Genome-wide copy number profiling on high-density bacterial artificial chromosomes, single-nucleotide polymorphisms, and oligonucleotide microarrays: a platform comparison based on statistical power analysis.

Recently, comparative genomic hybridization onto bacterial artificial chromosome (BAC) arrays (array-based comparative genomic hybridization) has proved to be successful for the detection of submicroscopic DNA copy-number variations in health and disease. Technological improvements to achieve a higher resolution have resulted in the generation of additional microarray platforms encompassing larger numbers of shorter DNA targets (oligonucleotides). Here, we present a novel method to estimate the ability of a microarray to detect genomic copy-number variations of different sizes and types (i.e. deletions or duplications). We applied our method, which is based on statistical power analysis, to four widely used high-density genomic microarray platforms. By doing so, we found that the high-density oligonucleotide platforms are superior to the BAC platform for the genome-wide detection of copy-number variations smaller than 1 Mb. The capacity to reliably detect single copy-number variations below 100 kb, however, appeared to be limited for all platforms tested. In addition, our analysis revealed an unexpected platform-dependent difference in sensitivity to detect a single copy-number loss and a single copy-number gain. These analyses provide a first objective insight into the true capacities and limitations of different genomic microarrays to detect and define DNA copy-number variations.     

Generation and application of dynamic standard reference intervals for analyzing results of comparative genomic hybridization

The present work was aimed at generating the dynamic standard reference intervals (DSRI) and their application for chromosomal-aberration (CA) analysis. The evaluation of the generated DSRI was performed using the DNA samples from four patients with already known CA. High-resolution comparative genomic hybridization analysis (HR-CGH) allowed us to not only identify all of the CAs that were not revealed by CGH, but also to detect the breakpoints and to determine the size of chromosomal imbalance.     

A simple method for fixation and microdissection of frozen fresh tissue sections for molecular cytogenetic analysis of cancers.

Microdissection has been widely used for procuring DNA from specific microscopic regions of formalin fixed, paraffin embedded tissue sections. We have developed a method for fixation and microdissection of frozen fresh biopsy tissue sections. Five micrometer frozen fresh tissue sections were fixed with ethanol and stored at room temperature. Well defined regions from hematoxylin and eosin (H & E) stained or unstained sections were briefly steamed and microdissected using a needle. The dissected tissue was digested with proteinase K and DNA was isolated. Whole genome amplifications were obtained by degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) from these samples. The reliability of this technique was demonstrated by comparing conventional comparative genomic hybridization (CGH) with DOP-PCR-CGH. The advantages of this method are that frozen fresh sections can be fixed easily and stored for more than 4 years, it is easy to microdissect and pick-up very minute regions (0.1 mm(2)), and it is rapid; microdissection and purification can be accomplished within 3 h. Using DNA from microdissected sections, DOP-PCR-CGH revealed genetic abnormalities more accurately than conventional CGH. Although this novel method was demonstrated using DOP-PCR-CGH, we believe that it will be useful for other genetic analyses of specific small regions and cell populations. We also observed whether storage time, H & E staining and crude DNA extracts affected the quality of amplified DNA. DNA integrity was maintained for at least 49 months in ethanol fixed sections that were stored at room temperature, but DNA was gradually degraded after one month if the ethanol fixed sections had been H & E stained and stored. When crude DNA extracts from H & E stained sections were used, the size of the DOP-PCR product was reduced. Our study suggests that ethanol fixed tissue sections may be stored at room temperature for at least 4 years without DNA degradation, the H & E stains may not affect the quality of amplified DNA, but H & E or other components in the staining process may reduce the size of DOP-PCR product, which is critical for the quality of CGH hybridization.     

Exon array CGH: detection of copy-number changes at the resolution of individual exons in the human genome

The development of high-throughput screening methods such as array-based comparative genome hybridization (array CGH) allows screening of the human genome for copy-number changes. Current array CGH strategies have limits of resolution that make detection of small (less than a few tens of kilobases) gains or losses of genomic DNA difficult to identify. We report here a significant improvement in the resolution of array CGH, with the development of an array platform that utilizes single-stranded DNA array elements to accurately measure copy-number changes of individual exons in the human genome. Using this technology, we screened 31 patient samples across an array containing a total of 162 exons for five disease genes and detected copy-number changes, ranging from whole-gene deletions and duplications to single-exon deletions and duplications, in 100% of the cases. Our data demonstrate that it is possible to screen the human genome for copy-number changes with array CGH at a resolution that is 2 orders of magnitude higher than that previously reported.     

Optimization of DOP-PCR amplification of DNA for high-resolution comparative genomic hybridization analysis

BACKGROUND: Whole-genome amplification of minute samples of DNA for the use in comparative genomic hybridization (CGH) analysis has found widespread use, but the method has not been well validated. METHODS: Four protocols for degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) and fluorescence labeling were applied to test DNA from normal and K-562 cells. The DNA products were used for CGH analysis. RESULTS: The DOP-PCR-amplified DNA from each protocol produced hybridizations with different qualities. These could be seen primarily as differences in background staining and signal-to-noise ratios, but also as characteristic deviations of normal/normal hybridizations. One DOP-PCR-protocol was further investigated. We observed concordance between CGH results using unamplified and DOP-PCR-amplified DNA. An example of an analysis of an invasive carcinoma of the breast supports the practical value of this approach. CONCLUSIONS: DOP-PCR-amplified DNA is applicable for high- resolution CGH, the results being similar to those of CGH using unamplified DNA.     

Utility of DNA amplified by degenerate oligonucleotide-primed PCR (DOP-PCR) from the total genome and defined chromosomal regions of field bean

Degenerate oligonucleotide primed (DOP)-PCR has emerged as a simple and rapid method for representative amplification of highly complex genomic DNA from humans, mice and Drosophila. The present paper describes the adaptation of this method for use on a plant species, Vicia faba, with a large genome (2C = 30 pg). Specific low-copy-number sequences as well as highly repeated sequences were detectable among DOP-PCR products obtained from small samples of purified genomic DNA (100 pg), DNA from 10 prophase nuclei, 10 flow-sorted chromosomes or 15 microdissected chromosome segments (satellites) following reamplification with sequence-specific primers and/or Southern hybridization. Biotinylated chromosome-specific DOP-PCR products were used for fluorescent in situ hybridization. All chromosomes showed hybridization signals, with the exception of regions containing Fok elements which are not present in the chromosomal DNA targeted by DOP-PCR.     

Detection of bacterial pathogens in municipal wastewater using an oligonucleotide microarray and real-time quantitative PCR

As a first step toward building a comprehensive microarray, two low density DNA microarrays were constructed and evaluated for the accurate detection of wastewater pathogens. The first one involved the direct hybridization of wastewater microbial genomic DNA to the functional gene probes while the second involved PCR amplification of 23S ribosomal DNA. The genomic DNA microarray employed 10 functional genes as detection targets. Sensitivity of the microarray was determined to be approximately 1.0 microg of Esherichia coli genomic DNA, or 2 x 10(8) copies of the target gene, and only E. coli DNA was detected with the microarray assay using municipal raw sewage. Sensitivity of the microarray was enhanced approximately by 6 orders of magnitude when the target 23S rRNA gene sequences were PCR amplified with a novel universal primer set and allowed hybridization to 24 species-specific oligonucleotide probes. The minimum detection limit was estimated to be about 100 fg of E. coli genomic DNA or 1.4 x 10(2) copies of the 23S rRNA gene. The PCR amplified DNA microarray successfully detected multiple bacterial pathogens in wastewater. As a parallel study to verify efficiency of the DNA microarray, a real-time quantitative PCR assay was also developed based on the fluorescent TaqMan probes (Applied Biosystems).     

Genome-wide detection of chromosomal imbalances in tumors using BAC microarrays

Chromosomal imbalances such as deletions and amplifications are common rearrangements in most tumors. Specific rearrangements are consistently associated with specific tumor types or stages, implicating the role of the genes in a region of chromosomal imbalance in tumor initiation and progression. The development of comparative genomic hybridization (CGH) has obviated the need to obtain metaphase spreads from tumors, so that the chromosomal imbalances in many solid tumors may be revealed using an extracted genomic DNA sample. However, the resolution of the cytogenetic method remains and the extreme technical difficulty of CGH has restricted its use. Conceptually, DNA microarray-based CGH is an obvious solution to all of the limitations of conventional CGH. Although arrays have been used for CGH studies, their success has been limited by poor specific signal-to-noise ratios. Here we demonstrate a microarray-based CGH method that allows reliable detection of chromosomal deletions and amplifications with high resolution. Our microarray system is fundamentally different from most current microarray technologies in that activated DNA is printed on natural glass surfaces while other systems almost exclusively focus on activating the surfaces, a strategy that invariably introduces hybridization backgrounds. The concept of using pre-modification may be generally applied for making arrays of other biological materials, as modifying the substrates will be more controllable in solution than on surfaces.     

Array-MAPH: a methodology for the detection of locus copy-number changes in complex genomes

High-throughput genome-wide screening methods to detect subtle genomic imbalances are extremely important for diagnostic genetics and genomics. Here, we provide a detailed protocol for a microarray-based technique, applying the principle of multiplex amplifiable probe hybridization (MAPH). Methodology and software have been developed for designing unique PCR-amplifiable sequences (400-600 bp) covering any genomic region of interest. These sequences are amplified, cloned and spotted onto arrays (targets). A mixture of the same sequences (probes) is hybridized to genomic DNA immobilized on a membrane. Bound probes are recovered and quantitatively amplified by PCR, labeled and hybridized to the array. The procedure can be completed in 4-5 working days, excluding microarray preparation. Unlike array-comparative genomic hybridization (array-CGH), test DNA of specifically reduced complexity is hybridized to an array of identical small amplifiable target sequences, resulting in increased hybridization specificity and higher potential for increasing resolution. Array-MAPH can be used for detection of small-scale copy-number changes in complex genomes, leading to genotype-phenotype correlations and the discovery of new genes.     

Allele-specific disparity in breast cancer

Background

Molecular alterations critical to development of cancer include mutations, copy number alterations (amplifications and deletions) as well as genomic rearrangements resulting in gene fusions. Massively parallel next generation sequencing, which enables the discovery of such changes, uses considerable quantities of genomic DNA (> 5 ug), a serious limitation in ever smaller clinical samples. However, a commonly available microarray platforms such as array comparative genomic hybridization (array CGH) allows the characterization of gene copy number at a single gene resolution using much smaller amounts of genomic DNA. In this study we evaluate the sensitivity of ultra-dense array CGH platforms developed by Agilent, especially that of the 1 million probe array (1 M array), and their application when whole genome amplification is required because of limited sample quantities.

Methods

We performed array CGH on whole genome amplified and not amplified genomic DNA from MCF-7 breast cancer cells, using 244 K and 1 M Agilent arrays. The ADM-2 algorithm was used to identify micro-copy number alterations that measured less than 1 Mb in genomic length.

Results

DNA from MCF-7 breast cancer cells was analyzed for micro-copy number alterations, defined as measuring less than 1 Mb in genomic length. The 4-fold extra resolution of the 1 M array platform relative to the less dense 244 K array platform, led to the improved detection of copy number variations (CNVs) and micro-CNAs. The identification of intra-genic breakpoints in areas of DNA copy number gain signaled the possible presence of gene fusion events. However, the ultra-dense platforms, especially the densest 1 M array, detect artifacts inherent to whole genome amplification and should be used only with non-amplified DNA samples.

Conclusions

This is a first report using 1 M array CGH for the discovery of cancer genes and biomarkers. We show the remarkable capacity of this technology to discover CNVs, micro-copy number alterations and even gene fusions. However, these platforms require excellent genomic DNA quality and do not tolerate relatively small imperfections related to the whole genome amplification.     

SNP genotyping on a genome-wide amplified DOP-PCR template

With the increasing demand for higher throughput single nucleotide polymorphism (SNP) genotyping, the quantity of genomic DNA often falls short of the number of assays required. We investigated the use of degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) to generate a template for our SNP genotyping methodology of fluorescence polarization template-directed dye-terminator incorporation detection. DOP-PCR employs a degenerate primer (5′-CCGACTCGAGNNNNNNATGTGG-3′) to produce non-specific uniform amplification of DNA. This approach has been successfully applied to microsatellite genotyping. We compared genotyping of DOP-PCR-amplified genomic DNA to genomic DNA as a template. Results were analyzed with respect to feasibility, allele loss of alleles, genotyping accuracy and storage conditions in a high-throughput genotyping environment. DOP-PCR yielded overall satisfactory results, with a certain loss in accuracy and quality of the genotype assignments. Accuracy and quality of genotypes generated from the DOP-PCR template also depended on storage conditions. Adding carrier DNA to a final concentration of 10 ng/µl improved results. In conclusion, we have successfully used DOP-PCR to amplify our genomic DNA collection for subsequent SNP genotyping as a standard process.     

Measurement of locus copy number by hybridisation with amplifiable probes

Despite its fundamental importance in genome analysis, it is only recently that systematic approaches have been developed to assess copy number at specific genetic loci, or to examine genomic DNA for submicroscopic deletions of unknown location. In this report we show that short probes can be recovered and amplified quantitatively following hybridisation to genomic DNA. This simple observation forms the basis of a new approach to determining locus copy number in complex genomes. The power and specificity of multiplex amplifiable probe hybridisation is demonstrated by the simultaneous assessment of copy number at a set of 40 human loci, including detection of deletions causing Duchenne muscular dystrophy and Prader–Willi/Angelman syndromes. Assembly of other probe sets will allow novel, technically simple approaches to a wide variety of genetic analyses, including the potential for extension to high resolution genome-wide screens for deletions and amplifications.     

Development of a DNA-labeling system for array-based comparative genomic hybridization.

Chromosomal amplifications and deletions are critical components of tumorigenesis and DNA copy-number variations also correlate with changes in mRNA expression levels. Genome-wide microarray comparative genomic hybridization (CGH) has become an important method for detecting and mapping chromosomal changes in tumors. Thus, the ability to detect twofold differences in fluorescent intensity between samples on microarrays depends on the generation of high-quality labeled probes. To enhance array-based CGH analysis, a random prime genomic DNA labeling method optimized for improved sensitivity, signal-to-noise ratios, and reproducibility has been developed. The labeling system comprises formulated random primers, nucleotide mixtures, and notably a high concentration of the double mutant exo-large fragment of DNA polymerase I (exo-Klenow). Microarray analyses indicate that the genomic DNA-labeled templates yield hybridization signals with higher fluorescent intensities and greater signal-to-noise ratios and detect more positive features than the standard random prime and conventional nick translation methods. Also, templates generated by this system have detected twofold differences in gene copy number between male and female genomic DNA and identified amplification and deletions from the BT474 breast cancer cell line in microarray hybridizations. Moreover, alterations in gene copy number were routinely detected with 0.5 microg of genomic DNA starting sample. The method is flexible and performs efficiently with different fluorescently labeled nucleotides. Application of the optimized CGH labeling system may enhance the resolution and sensitivity of array-based CGH analysis in cancer and medical genetic studies.     

Generation of chromosome paints: approach for increasing specificity and intensity of signals

Chromosome painting is a widely used technique, and the two principal means of generating probes for such experiments involve DNA isolation by chromosome flow sorting and by chromosome microdissection. Frequently, chromosome paints are bright and specific; however, on occasion, signals can be weak and nonspecific, particularly for microdissected probes. Reasons for this have been attributed to co-amplification of non-target DNA and the formation of primer concatamers during degenerate oligonucleotide primed (DOP)-PCR. Here we describe a technique of circumventing this problem by sequence enrichment. It involves co-hybridization of DOP-PCR biotinylated microdissected material and linkered genomic DNA. Biotinylated DNA fragments captured on streptavidin-coated paramagnetic beads are eluted and amplified by PCR using a single primer complementary to the linker arm.     

Long DOP-PCR of rare archival anthropological samples

The application of molecular DNA technologies to anthropological questions has meant that rare or archival samples of human remains, including blood, hair, and bone, can now be used as a source of material for genetic analysis. Often, these samples are irreplaceable, and/or yield very small quantities of DNA, so methods for preamplifying as much of the whole genome as possible would greatly enhance their usefulness. DOP-PCR (degenerate oligonucleotide-primed polymerase chain reaction) is an amplification method that uses a degenerate primer and very low initial annealing temperatures to amplify the whole genome. We adapted a published DOP-PCR protocol to long PCR enzyme and amplification conditions. The effectiveness of these modifications was tested by PCR amplification of DOP-PCR products at a mixture of genomic targets including 66 different microsatellites, 11 Alu insertion polymorphisms, and variable-length segments of the human lipoprotein lipase gene (LPL). The selected microsatellite markers were chosen to represent every chromosome, with expected product sizes ranging from 150 base pairs to 8,000 base pairs in length, while the 22 Alu insertion polymorphisms were selected to reveal biases in the recovery of alleles of different sizes. To determine nucleotide sequence variation, 2 kilobases (kb) of the LPL gene in 30 Mongolian individuals were sequenced. All gene-specific targets from DOP-PCR product template were amplified. No unexpected polymorphisms in the sequence results attributable to the DOP-PCR step were found, and 93% to 95% of Alu genotypes that have been amplified from total genomic DNA were replicated. The incorrect typings were all due to the preferential amplification of the shorter of two possible alleles in individuals heterozygous for an Alu insertion and were all correctly typed on subsequent reamplification of the gene-specific PCR products. This method of whole-genome amplification promises to be an efficient way to maximize the genetic use of rare anthropological samples.     

Two-dimensional DNA displays for comparisons of bacterial genomes

We have developed two whole genome-scanning techniques to aid in the discovery of polymorphisms as well as horizontally acquired genes in prokaryotic organisms.First, two-dimensional bacterial genomic display (2DBGD) was developed using restriction enzyme fragmentation to separate genomic DNA based on size, and then employing denaturing gradient gel electrophoresis (DGGE) in the second dimension to exploit differences in sequence composition. This technique was used to generate high-resolution displays that enable the direct comparison of >800 genomic fragments simultaneously and can be adapted for the high-throughput comparison of bacterial genomes. 2DBGDs are capable of detecting acquired and altered DNA, however, only in very closely related strains. If used to compare more distantly related strains (e.g. different species within a genus) numerous small changes (i.e. small deletions and point mutations) unrelated to the interesting phenotype, would encumber the comparison of 2DBGDs. For this reason asecond method, bacterial comparative genomic hybridization (BCGH), was developed to directly compare bacterial genomes to identify gain or loss of genomic DNA. BCGH relies on performing 2DBGD on a pooled sample of genomic DNA from 2 strains to be compared and subsequently hybridizing the resulting 2DBGD blot separately with DNA from each individual strain. Unique spots (hybridization signals) represent foreign DNA. The identification of novel DNA is easily achieved by excising the DNA from a dried gel followed by subsequent cloning and sequencing. 2DBGD and BCGH thus represent novel high resolution genome scanning techniques for directly identifying altered and/or acquired DNA. Published: June 15, 2003     

Human and mouse oligonucleotide-based array CGH

Array-based comparative genomic hybridization is a high resolution method for measuring chromosomal copy number changes. Here we present a validated protocol using in-house spotted oligonucleotide libraries for array comparative genomic hybridization (CGH). This oligo array CGH platform yields reproducible results and is capable of detecting single copy gains, multi-copy amplifications as well as homozygous and heterozygous deletions as small as 100 kb with high resolution. A human oligonucleotide library was printed on amine binding slides. Arrays were hybridized using a hybstation and analysed using BlueFuse feature extraction software, with >95% of spots passing quality control. The protocol allows as little as 300 ng of input DNA and a 90% reduction of Cot-1 DNA without compromising quality. High quality results have also been obtained with DNA from archival tissue. Finally, in addition to human oligo arrays, we have applied the protocol successfully to mouse oligo arrays. We believe that this oligo-based platform using ‘off-the-shelf’ oligo libraries provides an easy accessible alternative to BAC arrays for CGH, which is cost-effective, available at high resolution and easily implemented for any sequenced organism without compromising the quality of the results.     

Progress in the detection of human genome structural variations

The emerging of high-throughput and high-resolution genomic technologies led to the detection of submicroscopic variants ranging from 1 kb to 3 Mb in the human genome. These variants include copy number variations (CNVs), inversions, insertions, deletions and other complex rearrangements of DNA sequences. This paper briefly reviews the commonly used technologies to discover both genomic structural variants and their potential influences. Particularly, we highlight the array-based, PCR-based and sequencing-based assays, including array-based comparative genomic hybridization (aCGH), representational oligonucleotide microarray analysis (ROMA), multiplex amplifiable probe hybridization (MAPH), multiplex ligation-dependent probe amplification (MLPA), paired-end mapping (PEM), and next-generation DNA sequencing technologies. Furthermore, we discuss the limitations and challenges of current assays and give advices on how to make the database of genomic variations more reliable. Supported by the National High Technology Research and Development Program of China (Grant No. 2006AA020704).     

Painting of parental chromatin in Beta hybrids by multi-colour fluorescent in situ hybridization

Sugar beet (Beta vulgaris L.) is a relatively young crop and has a narrow gene pool. In order to introduce genetic variability into the crop, interspecific hybrids, selected from crosses with wild beets of the sections Corollinae and Procumbentes, have been generated. The introgressed B. procumbens chromatin carries resistance genes to beet cyst nematode Heterodera schachtii Schm. These lines are important for breeding of nematode-resistant sugar beet, while Corollinae species are potential donors of tolerance to biotic and abiotic stresses such as drought or saline soils. We have used in situ hybridization of genomic DNA to discriminate the parental chromosomes in these interspecific hybrids. Suppression of cross-hybridization by blocking DNA was not necessary indicating that the investigated Beta genomes contain sufficient species-specific DNA enabling the unequivocal determination of the genomic composition of the hybrids. Interspecific hybrid lines with an additional chromosome (2n = 18 + 1), chromosome fragment (2n = 18 + fragment) or translocation of B. procumbens (2n = 18) were analysed by genomic in situ hybridization (GISH) at mitosis and meiosis. Species-specific satellites and ribosomal genes used in combination with genomic DNA or in rehybridization experiments served as landmark probes for chromosome identification in hybrid genomes. The detection of a B. procumbens translocation of approx. I Mbp demonstrated the sensitivity and resolution of GISH and showed that this approach is a powerful method in genome analysis projects of the genus Beta.     

High frequency of common DNA copy number abnormalities detected by bacterial artificial chromosome array comparative genomic hybridization in 24 breast cancer cell lines

Breast cancer is a widespread disease in Japan and across the world. Breast cancer cells, as well as most other types of cancer cells, have diverse chromosomal aberrations. Clarifying the character of these chromosomal aberrations should contribute to the development of more suitable therapies, along with the predictions of metastasis and prognosis. Twenty-four breast cancer cell lines were analyzed by bacterial artificial chromosome (BAC) array comparative genomic hybridization (CGH). The array slide contained duplicate spots of 4030 BAC clone DNAs covering the entire human genome with 1 Mbp resolution. In all 24 breast cancer cell lines, frequent and significant amplifications as well as deletions were detected by BAC array CGH. Common DNA copy number gains, detected in 60% (above 15 cell lines) of the 24 breast cancer cell lines were found in 76 BAC clones, located at 1q, 5p, 8q, 9p, 16p, 17q, and 20q. Moreover, common DNA copy number loss was detected in 136 BAC clones, located at 1q, 2q, 3p, 4p, 6q, 8p, 9p, 11p, 13q, 17p, 18q, 19p, Xp, and Xq. The DNA copy number abnormalities found included abnormality of the well-known oncogene cMYC (8q24.21); however, most of them were not reported to relate to breast cancer. BAC array CGH has great potential to detect DNA copy number abnormalities, and has revealed that breast cancer cell lines have substantial heterogeneity.