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Array CGH. Louise McClelland 10 th November 2010. Introduction. Background Technical details Applications Interpreting CNV. Key terms. Array comparative genomic hybridisation ( aCGH ) Copy number variants (CNV) Catch all term for any copy number change Copy number polymorphisms (CNP)

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array cgh

Array CGH

Louise McClelland

10th November 2010

  • Background
  • Technical details
  • Applications
  • Interpreting CNV
key terms
Key terms
  • Array comparative genomic hybridisation (aCGH)
  • Copy number variants (CNV)
    • Catch all term for any copy number change
  • Copy number polymorphisms (CNP)
    • A CNV likely to be benign
  • BAC array
  • Oligoarray
  • Microdeletion
  • Microduplication
  • Frontline testing
array comparative genome hybridisation acgh
Array Comparative Genome Hybridisation (aCGH)
  • Technique for identifying loss or gain of subchromosomal band regions (microdeletions and duplications)
  • Compares differences between the patient DNA and a reference DNA
  • Based on the cytogenetic technique CGH
    • CGH the target ‘reference’ DNA is a metaphase chromosome
    • aCGH target reference DNA is normal genomic DNA in specific locations on a microarray
  • The 2003 Genetics white paper funding was used to establish many UK aCGH services
  • Patient DNA is fragmented, fluorescently labelled then hybridised to reference (“normal”) DNA which has an alternative fluorescent label
bac vs oligo
BAC vs Oligo
  • BAC – Large insert clones: ~100-200Kb
  • Oligo – Synthetic “in silico” probes; ~60-80 “mer”
  • BAC
    • Data is generally more “transparent” than oligoarrays
    • Accurate copy number determination from single data point
  • Oligos
    • Advantages
      • Higher theoretical resolution
      • Cheaper to produce
      • More flexible (more amenable to “multiple format design”, more options for probe selection)
    • Disadvantages
      • Smoothing algorithms required
      • Single data point can not be used for copy no.
      • Increased manipulation of data can introduce artefact



Karyotyping 3-10 Mb

Sequencing /MLPA





  • Balance required between sensitivity and specificity
  • BlueGnome
  • Agilent
  • Roche Nimblegen
hybridisation options
Hybridisation options
  • Dye swap
    • Hybridise patient DNA to reference ‘normal’ DNA
    • Repeat in opposite colours
    • 2 arrays required
  • Loop
    • 3 patient samples labelled to each of 2 colours
    • Each sample hybridised against the other 2
    • Only 1 array per patient
  • Patient/Patient
    • Hybridised patient to another phenotypically mismatched patient
    • The dye ratios inform ownership of any imbalances detected
    • This method is employed by the Guys lab
analysis normalisation
Analysis: Normalisation
  • Spatial ratio bias algorithm
    • Removes bias arising from hybridisation and scanning
      • e.g. scanner bias causing one side of array to be brighter
  • GC bias algorithm
    • Data tends to be noisy towards GC rich regions
    • Different clones corrected by GC bias algorithm differently depending on GC content
analysis exclusion
Analysis: Exclusion
  • Individual replicates with a substantially different log2 ratio to the other replicates
  • Low intensity spots with an inadequate signal to noise ratio
  • Spots with debris or uneven signal across spot surface
analysis quality control
Analysis: Quality control
  • 95% of clones must have worked to pass QC
  • For a copy number change to be significant it must exceed 3 SD from the mean
abnormal acgh results
Abnormal aCGH results
  • Due to the volume of data generated a high number of uncertain results may be obtained
  • An abnormal result should always be validated using an alternative method;
  • FISH
    • Gives positional information
    • Resolution can inhibit use in duplications (two fluorescent signals may appear as one because close together)
  • MLPA
    • Mental retardation kits P064-MR1, P094-MR2
    • Broad subtelomere kits P069, P036, P070
    • Centromere kits P181, P182
    • Microdeletion syndrome kits P245, P297
    • Reference kits P200, P300 (to be used with bespoke MLPA probes)
  • QF-PCR
applications in a diagnostic setting
Applications in a diagnostic setting
  • Currently used in combination with karyotyping for selective;
    • Constitutional postnatal cases
    • Fetal pathology cases
    • Leukaemia cases
  • Used for more widespread referrals
    • Prenatal testing of abnormal ultrasound cases (being validated)
    • Prognostics and residual disease monitoring in cancers
acgh frontline testing
aCGH: Frontline testing
  • For mental retardation (MR), autistic spectrum (ASD), multiple congenital abnormalities (MCA)
    • These referrals represent the largest referral number to cytogenetics
  • Advantages over conventional testing
    • Higher sensitivity than karyotyping
      • 15-20% compared to 3% if Down syndrome is excluded
    • Better resolution that FISH for reciprocal duplications of known microdeletions e.g.
      • 7q11 Williams Beuren
      • 17p11.2 Potocki-Lupski
  • Disadvantages
    • Increased unknown clinical significance CNV
    • Will not detect:
      • Truly balanced translocations
      • Low level mosaicism (BAC arrays can detect 10%, Oligos 20-30%)
recent publications
Recent publications
  • April 2010 – Ahn et al.
    • Guys lab
    • Their experience of using aCGH as a frontline test (since May 2008)
  • May 2010 – Miller et al.
    • Reviewed 33 studies of DD/ID, ASD, MCA, 21, 698 patients
  • Both papers suggest using oligoarrays
guys experience ahn et al 2010
Guys experience (Ahn et al., 2010)
  • Oligoarrays as first line test 1169 patients and 22% abnormality rate
  • 89% of abnormalities wouldn’t have been detected by karyotyping
  • 14% of imbalances detected fell within known sites of recurrent microdeletion/dups
  • Low parental sample received for follow up (40% of those requested)
  • Reduced costs by;
    • Patient/patient hybridisation method
      • Normal arrays – only 1 analysis was required
      • Risk of reciprocal imbalance in the two patients minimal (but don’t get a lot of clinical info)
    • Batching and robotics, streamlined IT systems
  • Offer aCGH at same cost as karyotype analysis
  • Karyotyping to remain for;
    • Turners, trisomy 13,18, 21, Kleinfelter
    • Distinguish free trisomy from translocateion associated trisomy
    • Multiple miscarriage – for balanced translocations
deciphering developmental disorders ddd project
Deciphering developmental disorders (DDD) project
  • 12,000 UK children with abnormal development
  • Recruited from all over the UK
  • Aim to transform clinical practice for children with abnormal development




Exome sequencing

Investigate and report back to Regional labs for confirmation work

Sequencing variant

interpreting cnvs
Interpreting CNVs
  • Pathogenic deletions more common than duplications – perhaps because duplications are harder to validate
  • Consider size of imbalance
    • Most pathogenic CNV >1 Mb and de novo (Miller et al., 2010)
  • Has a del/dup of any of the region been seen before
      • Can publish data in DECIPHER if consent is given
      • DECIPHER v.5 Haploinsufficiency LOD score (Huang et al .,2010 )
        • Help prioritize genes for investigation
    • Database of Genomic Variation (DGV)
      • Comprehensive info regarding benign and unlikely to be pathogenic CNVs
  • Ensembl search for genes in region
    • Any likely candidate genes for clinical features
    • Consider neighboring genes and regulatory elements
  • Literature search
    • ID overlapping del/dup?
      • Similar clinical features
    • Do literature search for each gene in / around region
  • Trio studies
    • Is the variant de novo
      • Exclusion of balanced parental rearrangements by FISH is recommended for apparently de novo abnormalities
    • If inherited, does the parent show any features

Frequency 1 in 5000

Most common microdeletion syndrome in Ahn et al., 2010 (15 cases, 0.62%)


“Our ability to discover genetic variation is running ahead of our ability to interpret them”

Huang et al., 2010

  • More unknown clinical significance variants; reporting and interpreting challenges 
  • This will improve as more data published more CNP will be confirmed etc
  • Change to infrastructure of diagnostic genetics labs
    • Reduce referral numbers for karyotyping, fragile X, PWS (will become a reflex tests)
    • More integration between molecular and cyto as karyotyping reduced
    • Increased demand on high spec hardware and IT storage solutions
  • ACC BPG for constitutional aCGH analysis
  • Ahn et al., Molecular Cytogenetics, 3, 9, 2010
  • Huang et al., PLOS, 6(10), e1001154, 2010
  • Miller et al., Am J Hum Genet, 86, 749-764, 2010
  • Oostlander et al., Clin Genet, 66, 488-495, 2004
  • Sagoo et al., Genet in Med, 11(3), 139-146, 2009