect low-level mosaicism (<15%)
5) Limited ability to detect multiple targets in a single assay due to spectral overlap of dyes

Question 12

What do we use QF-PCR for?

Answer 12

1) Can be used to aid interpretation of G-banding e.g. identification of marker chromosomes or interpretation of complex rearrangements
2) Prenatal aneuploidy screening
3) Detection of trisomy in pregnancy loss or PND
4) Post-transplant chimaerism monitoring

Question 13

What is Real-time qPCR?

Answer 13

• PCR amplification in which the amount of product is measured during each PCR cycle
• Uses fluorescent dyes (non-specific e.g. SYBR green) or probes (sequence specific e.g. Taqman)
• During the exponential phase the amount of amplified product is proportional to the amount of starting material.

Question 14

Advantages of real-time qPCR

Answer 14

1) Quantitative
2) Very high resolution
3) Rapid and easy to perform
4) Fast turnaround time
5) Requires little starting material
6) Single cell analysis possible
7) Detects UPD if methylation specific Res are used
8) Post-PCR processing is eliminated, reducing labour, cost and possibility of cross-contamination

Question 15

Disadvantages of real-time qPCR

Answer 15

1) No positional information obtained
2) Targeted test
3) Specialist equipment required – thermal cycler and optical instrument to measure fluorescence
4) Unlikely to detect low level mosaicism
5) Multiple reactions required to examine multiple loci
6) Sequence-specific probes expensive
7) Non-specific fluorescent dyes such as a SYBR green intercalate with any dsDNA which may lead to false positive signals

Question 16

What do we use real-time qPCR for?

Answer 16

1) Quantify gene expression (mRNA)
2) Minimal residual disease monitoring of gene fusion products e.g. PML-RARA in AML, BCR-ABL1 in CML
3) Detection of mutations e.g. JAK2 V617F, NPM1/FLT3-ITD
4) Detection of microdels/dups e.g. 22q11
5) Detection of subtel del/dup

Question 17

What is MLPA?

Answer 17

• PCR-based multiplex reaction allowing amplification of ~40-45 targets in a single reaction.
• Detects copy number imbalances in gDNA and RNA sequences
• Probes anneal to target and are then amplified and quantified

Question 18

Advantages of MLPA

Answer 18

1) Can test for ~40-50 different imbalances in one reaction
2) High throughput technique
3) High resolution– detects sequences of only ~60nt-can therefore detect dups/dels of a single exon
4) Fast turnaround time
5) Inexpensive
6) Requires little starting material
7) Detects methylation

Question 19

Disadvantages of MLPA

Answer 19

1) Targeted test
2) More labour-intensive than QF-PCR
3) Can’t be used for single cell screening i.e. PGD
4) No positional information obtained
5) Doesn’t detect balanced rearrangements
6) Cannot detect low-level mosaicism (<20-30%)&unreliable detection at higher levels
7) Unreliable for detection of maternal cell contamination
8) Cannot detect triploidy (69,XXX) and unreliable for other triploidy detection
9) Analysis gives average copy number per cell. Tumour analysis difficult if sample contains <50% cancer cells
10) SNPs under probes can prevent binding (false positive result)
11) Sensitive to contaminants
12) Not easily scaled up

Question 20

What are oligo arrays?

Answer 20

• Synthetic oligos used in hybridisation stage which are smaller than BAC clones therefore allowing greater coverage

Question 21

Advantages of oligo arrays

Answer 21

1) High resolution imbalance detection (~50-200Kb depending on coverage)
2) 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)
3) Simultaneously process large numbers of samples
4) Analysis of single cells possible (with whole genome amplification)
5) Uses DNA from uncultured or cultured material

Question 22

Disadvantages of Oligo arrays

Answer 22

1) No positional information obtained
2) Doesn’t detect balanced rearrangements/ploidy
3) Difficulty with interpreting some results-CNVs of unclear significance
4) Expensive
5) Labour intensive
6) Requires relatively large amounts of good quality DNA

Question 23

What do we use Oligo arrays for?

Answer 23

1) Genome screen of individuals with idiopathic mental retardation, dev. del, dysmorphism
2) Interpretation of abnormal karyotypes

Question 24

What are SNP arrays?

Answer 24

• Array-based technology as above but platform also has additional SNP specific oligos present throughout the genome

Question 25

Advantages of SNP arrays

Answer 25

1) High resolution
2) Can detect copy number changes and also genotype to reveal LOH and UPD

Question 26

Disadvantages of SNP arrays

Answer 26

1) Doesn’t detect balanced rearrangements
2) No positional/structural information
3) Coverage dependent on SNP locations
4) Difficulty with interpreting some results
5) Expensive
6) Labour intensive

Question 27

What do we use SNP arrays for?

Answer 27

1) Screen for LOH in cancers
2) Possible application to detect UPD
3) Could elucidate non-paternity or incidental findings

Question 28

What is NGS?

Answer 28

• A number of different methods are employed, but all involve step wise addition of nucleotides to fragmented DNA.
• WGS + WES have become primary strategies for NGS in CNV detection

Question 29

Advantages of NGS

Answer 29

1) Very high resolution (single base change detection)
2) Genome wide or targeted
3) Provides positional information
4) Detects UPD and LOH
5) High throughput
6) Detects balanced/unbalanced rearrangements (paired-end/mate-pair seq)
7) More tolerant of poor quality DNA
8) Detection of both single base variants and CNVs in a single assay
9) WES is lower cost, higher coverage + less complex data analysis than WGS
10) Exome represents a highly function enriched subset of human genome + CNV in exome are more likely to be disease causing aberrations than in nongenic regions

Question 30

Disadvantages of NGS

Answer 30

1) Difficulty with interpreting results-CNVs of unclear significance
2) Vast amount of data obtained
3) Requires a lot of starting material
4) Expensive
5) Labour intensive
6) Not suitable for small genes
7) Need to confirm abnormal results
8) WES data introduces biases due to hybridisation which doesn’t exist in WGS data

Question 31

What do we use NGS for?

Answer 31

1) Targeted genome resequencing to study a particular gene(s) or disease
2) Reduced representation sequencing to e.g. generate a SNP map of the human genome
3) Transcriptome sequencing to study gene expression profiling
4) Gene panels e.g.TruSight Cancer Panel (96 genes)
5) Whole exome sequencing – assessment of all coding regions (DDD project)
6) WES targeted to protein coding regions (less than 2% of the genome)
7) WES used for identifying clinically relevant aberrations in cancer which is challenging due to sequence data, WES technical problems andTumour complexity
8) Copy number detection is a relatively new use of this technology

Question 32

Principle of MLPA

Answer 32

• DNA is hybridised to probe sets
• Each probe set consists of 2 halves:
  1) target specific sequence (20-30 nucleotides) (blue) flanked by a universal primer (black).
  2) target specific sequence (25-43 nucleotides) (blue) flanked by a universal primer (black)
• BUT in-between is a random fragment of between 19-370 nucleotides, this is known as the stuffer sequence (green)
• The stuffer sequence is a different length for each probe pair and allows for the generation of different sized products for electrophoretic resolution.
• The target specific sequences are located directly adjacent to one another, therefore when the probes bind to the DNA they can be joined together using a ligase.
• This generates a contiguous probe flanked by universal primers.
• All ligated probes can then be amplified in a single PCR reaction using the same universal PCR primers.
• It is therefore the probes and not the target sequences that are amplified.
• Unbound probes will not be amplified, because they only contain one primer sequence.
• The amount of ligated probe produced is proportional to the copy number of the target region
• Following PCR amplification, comparing the relative peak heights to a control sample can indicate changes in copy number.

Question 33

MLPA technique

Answer 33

1) Denaturation of the genomic DNA
2) Hybridisation of the probes to the sample.
3) Ligation of the probes
4) PCR amplification of the ligated probes using universal primers
5) Separation of the amplified products by capillary electrophoresis
6) Analysis and quantification of ratios for each probe
- 0.5 (range 0.3-0.7) = HET deletion
- 1 (range 0.7-1.3) = Normal
- 1.5 (range 1.3-1.7) = HET duplication
- N.B. Control fragments for DNA amount (Q fragments), denaturation, and X and Y markers are included
- Also get MS-MLPA

Question 34

NGS for CNV detection

Answer 34

• Current NGS technologies generate billions of bases of accurate nucleotide sequences in short reads (50–250 bp)
• Several new tools have been developed to enable discovery of CNVs from NGS data
• Each of these tools have different strengths and weaknesses in their applicability and suitability for NGS data, and no single tool is capable of identifying the full range of DNA variation