Comparison of three single-cell whole genome amplification (WGA) methods for detection of genomic aberrations by array CGH: A step towards noninvasive prenatal diagnosis using intact fetal cells. A. Breman1, W. Bi1, C. A. Shaw1, I. Van den Veyver1,2, C. J. Shaw1, A. Stubbs1, M. Withers1, G. Fruhman1,2, A. Patel1, J. R. Lupski1, A. Beaudet1 1) Dept of Molecular & Human Genetics, Baylor College of Medicine, Houston, TX; 2) Dept of Obstetrics & Gynecology, Baylor College of Medicine, Houston, TX.

   Detection of genomic copy number abnormalities in a single cell using array CGH offers a promising non-invasive alternative for prenatal diagnosis. Since fetal cells are extremely rare in the maternal peripheral blood circulation, the genetic status of the fetus has to be derived from the analysis of a few or even a single fetal cell. We previously showed (PMID 22470934) that array CGH on whole genome amplification (WGA) DNA from a single cell could detect 1 Mb copy number changes using a custom array with focused regions of increased probe coverage. Recently, several new WGA technologies were described. The goal of this study was to compare three WGA methods for their ability to produce optimal array CGH results from single cells using a catalog array. Single cells from lymphoblastoid cell lines carrying various genomic abnormalities were analyzed using genome-wide oligonucleotide-based array CGH analysis with a SurePrint G3 Human CGH 180K Agilent array. Detection of copy number changes was compared across multiple WGA methodologies including PicoPLEX (Rubicon Genomics), Ampli1 (Silicon Biosystems) and Multiple Annealing and Looping Based Amplification Cycles (MALBAC; PMID 23258894). Our previously optimized conditions for single cell array CGH also included a two-step DNA purification method and closely matched pooled reference DNAs. We analyzed the single cell array data from each WGA method for the ability to detect copy number differences for the X chromosome with gender-mismatched cells as well as trisomy 21, DiGeorge syndrome deletion, CMT1A duplication and MECP2 duplication. We consistently detected dosage difference in sex chromosomes for gender mismatched hybridizations and for chromosome 21 in trisomy 21 cells. The 2.5 Mb DiGeorge syndrome deletion was also detectable in a single cell using all 3 WGA platforms, whereas detection of the 1.5 Mb CMT1A duplication and the 0.6 Mb MECP2 duplication was dependent on the algorithm used to identify copy number changes. These data suggest that aneuploidy and other genomic imbalances in a single cell can be detected by oligo-based array CGH when the WGA, purification and hybridization conditions are optimized, but that increased probe density may be necessary to consistently detect small copy number changes.

You may contact the first author (during and after the meeting) at