1 / 24

Affymetrix CytoScan HD array

Affymetrix CytoScan HD array. CytoScan HD vs current array. Current array (CGH based) patient + reference DNA required (two color) utilizes Cy dyes – ozone sensitive copy number probes only (135 K) CytoScan HD array (not CGH based) patient DNA only (single color)

galena-dunlap
Download Presentation

Affymetrix CytoScan HD array

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Affymetrix CytoScan HD array

  2. CytoScan HD vs current array • Current array(CGH based) • patient + reference DNA required (two color) • utilizes Cy dyes – ozone sensitive • copy number probes only (135 K) • CytoScan HD array (not CGH based) • patient DNA only (single color) • in silico reference based on >300 normal individuals and cell lines • utilizes phycoerythrin – not ozone sensitive • copy number probes (1.9 million) + SNP (750 K)

  3. Coverage • Average marker spacing: • ISCA genes – 384 bp • OMIM genes – 659 bp • X chromosome OMIM genes – 486 bp • RefSeq genes – 880 bp • Intergenic backbone – 1737 bp

  4. Single Nucleotide Polymorphisms (SNPs) ……..ATGC……… Allele A ……..ATAC……… Allele B

  5. SNP Non-polymorphic probes SNP Copy number + SNP arrays • SNPs limited to specific locations in genome – SNP • only arrays biased due to positional restrictions • Non-polymorphic (copy number) probes fill gaps to • allow broad coverage

  6. Improvements of CytoScan HD over Affy SNP 6.0 • Improved software • Much less noise • Probes empirically chosen based on performance • 20 million probes screened • All reagents centrally manufactured and provided as kits • Streamlined procedure – only onerestriction digest, ~half the steps, less hands-on time

  7. Other potential benefits of CytoScan • Affy filing for FDA clearance • CytoScan currently has best coverage on single array for both constitutional and neoplastic cases • Other large clinical labs switching to CytoScan (LabCorp, ARUP)

  8. Copy number + SNP arrays - detect copy number changes and allele frequencies • SNPs can detect uniparental isodisomy, consanguinity • more sensitive for detection of mosaicism • independent confirmation of copy number findings and better breakpoint determination

  9. Copy number + SNP array Copy # Allele peaks

  10. Normal Deletion Duplication A A A A A A B B B A A A B B B B B B A A A B B B A B B B A A A B AAA AA AA A AAB AB AB ABB B BB BB BBB Deletion Normal Duplication Normal

  11. SNP arrays more sensitive for detection of mosaicism Non-mosaic deletion Mosaic deletion

  12. CNC detection vs. reporting • Cytoscan software allows differential flagging in known clinically signficant critical regions vs. “backbone” regions • Can potentially detect smaller CNCs but doesn’t mean everything should be reported • Ex – LabCorp size cut-offs for reporting in backbone regions • Postnatal: >500 Kb gain, >200 Kb loss • Prenatal: >2 Mb gain, >1 Mb loss

  13. Uniparental disomy • Inheritance of two homologous chromosomes from one parent • isodisomy: two copies of the same homolog • heterodisomy: two different homologs • UPD mechanisms • meiotic non-disjunction with trisomy or monosomy rescue • post-zygotic mitotic recombination • Whole chromosome isodisomy vs. hetero/isodisomy

  14. Long continuous stretches of homozygosity (LCSH) with normal copy number Small deletion SNPs and consanguinity or UPD Chromosome 2

  15. Homozygous blocks of 1-3 Mb AA BB Copy number = 2 Normal allele homozygosity Whole chromosome isodisomy

  16. 13.5 Mb Copy # = 2 UPD or normal ?

  17. LabCorp studyPapenhausen et al. Am J Med Genet. 155A:757-68, 2011 • Homozygosity profiling by SNP array is screen for UPD • What LCSH size should be used as cut-off for recommending parental f/u for UPD? • Determined distribution of LCSH in patient population • Retrospectively analyzed eight confirmed UPD cases for LCSH

  18. Distribution of LCSH in 120 consecutive patients

  19. Eight known UPD cases • Two whole chromosome homozygosity • Six mixture of hetero/isodisomy • Single LCSH range: 13.5 – 48.4 Mb • One case with two LCSH of 11 and 11.2 Mb • Set LCSH UPD cut-off at >13.5 Mb (two LCSH with total of > 15 Mb) *LCSH in more than one chromosome = identity by descent

  20. Prospectively analyzed 13,000 patients by SNP array • 92 patients with UPD qualifying LCSH based on cut-offs • Parental f/u on 46 cases (mostly imprinted chromosomes) • Confirmed UPD in 29 cases • 14/30 whole chromosome isoUPD • 13/30 mixture of hetero/isoUPD • False-positive UPD 17 cases • Chromosome 3 and 11 pericentromeric region, 13q21

  21. LabCorp Study– other observations • False-positive cases had shorter average LCSH, greater freq near cen, no telomeric LCSH • No false-positive cases with qualifying telomeric LCSH • Sometimes see evidence of copy # mosaicism in trisomy/monosomy rescue; allele freq mosaicism in segmental UPD • Low likehood of false-negatives

  22. LabCorp current cut-offs for UPD (combined hetero/isodisomy or segmental UPD) • Single LCSH in one chromosome • >20 Mb interstitial or >10 Mb telomeric for non-imprinted chromosomes • >15 Mb interstitial or >8 Mb telomeric for known imprinted chromosomes

  23. SNP detection of consanguinity LCSH involving multiple chromosomes (regions of identity by descent)

  24. LabCorp cut-offs for consanguinityLCSH > 10 Mb

More Related