![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
|
Published online before print
August 9, 2006, 10.1101/gr.5402306 Genome Res. 16:1136-1148, 2006 ©2006 by Cold Spring Harbor Laboratory Press; ISSN 1088-9051/06 $5.00 OPEN ACCESS ARTICLE
Methods High-resolution genomic profiling of chromosomal aberrations using Infinium whole-genome genotyping1Illumina, Inc., San Diego, California 92121, USA; 2Baylor College of Medicine, Houston, Texas 77030, USA;
Array-CGH is a powerful tool for the detection of chromosomal aberrations. The introduction of high-density SNP genotyping technology to genomic profiling, termed SNP-CGH, represents a further advance, since simultaneous measurement of both signal intensity variations and changes in allelic composition makes it possible to detect both copy number changes and copy-neutral loss-of-heterozygosity (LOH) events. We demonstrate the utility of SNP-CGH with two Infinium whole-genome genotyping BeadChips, assaying 109,000 and 317,000 SNP loci, to detect chromosomal aberrations in samples bearing constitutional aberrations as well tumor samples at sub-100 kb effective resolution. Detected aberrations include homozygous deletions, hemizygous deletions, copy-neutral LOH, duplications, and amplifications. The statistical ability to detect common aberrations was modeled by analysis of an X chromosome titration model system, and sensitivity was modeled by titration of gDNA from a tumor cell with that of its paired normal cell line. Analysis was facilitated by using a genome browser that plots log ratios of normalized intensities and allelic ratios along the chromosomes. We developed two modes of SNP-CGH analysis, a single sample and a paired sample mode. The single sample mode computes log intensity ratios and allelic ratios by referencing to canonical genotype clusters generated from  120 reference samples, whereas the paired sample mode uses a paired normal reference sample from the same individual. Finally, the two analysis modes are compared and contrasted for their utility in analyzing different types of input gDNA: low input amounts, fragmented gDNA, and Phi29 whole-genome pre-amplified DNA.
A variety of chromosomal aberrations underlies developmental abnormalities (constitutional aberration) and cancer (acquired aberration) (Albertson and Pinkel 2003  ). Many of these aberrations are characterized by rearrangements in genomic DNA or changes in copy number such as deletions, duplications, and amplifications (Kallioniemi et al. 1992, 1994, 1996; Hayashizaki et al. 1993; Wang et al. 2002). Historically, two key techniques have been used to measure DNA copy number in DNA samples: comparative genomic hybridization (CGH) and loss of heterozygosity (LOH). CGH has been used extensively to detect amplifications and large homozygous deletions, and LOH has been used to detect regions of allelic homogeneity indicative of hemizygous deletions or copy-neutral LOH. LOH is typically assessed through the analysis of polymorphic genetic markers, traditionally either VNTRs or RFLPs (Singh et al. 1993; Dockhorn-Dworniczak et al. 1994), and more recently single nucleotide polymorphisms (SNPs) (Slater et al. 2005; Zheng et al. 2005). The importance of LOH is underscored by its extensive history in the discovery of many classical tumor-suppressor genes (TSGs) including RB1, WT1, and TP53, involved in the formation of retinoblastoma, Wilm's tumor, and Li-Fraumeni syndrome, respectively (Gray and Collins 2000; Hanahan and Weinberg 2000; Albertson and Pinkel 2003; Albertson et al. 2003).
CGH has been used widely to characterize DNA copy changes in tumors. Originally this technique was implemented using metaphase chromosomal spreads but has been adapted to array-CGH using BAC, cDNA, and oligonucleotide arrays (Solinas-Toldo et al. 1997
The ability to detect microdeletions and microduplications is essential in the study of constitutional disorders. Several disease states have been attributed to both microdeletion haploin-sufficiency and duplication-mediated overexpression in regions harboring known transcription factors and tumor-suppressor genes (Santarosa and Ashworth 2004
The ability of SNP-CGH to simultaneously measure both intensity differences and allelic ratios in a genomic sample allows both DNA copy number and copy-neutral LOH to be assessed. This is particularly important since copy-neutral LOH is receiving greater attention as a mechanism of possible tumor initiation (Langdon et al. 2006
In this study, we demonstrate the utility of SNP-CGH with two Infinium whole-genome genotyping BeadChips, Human-1 and the HumanHap300, assaying 109K and 317K SNP loci respectively, to detect chromosomal aberrations in constitutional and tumor samples. The Whole Genome Genotyping (WGG) assay has previously been described and is composed of four basic components: a whole-genome amplification, hybridization capture on a 50mer probe array (BeadChip), array-based primer extension SNP scoring, and immunohistochemistry-based signal amplification (Gunderson et al. 2005
We present two genomic plots, one of the log-normalized intensity ratio and the other of an allelic intensity ratio for a single representative SNP from the genome (Fig. 1). These two parameters originate from a polar coordinate transformation of two-channel (two alleles) intensity data. This transformation generates a normalized intensity value, called R, and an allelic intensity ratio, called theta (  ). The intensity comparison between a subject sample and a reference sample is conducted in either a single sample mode using canonical genotype clusters derived from a large training set of normal samples, or in a paired sample mode in which the subject and reference sample are from the same individual and run side by side in one experiment.
In the "single sample" analysis mode, the observed normalized intensity of the subject sample (Rsubject) is compared to the expected intensity (Rexpected) computed from a linear interpolation of the observed allelic ratio ( subject) with respect to the canonical genotype clusters (Fig. 1A). The three canonical genotype clusters were generated at one point in time by training on 120 normal samples, and serve as standards for all future experiments. In addition to computing Rexpected, the observed allelic intensity ratio (subject) is used to estimate a quantitative B allele frequency for the particular SNP in the given sample by using interpolation of the known B allele frequencies of the three canonical clusters (0, 0.5, and 1.0) (Fig. 1B). These two transformed parameters, log2 R ratio [log2(Rsubject/Rexpected)] and B allele frequency (AF), are then plotted along the entire genome for all SNPs on the array. It is these genomic profile plots that form the basis of detecting chromosomal aberrations with SNP-CGH.
X-copy cell lines as a model system for detection limits and sensitivity
We plotted the response of the log R ratio for both the X chromosome and an autosome of similar size, chromosome 10, for each X-copy number cell line. As expected, the log R ratio increases with increasing X-copy number for the X chromosome but not chromosome 10 (Fig. 2E). The corresponding standard deviation of the log R ratio is also shown. Statistical noise can be reduced by using a moving window average across neighboring SNPs. The corresponding log R ratio response plot is shown in Figure 2F for a 10-SNP moving average. In this case, the standard deviation of the moving average log R ratio is significantly reduced, and the effective SNR is correspondingly increased (Table 2). Interestingly, the response profile of the log R ratio is attenuated at higher copy levels potentially because of saturation effects in both the hybridization and immunohistochemistry staining steps in the assay. Receiver operator curves (ROC), which plot the true versus false positive rate for the detection of aberrations (deletions, duplications, and amplifications), were generated for both the raw X-copy data and data generated with a 5- and a 10-SNP moving average (see Supplemental material).
The SNR for a single measurement point is much higher for the allele frequency (AF) measurement than the log R ratio parameter. The combination of both the log R ratio and AF is quite powerful since the precision of the allelic ratio measurement allows robust detection of the aberration and the log R ratio can be used to interpret the nature of the aberration. Furthermore, the statistical confidence of detecting an aberration can be greatly increased by using a moving window average of neighboring SNPs. We derived a 10-SNP "rule-of-thumb" based on analysis of the ROCs for various numbers of SNPs in a moving median window; a window size of 10 generated the most ideal ROC curves (see Supplemental material). This window size of 10 also encompasses, on average, three to four heterozygous SNPs per window based on an average heterozygosity of 30%–40%. Based on only the log R ratio parameter, we achieved a deletion detection rate of 90% (true positives) with a 1% false-positive rate for a single SNP. When we used a 10-SNP moving median, we achieved a remarkable detection rate of 99.9% with a 1.2% false-positive rate. This 10-SNP rule implies that the 109K BeadChip (26 kb mean spacing; 13 kb median spacing) has an effective resolution <260 kb, and the 317K BeadChip (9 kb mean spacing) has an effective resolution of <90 kb. As such, we typically plot a smoothing function for the log R ratio profile using a moving average of 300 kb for the 109K array, and 100 kb for the 317K array.
Examples of common aberrations using HL-60
Using the 109K BeadChip, we successfully detected all of the aberrations previously detected by both platforms (SKY karyo-type and array-CGH) in the HL-60 cell line. Several types of such aberrations are shown in Figure 3. A 1.9-Mb amplification, denoted by a green arrow in Figure 3A, was found on chromosome 8. This amplification is detected by an increase in the log R ratio and a split in the heterozygous B allele frequency from a normal value of 0.5 (1:1 allelic ratio) to
Detection of constitutional chromosomal aberrations As illustrated by the previous examples, high-density SNP-CGH BeadChips should have the power to easily detect most common constitutional chromosomal aberrations. This has particular importance in the field of neo and postnatal analysis of chromosomal aberrations. Currently, most clinical tests of aberrations use a combination of karyotyping and fluorescence in situ hybridization (FISH). These karyotypes are useful for outlining the overall chromosomal structures and detecting aberrations including translocations and inversions. However, karyotyping resolution is limited to 10–20 Mb. As such, follow-up studies on patients usually employ FISH probes for detection of small deletions and duplications. Clinics currently use panels of FISH probes for 25 microdeletion syndromes including DiGeorge, Prader-Willi (Angleman), Williams-Beuren, and Wolf-Hirschhorn syndromes (Scambler et al. 1991; Driscoll et al. 1992; Clayton-Smith et al. 1993; Wright et al. 1997; Francke 1999). In contrast, genomic profiling with SNP-CGH arrays enables discovery of aberrations throughout the genome and allows for a precise localization of chromosomal breakpoints of those aberrations. To demonstrate the feasibility of using high-density Bead-Chips for detection of congenital chromosomal aberrations in patient samples, we conducted a blinded study on six DNA samples collected from patients with diagnosed developmental clinical phenotypes. The DNA was previously characterized by karyotype, FISH, and BAC array-CGH analysis. All documented chromosomal aberrations (n = 10) ranging in size from 72 kb (duplication) to entire chromosomes were detected on the 317K BeadChip. The 72-kb duplication was detected on the 109K primarily because of the split of the heterozygous cluster. The 109K array detected nine out of 10 aberrations (Fig. 4F), and the 317K detected the aberration missed by the 109K array (a 115-kb deletion) (Fig. 4G). An example of one such aberration, a small 1.5-Mb deletion on chromosome 22q11.2, detected by BAC array-CGH (Fig. 4A,B), FISH analysis (Fig. 4C), was also detected by SNP-CGH on the 109K and 317K (Fig. 4D,E, respectively). In the blinded study, numerous (n = 12) other small deletions and duplications ranging in size from 46 kb to 470 kb were discovered using either the 109K or 317K arrays (data not shown). These results demonstrate the utility of both the 109K and 317K Bead-Chips for the detection of various chromosomal aberrations in constitutional samples. In particular, the 317K array, with its greater density, allowed discovery of more small aberrations than the 109K.
Analyzing heterogeneous tumor samples Analysis of tumor samples obtained from patients typically involves some form of microdissection to extract a specific population of cells since contamination by surrounding tissues containing normal genomic DNA can result in decreased accuracy for LOH calls (Hata et al. 2006 ). Current estimates suggest that contamination with normal stroma from 10% to 50% of the total genomic DNA content can severely affect measurements of LOH. On the other hand, even in samples with high levels of normal tissues, it may still be possible to detect changes in copy number and homozygous deletions, but LOH resolution is still lost. With the development of laser capture microdissection (LCM), the ability to gather samples of high homogeneity increases, which should improve LOH measurements in the genome (Rook et al. 2004). Other techniques such as flow cytometry can also increase the accuracy of LOH calls in tumor samples (Abeln et al. 1994). We evaluated the effect of tumor heterogeneity on the detectability of various types of aberrations. This was accomplished by mixing gDNA harvested from cell line pairs (blood and tumor; ATCC: CRL-2325D, CRL-2324D) derived from the same patient in various ratios of 0%, 25%, 50%, 75%, and 100% tumor (Fig. 5A–E, respectively). The samples were combined and gDNA was amplified as described in the Methods section and then analyzed on 109K BeadChips. With relatively low levels of normal contamination (0%–25%) (see Fig. 5D), the allele frequency difference between tumor and normal is clearly distinguishable. Even in the case of 50% normal contamination, various aberrations can easily be detected (Fig. 5C); however, their interpretation remains ambiguous as to whether they are deletions or duplications (Fig. 5B). Finally, at 75% normal contamination, aberrations are not visible. At 100% tumor, homozygous deletions are easily detected in both the log R ratio and the allele frequency profile (Fig. 5E). Here, the AF profile exhibits "scatter" since the target loci are not present with homozygous deletions. Detection of homozygous deletions is made challenging in the presence of contaminating normal gDNA since the allele frequency is indistinguishable from normal; the only detectable signal is a slight dip in the log R ratio profile. In addition, since the AF profile is characteristically more responsive than the log R ratio, an LOH event or duplication may be occurring on the proximal region of the p-arm of chromosome 13 although no change in the log R ratio is observed (Fig. 5E). Overall, these results suggest that the fraction of normal tissue in heterogeneous tumor samples should be kept well below 50% for sensitive detection of aberrations with a goal of 25% or less.
Owing to the extensive aneuploidy often present in tumor cell lines that confounds normalization and interpretation of data, we also evaluated patient tumor samples containing endogenous levels of normal contamination. In cases with a modest number of aberrations, it can be straightforward to clearly detect LOH in samples containing 67% normal cells. Shown in Figure 5F is a profile from a paired colon tumor patient sample analyzed on the 317K BeadChip. The deflection downward in the log2 R ratio indicates a loss of copy number. In the AF profile, the heterozygous state splits into two populations (0.33 and 0.67), suggesting that this sample contains 67% normal cells since the heterozygotes have not completely collapsed to the homozygote axes. Regardless, in this case, the LOH event is clearly detectable even in the presence of a high percentage of normal cells. In spite of the challenges analyzing such samples, the ever-increasing density of array technology, such as the HumanHap550 BeadChip, should further improve the sensitivity with heterogeneous samples, especially since one can average over greater numbers of probes.
Effect of varying DNA input amount and fragmentation on SNP-CGH data
We suspected that the increase in log R ratio variation was due to systematic amplification bias. As such, we computed the regression r 2 for normalized intensity values between various input levels of gDNA (Fig. 6D). The r 2 correlation was high between similar input amounts of gDNA, but decreased dramatically between both high and low input amounts or between intact and degraded DNA (Fig. 6D). Here, we conclude that the variation levels can be greatly reduced in the log R ratio parameter when paired samples of similar quantity (defined as starting input amount) or fragment length are used, or when the canonical reference is generated from samples of similar quantity/quality to the subject sample. Regardless, these results suggest that only limited amounts of sample are required for LOH or copy number analyses if performed in the "paired analysis" mode. Currently, we use a canonical reference derived from "clustering" on 120 samples with a 1x input (750 ng in a full reaction; see Methods), and as such, single sample genomic profiling exhibits optimal performance when high-quality DNA is used at this input level. When using a lower input amount of gDNA, it is recommended that the user "re-cluster" on normal samples run at the corresponding reduced input levels of gDNA.
Single versus paired sample analysis To evaluate the differences between single and paired sample modes, we used paired breast tumor cell lines obtained from ATCC (CRL-2325D, CRL-2324D). From the analysis in the previous section on input amounts, we speculated that paired analysis would reduce the variability (standard deviation) in the log R ratio. We tested this effect by comparing the log R ratio variation generated when using the standard 750 ng of input gDNA versus 10 ng of input. In addition, we tested pre-amplification of gDNA with Phi29 (Repli-G; Qiagen) starting with 10 ng of input gDNA in the Phi29 WGA pre-reaction and using 750 ng of this product in the Infinium amplification reaction. We analyzed these samples on a 12-sample BeadChip using a 30,000 subset of loci from the 317K BeadChip. As expected, we find that paired analysis greatly decreases the variation in the log R ratio profile when using nonstandard DNA input (reduced amounts, phi29 pre-amplified, etc.) (Fig. 7B). In the single sample mode, the variation in the log R ratio across a region with normal genotypes on chromosome 6 is 0.422 for Phi29-amplified gDNA, 0.430 for 10-ng input DNA, and 0.236 for 750-ng input gDNA. In the paired sample mode, the variation in the log R ratio across the same region is significantly lower; 0.135 for Phi29-amplified gDNA, 0.162 for 10-ng input gDNA, and 0.106 for 750-ng input gDNA. In general, the data obtained with paired sample analysis are of higher quality than single sample analysis (Fig. 7B, see 750 ng). However, there are a few exceptions to this observation. If the reference sample is dramatically different in quality or quantity than the tumor sample, paired analysis may generate inferior results (data not shown). Thus it is of paramount importance when conducting paired sample analysis that the DNA samples be of similar quality as well as accurately quantified, preferably with a PicoGreen assay.
Our results demonstrate the utility of using Infinium SNP-CGH bead arrays for detection of chromosomal aberrations including both homozygous and hemizygous deletions, copy-neutral LOH, monoallelic duplications, and amplifications. A major strength of SNP-CGH arrays compared to conventional array-CGH is the ability to simultaneously profile the genome for both physical and genetic anomalies (Bignell et al. 2004 ; Zhao et al. 2004; Koed et al. 2005). Detection of copy-neutral events such as uniparental disomy (UPD), in which both alleles of a locus are derived from the same parent, is essential to understanding both developmental disorders (DeBaun et al. 2002; Fukuzawa et al. 2003; Bruce et al. 2005) as well as tumorigenesis.
A frequent cause of UPD in somatic cells (i.e., tumors) is rare but significant mitotic recombination events (Raghavan et al. 2005
We used an X chromosome titration model system to assess the signal to variability ratio of our Infinium SNP-CGH platform for various types of chromosomal aberrations. Averaging data from neighboring SNPs along the genome greatly increases the effective SNR. The average variation in the log2 intensity ratio for the Infinium SNP-CGH platform is relatively low ( Practical validation of the SNP-CGH platform was accomplished by a blinded study of six congenital samples containing various constitutional aberrations. We identified all previously documented chromosomal aberrations, as assessed by karyotype, FISH, and BAC array-CGH analysis. The detected aberrations ranged in size from a duplication of 72 kb to an entire duplicated chromosome. These results reveal the utility of using high-density SNP-CGH to screen for various chromosomal aberrations in constitutional samples. For future clinical applications, one can envision a custom SNP-CGH array containing perhaps from 20,000 to 50,000 elements designed to known regions of constitutional aberrations.
We find that the use of SNP-CGH for constitutional analysis of congenital samples is relatively straightforward. However, the heterogeneity typically found in tumor samples can make analysis and interpretation of results more challenging. Nonetheless, we have demonstrated sufficient sensitivity in mixed tumor–normal samples to detect single copy changes in tumor samples contaminated by as much as 50% normal background and LOH events in paired samples with In conclusion, we have shown that SNP-CGH arrays offer several distinct advantages over conventional array-CGH. SNPCGH arrays profile the genome for both physical and genetic anomalies allowing detection of copy-neutral genetic events. Additionally, these arrays provide allelic information on deletions, duplications, and amplifications, which is important since many high-level amplification events are monoallelic. Finally, a significant advantage of SNP-CGH bead arrays is their ease of manufacture and intrinsic scalability. Just recently, the density of SNPCGH beads has been extended to >550,000 SNPs (Illumina HumanHap550), and future innovations may drive this density even higher. Given these advantages, SNP-CGH may replace array-CGH as a standard for measuring genome-wide chromosomal aberrations.
High-density SNP genotyping arrays for SNP-CGH Illumina currently manufactures multiple formats of high-density SNP genotyping arrays (BeadChips); from 109K to 650K SNPs. A brief description of their content is provided in this section. All experimental processes for SNP-CGH described were completed with Infinium I and Infinium II WGG reagents from Illumina.
Sentrix Genotyping BeadChips
The 10K linkage multisample BeadChip contains uniformly spaced SNPs that are a subset of the SNPs from the 109K Bead-Chip, and the average spacing between each SNP probe is
DNA samples DNA from cell lines X (NA19160, male), 2X (NA12236, female), NA04626 (3X), and NA01416 (4X) were obtained from Coriell. The paired breast tumor cell lines (CRL-2325D and CRL-2324D) were obtained from American Type Culture Collection (ATCC). The acute promyelocytic leukemia cell line (HL-60) was also obtained from ATCC (ATCC No. HL60). The paired colon tumor genomic DNA was obtained from BioChain (A704198). For the DNA fragmentation experiments, cell line NA60136 was obtained from Coriell as was the cell line exhibiting the copy-neutral LOH event (NA09208). Genomic DNA from patient samples was isolated from peripheral blood lymphocytes, cultured amniocytes, or fibroblasts by using a PureGene DNA-purification kit according to the manufacturer's protocol (Gentra Systems).
Sample amplification and hybridization for BeadChips For the DNA fragmentation experiments, Coriell NA60136 was digested with 0.0125 U, 0.025 U, and 0.05 U of DNase I (Invitrogen), respectively. Briefly, 2 µg of gDNA was digested with DNase I in a 20-µL reaction, using 1-Phor-All Plus buffer and 0.5 mM CaCl2. The reactions were incubated at 37°C for 15 min, inactivated by 1 µL of 0.5 M EDTA, purified with MultiScreen PCR Plate (Millipore), resuspended in 40 µL of TE, and visualized on a 6% TBE/urea gel with SYBR Gold Staining (Invitrogen). For Phi29 amplifications, 10 µg of genomic DNA was amplified with the Repli-G kit from Qiagen (#59045) according to the manufacturer's protocol.
Infinium I (ASPE) and Infinium II (SBE) on BeadChips For the allele-specific primer extension (ASPE; Infinium I) assay, the BeadChip was first washed (Illumina RA1 and PB1) to remove unhybridized and nonspecifically hybridized DNA. Next the BeadChip was blocked for 10 min prior to addition of the extension mix (XB1). The extension step extends correctly matched probes hybridized to DNA on the BeadChip and incorporates biotin-labeled nucleotides (EMM). After extension, a formamide wash removed the hybridized DNA to reduce extraneous signal. The array then underwent a multilayer staining process to signal amplify and detect the incorporated label (LMM, ASM, and XB3). Finally, the BeadChips were washed (PB1) and dried before subsequent imaging. For the single-base extension (SBE) (Infinium II) assay, primers were extended with a polymerase and labeled nucleotide mix (TEM), and stained with repeated application of LTM (staining reagent) and ASM (anti-staining reagent). After staining was complete, the slides were washed with low salt wash buffer (PB1), immediately coated with XC4, and then imaged on the Illumina BeadArray Reader.
Imaging the BeadChip and data analysis
The data for each BeadChip is self-normalized using information contained within the array. This normalization algorithm removes outliers, adjusts for channel-dependent background and global intensity differences, and also scales the data. The X and Y color channels undergo an affine coordinate transformation to make the data appear as canonical as possible with the homozygotes lying along the transformed x-and y-axes. The following five steps are applied: (1) outlier removal; (2) a translation correction in which the asymptotes are fitted to candidate AA and BB homozygotes; the intersection of these fit lines defines the translated origin; (3) rotational correction: the angle of the AA homozygote asymptote with respect to the translated X-axis is used to define the rotational correction; (4) shear correction: the angle of the BB homozygote asymptote with respect to the translated and rotated y-axis is used to define the shear correction; (5) scaling correction: statistical centroids are computed for the candidate AA homozygotes to define an x-axis scaling parameter, and for candidate BB homozygotes to define a y-axis scaling parameter. The translated, rotated, shear-corrected data are normalized to a scale of
After normalization, the genotyping data are transformed to a polar coordinate plot of normalized intensity R = X norm + Y norm and allelic intensity ratio
The SNR for both the log R ratio and allele frequency (AF) were calculated with the following equations (where µX_XY represents the mean log R ratio intensity or mean heterozygous (nonhomozygous) AF for the X chromosome of the XY sample. The other parameters are similarly defined.
BAC array-CGH Patient genomic DNA was isolated as described above. Control DNA was from a healthy individual male and female using the same DNA extraction method. For CGH BAC-arrays, genomic DNA was fragmented to a smear between 600 and 2000 bp by sonication with a microcup horn (Sonics & Materials) and then purified (Zymo Research). The fragmented 500 ng of genomic DNA from patient and control was differentially labeled with cy3 and cy5 (Perkin Elmer) using Bioprime labeling (Invitrogen). To avoid dye bias, we performed dye swap experiments on each sample. Sixty micrograms of human cot-1 were added to each sample to block repetitive sequences (Invitrogen). The labeled DNA probe was dissolved in 30 µL of ULTRAhyb buffer (Ambion) and hybridized to the arrays in a sealed moisture hybridization chamber at 37°C in a rocking incubator for 22 h. After hybridization, washes were performed (2x SSC brief rinse, 50% formamide/2x SSC twice for 15 min, 0.5x SSC for 5 min, 0.07x SSC rinse twice, and nitrogen gas dry).
Arrays were scanned into 16-bit tiff image files with an Axon 4000B microarray scanner and quantified using GenePix Pro 6.0 (Axon Instruments). Data analysis was performed as previously described (Cheung et al. 2005
FISH analysis
We thank our many Illumina colleagues involved in the development of the Infinium genotyping products including those in assay development, high-throughput oligonucleotide synthesis, bead production, bioinformatics and software, marketing, and manufacturing. This work was supported, in part, by a grant from the NIH/NCI (2 R44 CA103406-02). Illumina, Sentrix, BeadArray, and Infinium are registered trademarks or trademarks of Illumina, Inc.
3 Corresponding author.  E-mail kgunderson{at}illumina.com; fax (858) 202-4680. Supplemental material is available online at http://www.genome.org. Article published online before print. Article and publication date are at http://www.genome.org/cgi/doi/10.1101/gr.5402306. Freely available online through the Genome Research Open Access option.
Abeln E.C., Corver W.E., Kuipers-Dijkshoorn N.J., Fleuren G.J., Cornelisse C.J. 1994. Molecular genetic analysis of flow-sorted ovarian tumour cells: Improved detection of loss of heterozygosity. Br. J. Cancer 70: 255–262.[Medline] Albertson D.G. and Pinkel D. 2003. Genomic microarrays in human genetic disease and cancer. Hum. Mol. Genet. 12: R145–R152. Albertson D.G., Collins C., McCormick F., Gray J.W. 2003. Chromosome aberrations in solid tumors. Nat. Genet. 34: 369–376.[CrossRef][Medline] Altshuler D., Brooks L.D., Chakravarti A., Collins F.S., Daly M.J., Donnelly P. 2005. A haplotype map of the human genome. Nature 437: 1299–1320.[CrossRef][Medline] Barrett M.T., Scheffer A., Ben-Dor A., Sampas N., Lipson D., Kincaid R., Tsang P., Curry B., Baird K., Meltzer P.S. et al. 2004. Comparative genomic hybridization using oligonucleotide microarrays and total genomic DNA. Proc. Natl. Acad. Sci. 101: 17765–17770. Bauters M., Van Esch H., Marynen P., Froyen G. 2005. X chromosome array-CGH for the identification of novel X-linked mental retardation genes. Eur. J. Med. Genet. 48: 263–275.[CrossRef][Medline] Bignell G.R., Huang J., Greshock J., Watt S., Butler A., West S., Grigorova M., Jones K.W., Wei W., Stratton M.R. et al. 2004. High-resolution analysis of DNA copy number using oligonucleotide microarrays. Genome Res. 14: 287–295. Brinkschmidt C., Christiansen H., Terpe H.J., Simon R., Boecker W., Lampert F., Stoerkel S. 1997. Comparative genomic hybridization (CGH) analysis of neuroblastomas—An important methodological approach in paediatric tumour pathology. J. Pathol. 181: 394–400.[CrossRef][Medline] Bruce S., Leinonen R., Lindgren C.M., Kivinen K., Dahlman-Wright K., Lipsanen-Nyman M., Hannula-Jouppi K., Kere J. 2005. Global analysis of uniparental disomy using high density genotyping arrays. J. Med. Genet. 42: 847–851. Cammarata M., Di Simone P., Graziano L., Giuffre M., Corsello G., Garofalo G. 1999. Rare sex chromosome aneuploidies in humans: Report of six patients with 48,XXYY, 49,XXXXY, and 48,XXXX karyotypes. Am. J. Med. Genet. 85: 86–87.[CrossRef][Medline] Cheung S.W., Shaw C.A., Yu W., Li J., Ou Z., Patel A., Yatsenko S.A., Cooper M.L., Furman P., Stankiewicz P. et al. 2005. Development and validation of a CGH microarray for clinical cytogenetic diagnosis. Genet. Med. 7: 422–432.[Medline] :Clayton-Smith J., Webb T., Cheng X.J., Pembrey M.E., Malcolm S. 1993. Duplication of chromosome 15 in the region 15q11–13 in a patient with developmental delay and ataxia with similarities to Angelman syndrome. J. Med. Genet. 30: 529–531.[Abstract] Cottier M., Tchirkov A., Perissel B., Giollant M., Campos L., Vago P. 2004. Cytogenetic characterization of seven human cancer cell lines by combining G- and R-banding, M-FISH, CGH and chromosome- and locus-specific FISH. Int. J. Mol. Med. 14: 483–495.[Medline] Curran S., McKay J.A., McLeod H.L., Murray G.I. 2000. Laser capture microscopy. Mol. Pathol. 53: 64–68. DeBaun M.R., Niemitz E.L., McNeil D.E., Brandenburg S.A., Lee M.P., Feinberg A.P. 2002. Epigenetic alterations of H19 and LIT1 distinguish patients with Beckwith-Wiedemann syndrome with cancer and birth defects. Am. J. Hum. Genet. 70: 604–611.[CrossRef][Medline] Dockhorn-Dworniczak B., Poremba C., Dantcheva R., Stucker A., Brommelkamp E., Blasius S., Mellin W., Roessner A., Yandell D.W., Bocker W. 1994. Rapid detection of loss of heterozygosity of chromosome 17p by polymerase chain reaction-based variable number of tandem repeat analysis and detection of single-strand conformation polymorphism of intragenic p53 polymorphisms. Virchows Arch. 424: 337–342. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Top
ABSTRACT
Results
) * arctan(Y norm/X norm), where X norm and Y norm represent transformed normalized signals from alleles A and B for a particular locus. The log R ratio of signal intensities is shown as log2(Rsubject/Rexpected) and is the base 2 logarithm of the ratio of observed intensity versus expected intensity (see Results section). Genomic profiles were created using the Illumina Genome Viewer (IGV) and Chromosome Browser (ICV) of Illumina's BeadStudio2.0 software, which plots SNP genotyping data to view, identify, and annotate chromosomal aberrations. 