Cgh array

Patient samples were flow sorted prior to genomic analysis using our published DNA content based protocols [21 (link)]. Briefly, DNAs were extracted using QIAGEN micro kits (Valencia, CA, USA). For each hybridization, 100 ng of genomic DNA from each sample and of pooled commercial reference (Promega, Madison, WI, USA) were amplified using the GenomiPhi amplification kit (GE Healthcare, Piscataway, NJ, USA). Subsequently, 1 ug of amplified sample and 1 ug of amplified reference template were digested with DNasel then labeled with Cy-5 dUTP and Cy-3 dUTP, respectively, using a BioPrime labeling kit (Invitrogen, Carlsbad, CA, USA). All labeling reactions were assessed using a Nanodrop assay (Nanodrop, Wilmington, DE, USA) prior to mixing and hybridization to CGH arrays with either 244,000 or 400,000 oligonucleotide features (Agilent Technologies, Santa Clara, CA, USA). The aCGH data have been deposited in the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (accession numbers GSE54328 and GSE21660).

DNA was DNAse I digested, labeled using a BioPrime Labeling Kit (Invitrogen) using Cy-5 dUTP for the sample and Cy-3 dUTP for the reference genome, hybridized to 400k comparative genomic hybridization (CGH) arrays (Agilent Technologies), scanned using an Agilent 2565C DNA scanner, and the images were analyzed with Agilent Feature Extraction v11.0 using default settings.

In all cases, CGH array (180K and 60K in cases 1 and 2, respectively) and SNP‐CGH array (180K in case 3) were performed according to standard manufacturer's protocols (Agilent Technologies, Santa Clara, CA). All nucleotide positions refer to the Human Genome, Feb 2009 Assembly (GRCh37, hg19). The arrays were analyzed using an Agilent scanner and Feature Extraction v.9.1 software (Agilent Technologies). A graphical overview of the results was obtained using CytoGenomics software.

Agilent CGH array was designed to query the entire length and extended regions of all FA and other inherited bone marrow failure syndrome genes. The FANCB (ENSG00000181544.13; ENST00000398334.5; NM_001018113.2) gene region on the array extended to 150 kb on either side of the gene interval. The experimental procedures are as described previously (Flynn et al., 2014). Briefly, genomic DNA from the patient and a reference male DNA were differentially labeled and hybridized to the array.

Exome sequencing was performed according to local protocols, namely, standard chromosome analysis on peripheral blood lymphocytes. Microdeletions were identified by array-CGH (Agilent, Santa Clara CA, USA) or SNP-array (Beadchip 850K, Illumina, San Diego, CA, USA) platforms at a resolution of 100 kb. Comprehensive open reading frame/splice site mutational analysis of clinical exomes were conducted through the Twist Human Core Exome Kit and sequenced on the Illumina NovaSeq6000 Platform. Lastly, an amplification study of the CGG repeats of the FMR1 gene for X fragile syndrome was carried out using RP-PCR. Further information is available upon request.