Breakpoint identification and smoothing of array comparative genomic hybridization data

SUMMARY: We describe a tool, called aCGH-Smooth, for the automated identification of breakpoints and smoothing of microarray comparative genomic hybridization (array CGH) data. aCGH-Smooth is written in visual C++, has a user-friendly interface including a visualization of the results and user-defined parameters adapting the performance of data smoothing and breakpoint recognition. aCGH-Smooth can handle array-CGH data generated by all array-CGH platforms: BAC, PAC, cosmid, cDNA and oligo CGH arrays. The tool has been successfully applied to real-life data. AVAILABILITY: aCGH-Smooth is free for researchers at academic and non-profit institutions at http://www.few.vu.nl/~vumarray/.     

Analysis of array CGH data for cancer studies using fused quantile regression

MOTIVATION: The identification of DNA copy number changes provides insights that may advance our understanding of initiation and progression of cancer. Array-based comparative genomic hybridization (array-CGH) has emerged as a technique allowing high-throughput genome-wide scanning for chromosomal aberrations. A number of statistical methods have been proposed for the analysis of array-CGH data. In this article, we consider a fused quantile regression model based on three motivations: (1) quantile regression may provide a more comprehensive picture for the ratio profile of copy numbers than the standard mean regression approach; (2) for simplicity, most available methods assume uniform spacing between neighboring clones, while incorporating the information of physical locations of clones may be helpful and (3) most current methods have a set of tuning parameters that must be carefully tuned, which introduces complexity to the implementation. RESULTS: We formulate the detection of regions of gains and losses in a fused regularized quantile regression framework, incorporating physical locations of clones. We derive an efficient algorithm that computes the entire solution path for the resulting optimization problem, and we propose a simple estimate for the complexity of the fitted model, which leads to convenient selection of the tuning parameter. Three published array-CGH datasets are used to demonstrate our approach. AVAILABILITY: R code are available at http://www.stat.lsa.umich.edu/~jizhu/code/cgh/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.     

Identification of Citrus sinensis BAC clones containing genes relevant to fruit quality by two-dimensional overgo hybridization

As a complement to whole-genome sequencing efforts, we are comparing targeted genomic regions among sweet orange cultivars to identify coding and conserved noncoding regions, including regulatory elements, responsible for biological features unique to this species. Here, we report the identification of 1,018 bacterial artificial chromosome (BAC) clones containing genes relevant to fruit quality from a Citrus sinensis cv. “Vaniglia” 19.3X BAC library by two-dimensional 9?×?9 overgo hybridization. To design the overgo probes, we used the “C38” expressed sequence tag assembly (http://harvest.ucr.edu/) and OligoSpawn software (http://138.23.178.42). For BAC library screening, we selected 81 overgo probes associated with unigenes that putatively code for enzymes relevant to fruit quality (flavonol, anthocyanin, carotenoid, cellulose, starch, ascorbic acid, aromatic amino acid, and lignin biosynthesis; sucrose catabolism; glycolysis; oxidative/nonoxidative pentose phosphate pathway; fatty acid biosynthesis and oxidation; Krebs cycle). Hybridization probes were pooled and hybridized in groups of intersecting rows and columns to high-density BAC filters, followed by a deconvolution process that established BAC-probe addresses. BAC addresses were obtained for 75 of the 81 overgo probes initially selected, for a total of 1,018 BAC clones, a number consistent with the depth of coverage of the BAC library. BAC end sequencing was carried out, and end-sequence pairs were mapped to their best location in the Citrus clementina genome sequence assembly using the comparative genomic database Phytozome (http://www.phytozome.net/). The BAC clones corresponding to each probe were mapped within the same scaffold as the target gene, demonstrating that the approach we used was successful in isolating the targeted genomic regions.     

ArrayCyGHt: a web application for analysis and visualization of array-CGH data

ArrayCyGHt is a web-based application tool for analysis and visualization of microarray-comparative genomic hybridization (array-CGH) data. Full process of array-CGH data analysis, from normalization of raw data to the final visualization of copy number gain or loss, can be straightforwardly achieved on this arrayCyGHt system without the use of any further software. ArrayCyGHt, therefore, provides an easy and fast tool for the analysis of copy number aberrations in any kinds of data format. AVAILABILITY: ArrayCyGHt can be accessed at http://genomics.catholic.ac.kr/arrayCGH/     

CGH, cDNA and tissue microarray analyses implicate FGFR2 amplification in a small subset of breast tumors.

Multiple regions of the genome are often amplified during breast cancer development and progression, as evidenced in a number of published studies by comparative genomic hybridization (CGH). However, only relatively few target genes for such amplifications have been identified. Here, we indicate how small-scale commercially available cDNA and CGH microarray formats combined with the tissue microarray technology enable rapid identification of putative amplification target genes as well as analysis of their clinical significance. According to CGH, the SUM-52 breast cancer cell line harbors several high-level DNA amplification sites, including the 10q26 chromosomal region where the fibroblast growth factor receptor 2 (FGFR2) gene has been localized. High level amplification of FGFR2 in SUM-52 was identified using CGH analysis on a microarray of BAC clones. A cDNA microarray survey of 588 genes showed >40-fold overexpression of FGFR2. Finally, a tissue microarray based FISH analysis of 750 uncultured primary breast cancers demonstrated in vivo amplification of the FGFR2 gene in about 1% of the tumors. In conclusion, three consecutive microarray (CGH, cDNA and tissue) experiments revealed high-level amplification and overexpression of the FGFR2 in a breast cancer cell line, but only a low frequency of involvement in primary breast tumors. Applied to a genomic scale with larger arrays, this strategy should facilitate identification of the most important target genes for cytogenetic rearrangements, such as DNA amplification sites detected by conventional CGH. Figures on http://www.esacp.org/acp/2001/22-4/heiskanen.htm     

A comparison study: applying segmentation to array CGH data for downstream analyses

MOTIVATION: Array comparative genomic hybridization (CGH) allows detection and mapping of copy number of DNA segments. A challenge is to make inferences about the copy number structure of the genome. Several statistical methods have been proposed to determine genomic segments with different copy number levels. However, to date, no comprehensive comparison of various characteristics of these methods exists. Moreover, the segmentation results have not been utilized in downstream analyses. RESULTS: We describe a comparison of three popular and publicly available methods for the analysis of array CGH data and we demonstrate how segmentation results may be utilized in the downstream analyses such as testing and classification, yielding higher power and prediction accuracy. Since the methods operate on individual chromosomes, we also propose a novel procedure for merging segments across the genome, which results in an interpretable set of copy number levels, and thus facilitate identification of copy number alterations in each genome. AVAILABILITY: http://www.bioconductor.org     

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.     

Clinical application of array-based comparative genomic hybridization for the identification of prognostically important genetic alterations in chronic lymphocytic leukemia

Genomic aberrations have increasingly gained attention as prognostic markers in B-cell chronic lymphocytic leukemia (CLL). Fluorescence in situ hybridization (FISH) has improved the detection rate of genomic alterations in CLL from approximately 50% using conventional cytogenetics to greater than 80%. More recently, array comparative genomic hybridization (CGH) has gained popularity as a clinical tool that can be applied to detect genomic gains and losses of prognostic importance in CLL. Array CGH and FISH are particularly useful in CLL because genomic gains and losses are key events with both biologic and prognostic significance, while balanced translocations have limited prognostic value. Although FISH has a higher technical sensitivity, it requires separate, targeted hybridizations for the detection of alterations at genomic loci of interest. Array CGH, on the other hand, has the ability to provide a genome-wide survey of genomic aberrations with a single hybridization reaction. Array CGH is expanding the known genomic regions of importance in CLL and allows these regions to be evaluated in the context of a genome-wide perspective. Ongoing clinical trials are evaluating the use of genomic aberrations as tools for risk-stratifying patients for therapy, thus increasing the need for reliable and high-yield methods to detect these genomic changes. In this review, we consider the use of array CGH as a clinical tool for the identification of genomic alterations with prognostic significance in CLL, and suggest ways to integrate this test into the clinical molecular diagnostic laboratory work flow.     

Genetics of early miscarriage

A miscarriage is the most frequent complication of a pregnancy. Poor chromosome preparations, culture failure, or maternal cell contamination may hamper conventional karyotyping. Techniques such as chromosomal comparative genomic hybridization (chromosomal‐CGH), array-comparative genomic hybridization (array-CGH), fluorescence in situ hybridization (FISH), multiplex ligation-dependent probe amplification (MLPA) and quantitative fluorescent polymerase chain reaction (QF-PCR) enable us to trace submicroscopic abnormalities. We found the prevalence of chromosome abnormalities in women facing a single sporadic miscarriage to be 45% (95% CI: 38–52; 13 studies, 7012 samples). The prevalence of chromosome abnormalities in women experiencing a subsequent miscarriage after preceding recurrent miscarriage proved to be comparable: 39% (95% CI: 29–50; 6 studies 1359 samples). More chromosome abnormalities are detected by conventional karyotyping compared to FISH or MLPA only (chromosome region specific techniques), and the same amount of abnormalities compared to QF-PCR (chromosome region specific techniques) and chromosomal‐CGH and array-CGH (whole genome techniques) only. Molecular techniques could play a role as an additional technique when culture failure or maternal contamination occurs: recent studies show that by using array-CGH, an additional 5% of submicroscopic chromosome variants can be detected. Because of the small sample size as well as the unknown clinical relevance of these molecular aberrations, more and larger studies should be performed of submicroscopic chromosome abnormalities among sporadic miscarriage samples. For recurrent miscarriage samples molecular technique studies are relatively new. It has often been suggested that miscarriages are due to chromosomal abnormalities in more than 50%, but the present review has determined that chromosomal and submicroscopic genetic abnormalities on average are prevalent in maximally half of the miscarriage samples. This article is part of a Special Issue entitled: Molecular Genetics of Human Reproductive Failure.     

A pseudolikelihood approach for simultaneous analysis of array comparative genomic hybridizations

DNA sequence copy number has been shown to be associated with cancer development and progression. Array-based comparative genomic hybridization (aCGH) is a recent development that seeks to identify the copy number ratio at large numbers of markers across the genome. Due to experimental and biological variations across chromosomes and hybridizations, current methods are limited to analyses of single chromosomes. We propose a more powerful approach that borrows strength across chromosomes and hybridizations. We assume a Gaussian mixture model, with a hidden Markov dependence structure and with random effects to allow for intertumoral variation, as well as intratumoral clonal variation. For ease of computation, we base estimation on a pseudolikelihood function. The method produces quantitative assessments of the likelihood of genetic alterations at each clone, along with a graphical display for simple visual interpretation. We assess the characteristics of the method through simulation studies and analysis of a brain tumor aCGH data set. We show that the pseudolikelihood approach is superior to existing methods both in detecting small regions of copy number alteration and in accurately classifying regions of change when intratumoral clonal variation is present. Software for this approach is available at http://www.biostat.harvard.edu/ approximately betensky/papers.html.     

Development of bioinformatics resources for display and analysis of copy number and other structural variants in the human genome

The discovery of an abundance of copy number variants (CNVs; gains and losses of DNA sequences >1 kb) and other structural variants in the human genome is influencing the way research and diagnostic analyses are being designed and interpreted. As such, comprehensive databases with the most relevant information will be critical to fully understand the results and have impact in a diverse range of disciplines ranging from molecular biology to clinical genetics. Here, we describe the development of bioinformatics resources to facilitate these studies. The Database of Genomic Variants (http://projects.tcag.ca/variation/) is a comprehensive catalogue of structural variation in the human genome. The database currently contains 1,267 regions reported to contain copy number variation or inversions in apparently healthy human cases. We describe the current contents of the database and how it can serve as a resource for interpretation of array comparative genomic hybridization (array CGH) and other DNA copy imbalance data. We also present the structure of the database, which was built using a new data modeling methodology termed Cross-Referenced Tables (XRT). This is a generic and easy-to-use platform, which is strong in handling textual data and complex relationships. Web-based presentation tools have been built allowing publication of XRT data to the web immediately along with rapid sharing of files with other databases and genome browsers. We also describe a novel tool named eFISH (electronic fluorescence in situ hybridization) (http://projects.tcag.ca/efish/), a BLAST-based program that was developed to facilitate the choice of appropriate clones for FISH and CGH experiments, as well as interpretation of results in which genomic DNA probes are used in hybridization-based experiments.     

ArrayFusion: a web application for multi-dimensional analysis of CGH, SNP and microarray data

ArrayFusion annotates conventional CGH results and various types of microarray data from a range of platforms (cDNA, expression, exon, SNP, array-CGH and ChIP-on-chip) and converts them into standard formats which can be visualized in genome browsers (Affymetrix Integrated Genome Browser and GBrowse in the HapMap Project). Converted files can then be imported simultaneously into a single genome browser to benefit a collective interpretation between different array results. ArrayFusion therefore provides a new type of tool facilitating the integration of CGH and array results to provide new experimental directions. AVAILABILITY: http://microarray.ym.edu.tw/tools/arrayfusion     

CGHcall: calling aberrations for array CGH tumor profiles

CGHcall achieves high calling accuracy for array CGH data by effective use of breakpoint information from segmentation and by inclusion of several biological concepts that are ignored by existing algorithms. The algorithm is validated for simulated and verified real array CGH data. By incorporating more than three classes, CGHcall improves detection of single copy gains and amplifications. Moreover, it allows effective inclusion of chromosome arm information. AVAILABILITY: An R-package (GUI), a manual and an example data set are available at http://www.few.vu.nl/~mavdwiel/CGHcall.html. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.     

De novo balanced complex chromosome rearrangement (CCR) involving chromosome 8, 11 and 16 in a boy with mild developmental delay and psychotic disorder

Congenital Complex Chromosome rearrangements (CCRs) compatible with life are rare in humans. We report a de novo CCR involving chromosomes 8, 11 and 16 with 4 breakpoints in a patient with mild dysmorphic features, acquisition delay and psychotic disorder. Conventional cytogenetic analysis revealed an apparently balanced 8;16 translocation. Further FISH analysis with WCP 8 and WCP 16 probes revealed the presence of a third chromosome involved in the translocation. The multicolour karyotype confirmed the complexity of the rearrangement and showed that the derivative chromosome 8 was composed of 3 distinct segments derived from chromosomes 8, 16 and 11. The breakpoints of this complex rearrangement were located at 8q21, 11q14, 11q23 and 16q12. Comparative genomic hybridization (CGH) and array-CGH were performed to investigate the possibility of any genomic imbalance as a result of the complex rearrangement. No imbalance was detected by these two techniques. Our study showed: i) the necessity to confirm reciprocal translocations with FISH using painting probes, particularly when the karyotype resolution is weak; ii) the usefulness of multicolour karyotype for the characterization of structural chromosomal rearrangements, particularly when they are complex; iii) the usefulness of CGH and array-CGH in cases of abnormal phenotype and apparently balanced rearrangement in order to explore the breakpoints and to detect additional imbalances.     

Effects of chromosomal variations on pharmacokinetic activity of zolpidem in healthy volunteers: an array-based comparative genomic hybridization study

Zolpidem has been known as a very safe and effective hypnotic drug used to treat a variety of patients with insomnia. Even though the same dose of the medicine is administered to each patient, the blood level of zolpidem and the time required to obtain peak concentration are not consistent among different people. We evaluated the relationship between the peak concentrations of zolpidem and chromosomal imbalances using a high-resolution genome-wide array-based comparative genomic hybridization (CGH) in 16 healthy volunteers in order to detect the genetic factors underlying the variations. The present study showed that chromosomal losses were detected in the 4q35.2, 9p13.1 and 9p12 regions, and those gains were indicated in the 2p14, 11q13.4 and 15q11.2 regions. The abnormal regions were confirmed by fluorescence in situ hybridization (FISH) and real-time PCR. It is suggested that array-CGH analysis may be used as a measure for pharmacogenomic applications in the patients with insomnia and for further exploration of candidate genomic regions implicated in sleep disturbances.     

Classification and feature selection algorithms for multi-class CGH data

Recurrent chromosomal alterations provide cytological and molecular positions for the diagnosis and prognosis of cancer. Comparative genomic hybridization (CGH) has been useful in understanding these alterations in cancerous cells. CGH datasets consist of samples that are represented by large dimensional arrays of intervals. Each sample consists of long runs of intervals with losses and gains. In this article, we develop novel SVM-based methods for classification and feature selection of CGH data. For classification, we developed a novel similarity kernel that is shown to be more effective than the standard linear kernel used in SVM. For feature selection, we propose a novel method based on the new kernel that iteratively selects features that provides the maximum benefit for classification. We compared our methods against the best wrapper-based and filter-based approaches that have been used for feature selection of large dimensional biological data. Our results on datasets generated from the Progenetix database, suggests that our methods are considerably superior to existing methods. AVAILABILITY: All software developed in this article can be downloaded from http://plaza.ufl.edu/junliu/feature.tar.gz.     

Detection of gene copy number changes in CGH microarrays using a spatially correlated mixture model

MOTIVATION: Comparative genomic hybridization array experiments that investigate gene copy number changes present new challenges for statistical analysis and call for methods that incorporate spatial dependence between sequences along the chromosome. For this purpose, we propose a novel method called CGHmix. It is based on a spatially structured mixture model with three states corresponding to genomic sequences that are either unmodified, deleted or amplified. Inference is performed in a Bayesian framework. From the output, posterior probabilities of belonging to each of the three states are estimated for each genomic sequence and used to classify them. RESULTS: Using simulated data, CGHmix is validated and compared with both a conventional unstructured mixture model and with a recently proposed data mining method. We demonstrate the good performance of CGHmix for classifying copy number changes. In addition, the method provides a good estimate of the false discovery rate. We also present the analysis of a cancer related dataset. SUPPLEMENTARY INFORMATION: http://www.bgx.org.uk/papers.html     

Mediante: a web-based microarray data manager

Mediante is a MIAME-compliant microarray data manager that links together annotations and experimental data. Developed as a J2EE three-tier application, Mediante integrates a management system for production of long oligonucleotide microarrays, an experimental data repository suitable for home made or commercial microarrays, and a user interface dedicated to the management of microarrays projects. Several tools allow quality control of hybridizations and submission of validated data to public repositories. AVAILABILITY: http://www.microarray.fr. SUPPLEMENTARY INFORMATION: http://www.microarray.fr/SP/lebrigand2007/