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CGHScan: finding variable regions using high-density microarray comparative genomic hybridization data
Bradley D Anderson, Michael C Gilson, Abigail A Scott, Bryan S Biehl, Jeremy D Glasner, Gireesh Rajashekara, Gary A Splitter, Nicole T Perna
BMC Genomics , 2006, DOI: 10.1186/1471-2164-7-91
Abstract: We present an algorithm for analyzing microarray hybridization data to aid identification of regions that vary between an unsequenced genome and a sequenced reference genome. The program, CGHScan, uses an iterative random walk approach integrating multi-layered significance testing to detect these regions from comparative genomic hybridization data. The algorithm tolerates a high level of noise in measurements of individual probe intensities and is relatively insensitive to the choice of method for normalizing probe intensity values and identifying probes that differ between samples. When applied to comparative genomic hybridization data from a published experiment, CGHScan identified eight of nine known deletions in a Brucella ovis strain as compared to Brucella melitensis. The same result was obtained using two different normalization methods and two different scores to classify data for individual probes as representing conserved or variable genomic regions. The undetected region is a small (58 base pair) deletion that is below the resolution of CGHScan given the array design employed in the study.CGHScan is an effective tool for analyzing comparative genomic hybridization data from high-density microarrays. The algorithm is capable of accurately identifying known variable regions and is tolerant of high noise and varying methods of data preprocessing. Statistical analysis is used to define each variable region providing a robust and reliable method for rapid identification of genomic differences independent of annotated gene boundaries.Comparative genomic hybridization (CGH) is a powerful technique to determine the differences between the genomes of different cell types or organisms. Typically, genomic DNA from known (sequenced) and experimental (unsequenced) genomes is labeled and hybridized to an array of DNA sequences prepared from the known genome. Intensities of hybridization of the two samples are compared to determine the relative copy number of each targ
Experimental analysis of oligonucleotide microarray design criteria to detect deletions by comparative genomic hybridization
Stephane Flibotte, Donald G Moerman
BMC Genomics , 2008, DOI: 10.1186/1471-2164-9-497
Abstract: We have quantified the effects of filtering various oligonucleotide properties by measuring the resolving power for detecting deletions in the human and C. elegans genomes using NimbleGen microarrays. Approximately twice as many oligonucleotides are typically required to be affected by a deletion in human DNA samples in order to achieve the same statistical confidence as one would observe for a deletion in C. elegans. Surprisingly, the ability to detect deletions strongly depends on the oligonucleotide 15-mer count, which is defined as the sum of the genomic frequency of all the constituent 15-mers within the oligonucleotide. A similarity level above 80% to non-target sequences over the length of the probe produces significant cross-hybridization. We recommend the use of a fairly large melting temperature window of up to 10°C, the elimination of repeat sequences, the elimination of homopolymers longer than 5 nucleotides, and a threshold of -1 kcal/mol on the oligonucleotide self-folding energy. We observed very little difference in data quality when varying the oligonucleotide length between 50 and 70, and even when using an isothermal design strategy.We have determined experimentally the effects of varying several key oligonucleotide microarray design criteria for detection of deletions in C. elegans and humans with NimbleGen's CGH technology. Our oligonucleotide design recommendations should be applicable for CGH analysis in most species.In human health research microarray comparative genomic hybridization (CGH) has become a powerful technique to investigate DNA copy number variants (CNVs) in healthy subjects [1,2] and genomic aberrations associated with various diseases and syndromes [3,4]. Furthermore, CGH is now frequently used to analyze the genome of strains of interest in various model organisms [5,6]. On some oligonucleotide microarray platforms individual researchers can design their own specialized microarrays for very specific experiments. Basically, the
Application of Microarray-Based Comparative Genomic Hybridization in Prenatal and Postnatal Settings: Three Case Reports  [PDF]
Jing Liu,Francois Bernier,Julie Lauzon,R. Brian Lowry,Judy Chernos
Genetics Research International , 2011, DOI: 10.4061/2011/976398
Abstract: Microarray-based comparative genomic hybridization (array CGH) is a newly emerged molecular cytogenetic technique for rapid evaluation of the entire genome with sub-megabase resolution. It allows for the comprehensive investigation of thousands and millions of genomic loci at once and therefore enables the efficient detection of DNA copy number variations (a.k.a, cryptic genomic imbalances). The development and the clinical application of array CGH have revolutionized the diagnostic process in patients and has provided a clue to many unidentified or unexplained diseases which are suspected to have a genetic cause. In this paper, we present three clinical cases in both prenatal and postnatal settings. Among all, array CGH played a major discovery role to reveal the cryptic and/or complex nature of chromosome arrangements. By identifying the genetic causes responsible for the clinical observation in patients, array CGH has provided accurate diagnosis and appropriate clinical management in a timely and efficient manner. 1. Introduction Genomic disorders, resulting from DNA rearrangements involving region-specific repeat sequences, are caused by abnormal dosage of one or more genes located within the rearranged genomic fragments. Cytogenetic analysis has been a useful diagnostic tool for this disease category especially in idiopathic developmental delay/mental retardation, multiple congenital anomalies, dysmorphism, and pregnancy at risk for chromosomal abnormalities. However, the limitation of band resolution in the conventional cytogenetic methodology karyotype (5–10?Mb) has prompted the development of technologies which can identify previously unrecognized chromosomal anomalies. Since Solinas-Toldo et al. published the first article on array-based comparative genome hybridization (array CGH) in 1997 [1], this technique has become one of the fastest growing ones due to its ability to screen a sample for thousands to millions of different loci at once. The array CGH platforms used for clinical diagnosis are able to detect nonmosaic and mosaic aneuploidies, subtelomeric imbalances, known microdeletion/microduplication syndromes, and other unique unbalanced chromosomal rearrangements [2–4]. The detection rate has been improved to 10–16% in patients with a normal karyotype [5, 6]. In addition, array CGH is able to uncover numerous copy number variations (CNVs) of not-yet-known clinical significance scattered throughout the human genome. In pediatric patients with idiopathic developmental delay and dysmorphic features, it is difficult to come up with a
Significance of genomic instability in breast cancer in atomic bomb survivors: analysis of microarray-comparative genomic hybridization
Masahiro Oikawa, Koh-ichiro Yoshiura, Hisayoshi Kondo, Shiro Miura, Takeshi Nagayasu, Masahiro Nakashima
Radiation Oncology , 2011, DOI: 10.1186/1748-717x-6-168
Abstract: Tumor DNA was extracted from FFPE tissues of invasive ductal cancers from 15 survivors who were exposed at 1.5 km or less from the hypocenter and 13 calendar year-matched non-exposed patients followed by aCGH analysis using a high-density oligonucleotide microarray. The total length of copy number aberrations (CNA) was used as an indicator of GIN, and correlation with clinicopathological factors were statistically tested.The mean of the derivative log ratio spread (DLRSpread), which estimates the noise by calculating the spread of log ratio differences between consecutive probes for all chromosomes, was 0.54 (range, 0.26 to 1.05). The concordance of results between aCGH and fluorescence in situ hybridization (FISH) for HER2 gene amplification was 88%. The incidence of HER2 amplification and histological grade was significantly higher in the A-bomb survivors than control group (P = 0.04, respectively). The total length of CNA tended to be larger in the A-bomb survivors (P = 0.15). Correlation analysis of CNA and clinicopathological factors revealed that DLRSpread was negatively correlated with that significantly (P = 0.034, r = -0.40). Multivariate analysis with covariance revealed that the exposure to A-bomb was a significant (P = 0.005) independent factor which was associated with larger total length of CNA of breast cancers.Thus, archival FFPE tissues from A-bomb survivors are useful for genome-wide aCGH analysis. Our results suggested that A-bomb radiation may affect the increased amount of CNA as a hallmark of GIN and, subsequently, be associated with a higher histologic grade in breast cancer found in A-bomb survivors.Genomic instability (GIN) is an important hallmark of an enhanced carcinogenic process in human. Although there are various forms of GIN, many cancer cells show higher rates of chromosomal instability, which means changes in chromosome structure and number, compared with normal cells [1]. Recent cytogenetic analysis revealed that there were equal
Detection of pathogenic copy number variants in children with idiopathic intellectual disability using 500 K SNP array genomic hybridization
JM Friedman, Shelin Adam, Laura Arbour, Linlea Armstrong, Agnes Baross, Patricia Birch, Cornelius Boerkoel, Susanna Chan, David Chai, Allen D Delaney, Stephane Flibotte, William T Gibson, Sylvie Langlois, Emmanuelle Lemyre, H Irene Li, Patrick MacLeod, Joan Mathers, Jacques L Michaud, Barbara C McGillivray, Millan S Patel, Hong Qian, Guy A Rouleau, Margot I Van Allen, Siu-Li Yong, Farah R Zahir, Patrice Eydoux, Marco A Marra
BMC Genomics , 2009, DOI: 10.1186/1471-2164-10-526
Abstract: We performed 500 K Affymetrix GeneChip? array genomic hybridization in 100 idiopathic intellectual disability trios, each comprised of a child with intellectual disability of unknown cause and both unaffected parents. We found pathogenic genomic imbalance in 16 of these 100 individuals with idiopathic intellectual disability. In comparison, we had found pathogenic genomic imbalance in 11 of 100 children with idiopathic intellectual disability in a previous cohort who had been studied by 100 K GeneChip? array genomic hybridization. Among 54 intellectual disability trios selected from the previous cohort who were re-tested with 500 K GeneChip? array genomic hybridization, we identified all 10 previously-detected pathogenic genomic alterations and at least one additional pathogenic copy number variant that had not been detected with 100 K GeneChip? array genomic hybridization. Many benign copy number variants, including one that was de novo, were also detected with 500 K array genomic hybridization, but it was possible to distinguish the benign and pathogenic copy number variants with confidence in all but 3 (1.9%) of the 154 intellectual disability trios studied.Affymetrix GeneChip? 500 K array genomic hybridization detected pathogenic genomic imbalance in 10 of 10 patients with idiopathic developmental disability in whom 100 K GeneChip? array genomic hybridization had found genomic imbalance, 1 of 44 patients in whom 100 K GeneChip? array genomic hybridization had found no abnormality, and 16 of 100 patients who had not previously been tested. Effective clinical interpretation of these studies requires considerable skill and experience.Chromosomal imbalance has been recognized as the most frequent cause of intellectual disability (ID) for 50 years [1-3]. Until recently, most of this genomic imbalance was diagnosed by cytogenetic analysis, but studies over the past few years have found that ID is caused by constitutional gains or losses of submicroscopic genomic segme
Microarray comparative genomic hybridization detection of chromosomal imbalances in uterine cervix carcinoma
Alfredo Hidalgo, Michael Baudis, Iver Petersen, Hugo Arreola, Patricia Pi?a, Guelaguetza Vázquez-Ortiz, Dulce Hernández, José González, Minerva Lazos, Ricardo López, Carlos Pérez, José García, Karla Vázquez, Brenda Alatorre, Mauricio Salcedo
BMC Cancer , 2005, DOI: 10.1186/1471-2407-5-77
Abstract: In order to further define specific DNA copy number changes in cervical cancer, we analyzed 20 cervical samples (3 pre-malignant lesions, 10 invasive tumors, and 7 cell lines), using the GenoSensor microarray CGH system to define particular genetic targets that suffer copy number changes.The most common DNA gains detected by array CGH in the invasive samples were located at the RBP1-RBP2 (3q21-q22) genes, the sub-telomeric clone C84C11/T3 (5ptel), D5S23 (5p15.2) and the DAB2 gene (5p13) in 58.8% of the samples. The most common losses were found at the FHIT gene (3p14.2) in 47% of the samples, followed by deletions at D8S504 (8p23.3), CTDP1-SHGC- 145820 (18qtel), KIT (4q11-q12), D1S427-FAF1 (1p32.3), D9S325 (9qtel), EIF4E (eukaryotic translation initiation factor 4E, 4q24), RB1 (13q14), and DXS7132 (Xq12) present in 5/17 (29.4%) of the samples.Our results confirm the presence of a specific pattern of chromosomal imbalances in cervical carcinoma and define specific targets that are suffering DNA copy number changes in this neoplasm.Uterine cervix carcinoma (UCC) represents the second cause of death among the female population worldwide. The fact that more than 99% of all the cervical invasive tumors are positive for infection with high risk human papillomavirus (HPV) suggests that this is one of the most important factors for the development of this neoplasm [1,2]. These viruses can induce cellular transformation by several mechanisms; the viral oncoproteins E6 and E7 can interact with cellular proteins involved in important cellular functions, such as tumor suppression, apoptosis, cell cycle control, genomic instability, transcriptional regulation and immune evasion [3].The induction of genomic instability by HPV seems to be particularly important for the establishment and development of an invasive tumor [4,5] since this increased genomic plasticity would generate cellular clones with enhanced transforming and invasive potential [6].Metaphase comparative genomic hyb
GeneCount: genome-wide calculation of absolute tumor DNA copy numbers from array comparative genomic hybridization data
Heidi Lyng, Malin Lando, Runar S Br?vig, Debbie H Svendsrud, Morten Johansen, Eivind Galteland, Odd T Brustugun, Leonardo A Meza-Zepeda, Ola Myklebost, Gunnar B Kristensen, Eivind Hovig, Trond Stokke
Genome Biology , 2008, DOI: 10.1186/gb-2008-9-5-r86
Abstract: Array comparative genomic hybridization (aCGH) is widely used for genome-wide mapping of DNA copy number changes in malignant cells [1,2]. Genetic gains and losses impact gene expression levels, and thereby promote tumor growth and progression [3-5]. Numerous clinical studies have been performed to find tumor characteristics and to classify patients with respect to their prognosis based on the copy number changes [6,7]. The usefulness of the aCGH data is limited, however, because only relative and not absolute copy numbers are achieved, making the interpretation of the data and comparisons across experiments difficult. Absolute DNA copy numbers can be obtained only on a single gene basis by the use of fluorescence in situ hybridization (FISH). Development of genome-wide methods for this purpose would enable generation of universal gene copy number databases of individual diseases that could be utilized more widely, as is the goal of several public repositories like the Mitelman Database of Chromosome Aberrations in Cancer [8].The relative values achieved in aCGH experiments are influenced by the total DNA content (ploidy) of the tumor cells, the proportion of normal cells in the sample, and the experimental bias, in addition to the DNA copy numbers. The values are presented as intensity ratios between tumor and normal DNA [2]. The data are normalized so that the ratio of 1.0 is the baseline for the analysis, and corresponds to two DNA copies in near diploid (2n) tumors. The copy number changes are identified from the ratios deviating from the baseline, using statistical methods for ratio smoothing and breakpoint detection [9-12]. To assign an absolute copy number to each ratio level identified by the statistical analysis and thereby score genetic aberrations are, however, challenging. In aneuploid tumors with gross alterations in the DNA content, the baseline represents a copy number other than 2, like 3 or 4 in tri- or tetraploid tumors, or a non-integer value when
Microarray MAPH: accurate array-based detection of relative copy number in genomic DNA
Brian Gibbons, Parikkhit Datta, Ying Wu, Alan Chan, John AL Armour
BMC Genomics , 2006, DOI: 10.1186/1471-2164-7-163
Abstract: In this study we showed that microarray MAPH measurement of PMP22 gene dosage correlates well with PMP22 gene dosage determined by capillary MAPH and that copy number was accurately reported in analyses of DNA from 38 individuals, 12 of which were known to have Charcot-Marie-Tooth disease type 1A (CMT1A).Measurement of microarray-based endpoints for MAPH appears to be of comparable accuracy to electrophoretic methods, and holds the prospect of fully exploiting the potential multiplicity of MAPH. The technology has the potential to simplify copy number assays for genes with a large number of exons, or of expanded sets of probes from dispersed genomic locations.The role of submicroscopic DNA copy number variation in genetic pathologies has been established now for two decades [1]. Early investigations recognised the importance of deletions or duplications in specific genes as causative mutations in clinical conditions, such as BRCA1 in familial breast and ovarian cancer [2] and DMD in Duchenne/Becker muscular dystrophy [3]. Furthermore, subtelomeric chromosomal rearrangements leading to copy number changes have been associated with learning disability and other developmental abnormalities [4]. Recent advances in the diagnostic technologies applied to such conditions have also proven to be useful tools in elucidating the dynamic model of the human genome, with copy number variation recently recognised as an important component of human polymorphism [5-8].As the significance of copy number variation in genetic analysis becomes more widely recognised so does the need to improve and extend the range of techniques available. In particular, for scanning multi-exon genes for deletions or duplications, MAPH and MLPA [9] have been used to assess the dosage of small (about 100 bp) regions to high accuracy (reliably discriminating 3 copies from 2), properties not easily implemented using, for example, current array-CGH methods. One feature shared by both Multiplex Amplifiable Pr
Detection of copy number variations in rice using array-based comparative genomic hybridization
Ping Yu, Caihong Wang, Qun Xu, Yue Feng, Xiaoping Yuan, Hanyong Yu, Yiping Wang, Shengxiang Tang, Xinghua Wei
BMC Genomics , 2011, DOI: 10.1186/1471-2164-12-372
Abstract: To detect CNVs, we used a set of NimbleGen whole-genome comparative genomic hybridization arrays containing 718,256 oligonucleotide probes with a median probe spacing of 500 bp. We compiled a high-resolution map of CNVs in the rice genome, showing 641 CNVs between the genomes of the rice cultivars 'Nipponbare' (from O. sativa ssp. japonica) and 'Guang-lu-ai 4' (from O. sativa ssp. indica). The CNVs identified vary in size from 1.1 kb to 180.7 kb, and encompass approximately 7.6 Mb of the rice genome. The largest regions showing copy gain and loss are of 37.4 kb on chromosome 4, and 180.7 kb on chromosome 8. In addition, 85 DNA segments were identified, including some genic sequences. Contracted genes greatly outnumbered duplicated ones. Many of the contracted genes corresponded to either the same genes or genes involved in the same biological processes; this was also the case for genes involved in disease and defense.We detected CNVs in rice by array-based comparative genomic hybridization. These CNVs contain known genes. Further discussion of CNVs is important, as they are linked to variation among rice varieties, and are likely to contribute to subspecific characteristics.Copy number variations (CNVs), or copy number polymorphisms (CNPs), are forms of structural variation (SV) that are alterations in DNA resulting in the cell having an abnormal number of copies of one or more segments of DNA. A CNV is a DNA segment ranging from 1 kb to 3 Mb that has been deleted, inserted, or duplicated, on certain chromosomes [1,2]. In particular, segmental duplications (SDs) were demonstrated to be one of the major catalysts and hotspots for CNV formation [3-5]. A CNV was described as early as 1936, with the duplication of the Bar gene in Drosophila melanogaster [6]. Recently, many studies have discovered CNVs in humans [7-9], chimpanzee [10], dog [11], cattle [12], rat [13], mice [14], Drosophila [15], yeast [16], E. coli [17], and maize [18,19]. CNVs can be detected using cyto
Assessment of algorithms for high throughput detection of genomic copy number variation in oligonucleotide microarray data
ágnes Baross, Allen D Delaney, H Irene Li, Tarun Nayar, Stephane Flibotte, Hong Qian, Susanna Y Chan, Jennifer Asano, Adrian Ally, Manqiu Cao, Patricia Birch, Mabel Brown-John, Nicole Fernandes, Anne Go, Giulia Kennedy, Sylvie Langlois, Patrice Eydoux, JM Friedman, Marco A Marra
BMC Bioinformatics , 2007, DOI: 10.1186/1471-2105-8-368
Abstract: We evaluated four publicly available software packages for high throughput copy number analysis using synthetic and empirical 100 K SNP array data sets, the latter obtained from 107 mental retardation (MR) patients and their unaffected parents and siblings. We evaluated the software with regards to overall suitability for high-throughput 100 K SNP array data analysis, as well as effectiveness of normalization, scaling with various reference sets and feature extraction, as well as true and false positive rates of genomic copy number variant (CNV) detection.We observed considerable variation among the numbers and types of candidate CNVs detected by different analysis approaches, and found that multiple programs were needed to find all real aberrations in our test set. The frequency of false positive deletions was substantial, but could be greatly reduced by using the SNP genotype information to confirm loss of heterozygosity.Chromosomal abnormalities frequently contribute to human disorders including cancer [1-3] and mental retardation (MR) [4-6], and characterization of these DNA alterations is important for both diagnosis and understanding of disease mechanisms. A surprising recent finding has been the extent to which genomic copy number variants (CNVs) also exist in the normal population [7-13]. Such variation may represent an important class of mutations that predispose to disease.Conventional cytogenetic studies such as karyotyping are routinely used to detect genomic deletions and duplications involving more than 5–10 Mb, but detection of submicroscopic aberrations requires higher resolution approaches. Oligonucleotide microarray technologies offer high resolution, scalable methods for whole genome screening and can detect previously unidentified CNVs [6,14-17]. Among these approaches, the Affymetrix GeneChip? Mapping Assay [18,19] is increasingly used for detecting CNVs in human DNA. This method involves a whole genome sampling analysis (WGSA) combined with hig
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