Agilent genomic workbench 7

Array-CGH analyses were achieved by applying the Human Genome Array-CGH 8x60K Microarray (Agilent Technologies, Palo Alto, CA, USA), with a typical probe spacing of around 55 Kb. The arrays were performed using Agilent Reference DNAs, analyzed with the Agilent Microarray Scanner Feature Extraction Software v11.5, and Agilent Genomic Workbench 7.0.4.0 software using the ADM-2 algorithm. Genomic positions of the rearrangements refer to the public UCSC database GRCh37/hg19. Inheritance of non-polymorphic CNVs was evaluated with multiplex ligation-dependent probe amplification or real-time PCR assays.

Test and reference signals were calculated for each probe and internal normalization equalized the systematic difference between the two fluorochromes. The base-2 log of the Cy3/Cy5 ratio (LRR) was plotted against chromosomal coordinates. Probes covering NOD or BALB/c germline deletions were eliminated. Regions bearing copy-number aberrations were identified using two independent algorithms, Agilent Genomic Workbench 7.0 (Agilent Technologies) (Roy and Motsinger Reif 2013 (link)) implementing ADM-2 algorithm and DNAcopy software implementing the circular binary segmentation (CBS) algorithm (Venkatraman and Olshen 2007 (link)). In ADM-2 (threshold 6.0), we applied Agilent Technologies recommended filtering standards of a minimum of three aberrant consecutive probes. The mouse genome UCSC mm9 NCBI build 37/July 2007 was used to match the indexing of the Agilent probes (converting coordinates did not affect the findings).
CNAs were kept as true if called by both algorithms ADM-2 (LRR ≥ |0.25|, P-value ≤ 2.90 × 10⁻⁰⁷, Bonferroni threshold) and CBS (LRR|0.25|, P-value ≤ 1 × 10⁻⁰⁴). The lower statistical significance threshold for CBS is justified by the vastly smaller number of hypotheses tested (number of ADM-2-called CNAs).
Tissue-specificity of the expression levels of the genes involved was obtained from mouse and human BioGPS data, 2018 (Wu et al. 2016 (link)).

Copy number variants (CNVs) were tested with a customized 2 × 400K array (Agilent Technologies, Santa Clara, CA, USA) with genomewide coverage and enriched probes in >300 genes linked to skeletal disease, as described.⁽²⁶⁾ On average, the array covers targeted gene areas with one probe per 100 base pairs (bp) in coding regions and one per 500 in introns and untranslated regions (UTRs). We performed the tests with standard procedures and analyzed results with Agilent Genomic Workbench 7.0 (Agilent Technologies).

Array comparative genomic hybridization (aCGH, 180K, Agilent, United States) was performed in both patients according to the recommendations of the manufacturer. The analysis was carried out using Agilent Genomic Workbench 7.0 (Agilent Technologies, Santa Clara, CA, United States).
Real-time quantitative PCR (RT-qPCR) of TBCK was performed to detect the predicted small deletion in the region chr4:107,071,580−107,113,380. Six primer pairs were designed on Primer-BLAST (Supplementary Table 2). Relative quantification was carried out by normalization to GAPDH, and quantification data were calibrated relative to a control without any known CNV in TBCK (D’haene et al., 2010 (link)).

Array − CGH analysis on both patients and their parents was carried out using the SurePrint G3 Custom CGH Microarray, 8 × 60 K 4 × 180 K (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s protocol version 7.1, using appropriate Agilent Reference DNAs (Euro male and Euro female). The arrays were analyzed with the Agilent Microarray Scanner, Feature Extraction Software version 11.5, and Agilent Genomic Workbench 7.0.