A pool of suitable oligonucleotide probes from ab initio intervals was designed with PICKY (30), which matches melting temperatures to avoid complementarity between probes and stable hairpin formation. Default parameters were modified as follows: left selection boundary 200, right selection boundary 200, maximum oligonucleotide size 60, maximum match length 20, minimum match length 17 and probes per gene 5. PICKY-suggested 2 057 653 coordinate-defined probes from 513 689 ab initio sc intervals.
A subset of these probe sequences was selected to populate a custom genome-wide 4x44K array. To minimize cross-hybridization of ab initio probes to repetitive sequences within the labelled genomic sample, oligonucleotides were chosen complimentary to genomic targets whose distance to an adjacent conserved repetitive element exceeded the length of the labelled extension products. Products were <300 nt. Oligonucleotide targets and adjacent repeat elements were separated by at least 300 nt, for repetitive sequences with <30% divergence (higher divergence sequences were tolerated). For purposes of comparison, ab initio oligonucleotide targets were paired with Agilent Technologies Human Catalog CGH 4 × 44K microarray (Agilent 44K) genomic probe sequences in closest genomic proximity to ensure similar distributions. Where possible, gene coverage was maximized. The Galaxy metaserver (https://main.g2.bx.psu.edu) was used to ‘fetch’ the closest non-overlapping feature for every interval, ‘subtract’ intervals present in the ab initio and Agilent 44K oligonucleotide sets and determine the base ‘coverage’ of all intervals. We first determined the distance in nt of the closest repeat masked repetitive element to each probe. Oligonucleotides within 300 nt of a repeat were subtracted from the set. The closest ab initio probe to a corresponding sequence on the Agilent 44K array was fetched. The distance between ab initio probes and adjacent repeat elements was then maximized on the custom designed microarray by selecting oligonucleotides central to each ab initio interval. Gene coverage, which was determined from the proximity of probes to known NCBI RefSeq gene sequences, demonstrated that the paired set of ab initio probes did not cover all known genes (31). Gene coverage in the custom microarray was improved by adding 1510 probes within or adjacent to the missing genes.
Ab initio normalization and replicate probes were also selected in close proximity of the corresponding Agilent probes. Both the custom designed ab initio 44K and commercial Agilent 44K microarrays were manufactured by Agilent. We hybridized them with genomic DNA from HapMap family trios (YRI: GM19143/GM19144/GM19415, and CEU: GM07019/GM07056/GM07022). DNA from the offspring (GM19145/GM07019) was used as the reference sample and co-hybridized with either the maternal (GM19143/GM07056) or paternal (GM19144/GM07022) sample on two replicate sectors of each array. To produce extension products <300 nt, DNA was subjected to heat fragmentation (98°C for 10′) before labelling and sized by electrophoresis. Pairs of genomic DNA samples (0.5 µg each) were individually enzymatically labelled using 5′-terminally labelled, fluorescent random nonamers (either Cy3 or Cy5 from IDT) with 5′→3′-exo- Klenow DNA polymerase (New England Biolabs), then mixed and co-hybridized according to the Agilent Oligonucleotide Array-Based CGH for Genomic DNA Analysis Protocol (v6.2). Microarrays were scanned and quantified with Agilent Feature Extraction software (v10.5.1.1). Hybridization intensities of Agilent’s non-human control sequences were used to correct for background fluorescence. The coefficients of variation [CV = |(Log2 ratio or signal intensity) standard deviation|/mean] were calculated from replicate spot intensities of each autosomal probe sequence on the same microarray platform. Identical probe sequences were replicated within the same and on different sectors on the array, enabling comparisons of both inter- and intra-array reproducibility on each platform. Full paper
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