Blocking agent

Genomic DNA was isolated using a method described by Pitcher et al. [42 (link)]. A total of 500 ng of genomic DNA from each Yersinia strain was fluorescently labeled with the BioPrime ArrayCGH labeling module (Invitrogen, Carlsbad, CA, USA) using either Cy3 or Cy5 (GE Healthcare, Buckinghamshire, UK). For each hybridization, one Cy3-labeled and one Cy5-labeled DNA sample were combined. The mixture was purified with a DNA purification kit (QIAquick PCR Purification Kit, Qiagen, Hilden, Germany) according to the manufacturer's instructions. The concentration of DNA and the incorporation of the dye were checked with the Nanodrop device (Nanodrop Technologies, Wilmington, MA, USA) before and after labeling. The differently labeled DNA sample pairs to be hybridized into one of the eight subarrays on each array slide were randomly selected.
A volume of 2.2 μL salmon sperm DNA (1 mg/mL) was added to 17.8 μL of labeled combined sample solution, and the mixture was heated at 95°C for 2 minutes for denaturation. A volume of 5 μL of 10x blocking agent (Agilent) and 25 μL of 2xGE (HI-RPI) hybridization buffer (Agilent) was added. A total of 45 μL of the solution was hybridized on each microarray at 65°C for 16 hours. The arrays were washed twice for 1 minute with Wash Buffer 1 (Agilent) and then for 1 minute with prewarmed Wash Buffer 2 (Agilent).

Target capture probes were used to enrich libraries using in-solution Hybridization [16 ,28 ]. Each barcoded library was mixed with 2x hybridization buffer (Agilent Technologies), 10x blocking agent (Agilent Technologies), blocking oligonucleotides, human Cot-1 (Invitrogen, Carlsbad, CA, USA) and salmon sperm DNA (Invitrogen) [16 ,25 (link),28 ,29 ]. The libraries were then incubated with the biotinylated capture probes for 48 hours at 66°C and were eluted by heating [16 ,27 ]. Enriched fetal-specific regions were amplified for 12 cycles using Herculase II Fusion Enzyme kit (Agilent Technologies) and outward bound adaptor primers [27 ]. Following quantification with the KAPA Library Quantification Kit Illumina (KAPA Biosystems, Boston, MA, USA) the amplified post-captured libraries were sequenced on a HiSeq 2500 sequencing system platform (Illumina, San Diego, USA).

The procedures for DNA digestion, labeling, and hybridization for the oligo arrays were performed according to the standard Agilent protocol v7.1, with minor modifications. Briefly, 0.25 µg of patient genomic DNA and 0.25 µg of pooled gender-matched reference DNA (Promega) were labeled with Cyanine 5 (Cy5) or Cyanine 3 (Cy3) dyes (Agilent). Following purification with Amicon 30 kDa filters (Millipore), the labeled DNA yield and dye incorporation were measured using an ND-2000 spectrophotometer (NanoDrop). The Cy3-and Cy5-labeled samples along with 2 µg human Cot-1 DNA (Invitrogen), 10X blocking agent (Agilent) and 2X Hi-RPM Buffer (Agilent) were added and hybridized together at 65°C on the CancerArray (Ambry Genetics) for 24 h in a rotisserie oven at 20 rpm. Slides were washed according to the manufacture's protocol and scanned at 3 µm resolution on an Agilent G2565CA high-resolution DNA microarray scanner.