Cytoscan hd microarray

DNA samples from all cases were extracted from peripheral blood with standard procedures. CHD patients at BCH were tested on the Agilent 244 K comparative genomic hybridization (CGH) array platform or a 4 × 180 K SNP + CGH microarray in a clinical diagnostic setting. CNVs were identified and evaluated as previously described
[23 ].
CHD cases at SCMC were tested on the Affymetrix Cytoscan™ HD microarray platform in a research setting. Data was visualized and analyzed by Chromosome Analysis Suite (ChAS) software package (Affymetrix, USA) with a minimal cutoff of 20 consecutive markers for CNV calling. All CNVs reported are based on NCBI human genome build 37 (hg 19).
Detected CNVs were evaluated through a filtering procedure and classified into five categories based on the ACMG guideline
[24 (link)] (for details see Additional file
1).

Clinical CMA was performed using an optimized, ultra-high-resolution custom Affymetrix microarray that includes all functional content present on the Affymetrix CytoScan-HD microarray with probes added to improve detection of copy number variants associated with pediatric neurodevelopmental disorders [4 ]. DNA samples (250 ng) were digested, processed, labeled and hybridized to microarrays using the manufacturer’s recommended protocol (Affymetrix, Inc., Santa Clara, CA). Array results were interpreted by ABMGG-certified clinical cytogeneticists using the Chromosome Analysis Suite v2.0.1 software (Affymetrix, Inc., Santa Clara, CA). To estimate the percentage of genome homozygosity, all ROH regions greater than 3 Mb are totaled for the autosomes (X chromosome excluded to avoid male/female bias) and the total is divided by the total size of the autosomes. Samples were randomly selected for exome sequencing if the report of the clinical cytogeneticist stated ROH to be a potential concern. ROH were considered as a potential concern by the clinical cytogeneticist if the total ROH encompasses greater than 3% of the genome. Additionally, for cases with ROH between 1 and 3%, the cytogeneticist compared the ROH of the patient with common ROH in control populations and decides to flag these as concerning if there are significant disparities.

DNA samples of patients negative for the G2019S mutation were then screened for exon rearrangements using Affymetrix Cytoscan HD microarray according to the manufacturer’s protocol. With a median inter-marker distance of 500–600 bp, CytoScan HD offers the highest physical coverage of the genome for detecting human chromosomal abnormalities. Indeed, these chips include 750,000 single-nucleotide polymorphism (SNP) and 2.6 million copy number variation (CNV) markers that enable high-resolution (25–50 kb resolution) detection of CNVs, region of homozygosity (ROH), uniparental disomy, and low-level mosaicism. Briefly, 250 ng of DNA samples were digested with Nsp1, amplified with TITANIUM Taq DNA polymerase (Clontech, Mountain View, CA, USA), fragmented with Affymetrix fragmentation reagent, and labeled with biotin end-labeled nucleotides. DNAs were hybridized to the microarrays for 16 h, washed and stained on the GeneChip Fluidics Station 450, and scanned on the GeneChip Scanner 3000 7G (Affymetrix). Data analysis was performed using Chromosome Analysis Suite software version 1.2.2 (Affymetrix). Data were considered significant only when they met the quality control criteria set by the manufacturer [the Median Absolute Pairwise Difference scores (MAPD) < 0.25, the Waviness Standard Deviation (WSD) < 0.12, and the SNP Quality Control (SNP-QC) > 0.15].

CMA was performed using peripheral blood and examined with Cytoscan HD microarray (Affymetrix, Santa Clara, CA). This array consists of 2,696,550 oligonucleotide probes, including 1,953,246 distinctive non-polymorphic oligonucleotide probes, and 743,304 single nucleotide polymorphism probes. Genomic DNA was extracted and purified from whole blood sample using Gentra Puregene Blood Kit (Qiagen Inc., Valencia, CA). Procedures for DNA digestion, adapter ligation, polymerase chain reaction (PCR), amplicon DNA fragmentation, labeling, and hybridization of the arrays were performed according to manufacturer’s instructions (Affymetrix, Santa Clara, CA). Results were investigated using the Chromosome Analysis Suite (ChAS; Affymetrix, Santa Clara, CA). The settings for smallest copy number variation (CNV) regions in ChAS were 25 kb and 25 probes for losses, and 50 kb and 50 probes for gains. Genomic linear positions are given relative to NCBI build 37 (hg19) [12 (link)]. Results were interpreted based on published literature, publicly available databases, and by investigating gene content following practice guidelines [13 (link),14 (link)].

Tumor and matched lymphocyte DNA were hybridized to the CytoScan HD microarray (Affymetrix, UK) and analyzed as described [15 (link)]. Briefly, three different software were used for calling and filtering copy number aberrations (CNA) and copy number neutral runs of homozygosity (CNN-ROH); (1) Chromosome analysis suite v3.2 (ChAS, Affymetrix, UK), (2) Rawcopy [23 (link)] and (3) Nexus Copy Number (BioDiscovery, El Segundo, CA, USA). For estimating aberrant cell fraction and allele specific copy number profiles in the tumors, the Allele-Specific Copy number Analysis of Tumors 2.5.2 (ASCAT) software was used [36 (link)]. Clustering of the sample set based on CNA profiles was done with Rawcopy using the hclust R package as well as with the built-in complete linkage hierarchical clustering algorithm in Nexus Copy Number. Called variant segments from the CytoScan microarray were used for generating input for the Chromothripsis-like pattern (CTLP) scanner [39 (link)]. Identification and calculation of likelihood ratios of CTLP present in the samples were done using the website http://cgma.scu.edu.cn/CTLPScanner/ with default parameters except for the parameter of Log₂ signal value difference between two adjacent segments that was set to 0.25.