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Accurate and exact CNV identification from targeted high-throughput sequence data
Alex S Nord, Ming Lee, Mary-Claire King, Tom Walsh
BMC Genomics , 2011, DOI: 10.1186/1471-2164-12-184
Abstract: Sequencing data is first scanned for gains and losses using a comparison of normalized coverage data between samples. CNV calls are confirmed by testing for a signature of sequences that span the CNV breakpoint. With our method, CNVs can be identified regardless of whether breakpoints are within regions targeted for sequencing. For CNVs where at least one breakpoint is within targeted sequence, exact CNV breakpoints can be identified. In a test data set of 96 subjects sequenced across ~1 Mb genomic sequence using multiplexing technology, our method detected mutations as small as 31 bp, predicted quantitative copy count, and had a low false-positive rate.Application of this method allows for identification of gains and losses in targeted sequence data, providing comprehensive mutation screening when combined with a short read aligner.Massively parallel sequencing technology can be used to efficiently interrogate multiple targeted genomic regions for clinically relevant mutations [1]. The use of short unique sequence indexes, or barcodes, increases sequencing throughput and allows analysis of multiple samples in a single sequencing run. Application of this technology will allow cost-efficient screening of genes known or suspected to harbor clinically relevant pathogenic mutations. While small mutations are readily detectable with current analysis platforms [2,3], methods for the identification of copy-number variants (CNVs) are not well established for targeted data. The ability to detect the full spectrum of mutations is critical to the success of targeted sequencing projects.Current methods for detecting structural variation from massively parallel data use either paired-end mapping or depth of coverage methods (see [4] for a recent review). Paired-end mapping methods perform well for mate-pair sequence data and have the advantage of identifying both balanced (e.g. translocations or inversions) and unbalanced (deletions and duplications) structural variation. Compre
Comprehensive Molecular Diagnosis of Bardet-Biedl Syndrome by High-Throughput Targeted Exome Sequencing  [PDF]
Dong-Jun Xing, Hong-Xing Zhang, Na Huang, Kun-Chao Wu, Xiu-Feng Huang, Fang Huang, Yi Tong, Chi-Pui Pang, Jia Qu, Zi-Bing Jin
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0090599
Abstract: Bardet-Biedl syndrome (BBS) is an autosomal recessive disorder with significant genetic heterogeneity. BBS is linked to mutations in 17 genes, which contain more than 200 coding exons. Currently, BBS is diagnosed by direct DNA sequencing for mutations in these genes, which because of the large genomic screening region is both time-consuming and expensive. In order to develop a practical method for the clinic diagnosis of BBS, we have developed a high-throughput targeted exome sequencing (TES) for genetic diagnosis. Five typical BBS patients were recruited and screened for mutations in a total of 144 known genes responsible for inherited retinal diseases, a hallmark symptom of BBS. The genomic DNA of these patients and their families were subjected to high-throughput DNA re-sequencing. Deep bioinformatics analysis was carried out to filter the massive sequencing data, which were further confirmed through co-segregation analysis. TES successfully revealed mutations in BBS genes in each patient and family member. Six pathological mutations, including five novel mutations, were revealed in the genes BBS2, MKKS, ARL6, MKS1. This study represents the first report of targeted exome sequencing in BBS patients and demonstrates that high-throughput TES is an accurate and rapid method for the genetic diagnosis of BBS.
High-Throughput Targeted Repeat Element Bisulfite Sequencing (HT-TREBS): Genome-Wide DNA Methylation Analysis of IAP LTR Retrotransposon  [PDF]
Muhammad B. Ekram, Joomyeong Kim
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0101683
Abstract: In vertebrates, DNA methylation-mediated repression of retrotransposons is essential for the maintenance of genomic integrity. In the current study, we developed a technique termed HT-TREBS (High-Throughput Targeted Repeat Element Bisulfite Sequencing). This technique is designed to measure the DNA methylation levels of individual loci of any repeat families with next-generation sequencing approaches. To test the feasibility of HT-TREBS, we analyzed the DNA methylation levels of the IAP LTR family using a set of 12 different genomic DNA isolated from the brain, liver and kidney of 4 one-week-old littermates of the mouse strain C57BL/6N. This technique has successfully generated the CpG methylation data of 5,233 loci common in all the samples, representing more than 80% of the individual loci of the five targeted subtypes of the IAP LTR family. According to the results, approximately 5% of the IAP LTR loci have less than 80% CpG methylation levels with no genomic position preference. Further analyses of the IAP LTR loci also revealed the presence of extensive DNA methylation variations between different tissues and individuals. Overall, these data demonstrate the efficiency and robustness of the new technique, HT-TREBS, and also provide new insights regarding the genome-wide DNA methylation patterns of the IAP LTR repeat elements.
Effects of scanning sensitivity and multiple scan algorithms on microarray data quality
Andrew Williams, Errol M Thomson
BMC Bioinformatics , 2010, DOI: 10.1186/1471-2105-11-127
Abstract: Of the individual scans, the low scan exhibited the lowest background signal, the highest signal-to-noise ratio, and equivalent reproducibility to the medium and high scans. Most multiple scan approaches increased the range of probe intensities compared to the individual scans, but did not increase the dynamic range (the proportion of useable data). Approaches displayed striking differences in the background signal and signal-to-noise ratio. However, increased probe intensity range and improved signal-to-noise ratios did not necessarily correlate with improved reproducibility. Importantly, for one multiple scan method that combined 3 scans, reproducibility was significantly improved relative to individual scans and all other multiple scan approaches. The same method using 2 scans yielded significantly lower reproducibility, attributable to a lack-of-fit of the statistical model.Our data indicate that implementation of a suitable multiple scan approach can improve reproducibility, but that model validation is critical to ensure accurate estimates of probe intensity.DNA microarrays allow analysis of genome-wide gene expression. While an entire transcriptome can theoretically be quantified on a single array, in practice a proportion of probes analysed will not provide quantifiable signal. For example, when scanning any complex biological sample hybridized to a microarray, low copy number genes may emit low fluorescence signals not detectable above background; conversely, high copy number genes may emit fluorescence signals that are saturated. To maximize the amount of data acquired from a single microarray scan, the user attempts to generate a scan that spans the entire intensity range by selecting appropriate photo-multiplier tube (PMT) settings. In selecting the PMT of the scanner, the user has two major concerns: quantification error associated with image analysis (e.g., distinguishing signal from background) and signal saturation associated with the selection of sc
Targeted high throughput sequencing in clinical cancer Settings: formaldehyde fixed-paraffin embedded (FFPE) tumor tissues, input amount and tumor heterogeneity
Martin Kerick, Melanie Isau, Bernd Timmermann, Holger Sültmann, Ralf Herwig, Sylvia Krobitsch, Georg Schaefer, Irmgard Verdorfer, Georg Bartsch, Helmut Klocker, Hans Lehrach, Michal R Schweiger
BMC Medical Genomics , 2011, DOI: 10.1186/1755-8794-4-68
Abstract: Using identification of genetic variations in prostate cancer as an example we address three crucial challenges in the field of targeted re-sequencing: Small nucleotide variation (SNV) detection in samples of formalin-fixed paraffin embedded (FFPE) tissue material, minimal amount of input sample and sampling in view of tissue heterogeneity.We show that FFPE tissue material can supplement for fresh frozen tissues for the detection of SNVs and that solution-based enrichment experiments can be accomplished with small amounts of DNA with only minimal effects on enrichment uniformity and data variance.Finally, we address the question whether the heterogeneity of a tumor is reflected by different genetic alterations, e.g. different foci of a tumor display different genomic patterns. We show that the tumor heterogeneity plays an important role for the detection of copy number variations.The application of high throughput sequencing technologies in cancer genomics opens up a new dimension for the identification of disease mechanisms. In particular the ability to use small amounts of FFPE samples available from surgical tumor resections and histopathological examinations facilitates the collection of precious tissue materials. However, care needs to be taken in regard to the locations of the biopsies, which can have an influence on the prediction of copy number variations. Bearing these technological challenges in mind will significantly improve many large-scale sequencing studies and will - in the long term - result in a more reliable prediction of individual cancer therapies.According to the world health organization (WHO) malignant neoplasms are the most common cause of death worldwide in 2010 [1]. We now know that human solid tumors, which account for the majority of all human cancers, result from the accumulation of numerous genetic and epigenetic alterations that finally lead to the deregulation of protein-encoding genes [2-10].Previous efforts to identify protein-enco
CASCADE_SCAN: mining signal transduction network from high-throughput data based on steepest descent method
Kai Wang, Fuyan Hu, Kejia Xu, Hua Cheng, Meng Jiang, Ruili Feng, Jing Li, Tieqiao Wen
BMC Bioinformatics , 2011, DOI: 10.1186/1471-2105-12-164
Abstract: We propose a new approach, namely CASCADE_SCAN, for mining signal transduction networks from high-throughput data based on the steepest descent method using indirect protein-protein interactions (PPIs). This method is useful for actual biological application since the given proteins utilized are no longer confined to membrane receptors or transcription factors as in existing methods. The precision and recall values of CASCADE_SCAN are comparable with those of other existing methods. Moreover, functional enrichment analysis of the network components supported the reliability of the results.CASCADE_SCAN is a more suitable method than existing methods for detecting underlying signaling pathways where the membrane receptors or transcription factors are unknown, providing significant insight into the mechanism of cellular signaling in growth, development and cancer. A new tool based on this method is freely available at http://www.genomescience.com.cn/CASCADE_SCAN/ webcite.Signal transduction plays an essential role in cell response to environment changes. This biological process is usually characterized by phosphorylation/dephosphorylation of some key proteins (e.g. kinases) and generally involves a signal cascade. The signal transduction process often starts from a membrane protein (usually a membrane surface receptor), spans a series of intercellular signaling proteins and then transfers to transcription factors in the nucleus, subsequently raising the expression of downstream genes. Studies demonstrate that many important cellular processes such as cell proliferation, differentiation, cell cycle control and cellular responses to nutrient limiting conditions are involved in different signaling pathways [1,2]. For example, Yokoi et al [3] demonstrated that hyperglycemia mediates endothelial cell senescence through the ASK1 signaling pathway. Tang et al [4] showed that the receptor kinase BRI1 and BR-signaling kinases (BSKs) mediate growth regulation related signal tran
Tag Me Maybe: Perceptions of Public Targeted Sharing on Facebook  [PDF]
Saiph Savage,Andres Monroy-Hernandez,Kasturi Bhattacharjee,Tobias Hollerer
Computer Science , 2015,
Abstract: Social network sites allow users to publicly tag people in their posts. These tagged posts allow users to share to both the general public and a targeted audience, dynamically assembled via notifications that alert the people mentioned. We investigate people's perceptions of this mixed sharing mode through a qualitative study with 120 participants. We found that individuals like this sharing modality as they believe it strengthens their relationships. Individuals also report using tags to have more control of Facebook's ranking algorithm, and to expose one another to novel information and people. This work helps us understand people's complex relationships with the algorithms that mediate their interactions with each another. We conclude by discussing the design implications of these findings.
Correcting nonlinear drift distortion of scanning probe microscopy from image pairs with orthogonal scan directions  [PDF]
Colin Ophus,Chris T Nelson,Jim Ciston
Physics , 2015,
Abstract: Unwanted motion of the probe with respect to the sample is a ubiquitous problem in scanning probe microscopy, causing both linear and nonlinear artifacts in experimental images. We have designed a procedure to correct these artifacts by using orthogonal scan pairs to align each measurement line-by-line along the slow scan direction. We demonstrate the accuracy of our algorithm on both synthetic and experimental data and provide an implementation of our method.
Application of Single Scan Differential Scanning Calorimetry Technique for Determination of Kinetic Parameters of Crystallisation in Se-Sb-Ag  [PDF]
Nidhi Yaduvanshi, Deepak Kumar, Nikhil Rastogi, Ashok Kumar
Journal of Crystallization Process and Technology (JCPT) , 2019, DOI: 10.4236/jcpt.2019.91001
Abstract:
A single scan has been performed in Differential Scanning Calorimetry (DSC) at a heating rate of 15oC/min under non-isothermal conditions to investigate the crystallization kinetics of glassy Se90Sb10-xAgx alloys (where x = 2, 4, 6, 8). For this purpose, Handerson’s theory based on non-isothermal method for thermal analysis of single-scan DSC data has been used. The activation energy of crystallization and order parameter has been determined and composition dependence of these parameters has been discussed.
A high-throughput protocol for mutation scanning of the BRCA1 and BRCA2 genes
Heather L Hondow, Stephen B Fox, Gillian Mitchell, Rodney J Scott, Victoria Beshay, Stephen Q Wong, kConFab Investigators, Alexander Dobrovic
BMC Cancer , 2011, DOI: 10.1186/1471-2407-11-265
Abstract: Assays for the analysis of all coding regions and intron-exon boundaries of BRCA1 and BRCA2 were designed, and optimised. A final set of 94 assays which ran under identical amplification conditions were chosen for BRCA1 (36) and BRCA2 (58). Significant attention was placed on primer design to enable reproducible detection of mutations within the amplicon while minimising unnecessary detection of polymorphisms. Deoxyinosine residues were incorporated into primers that overlay intronic polymorphisms. Multiple 384 well plates were used to facilitate high throughput.169 BRCA1 and 239 BRCA2 known sequence variants were used to test the amplicons. We also performed an extensive blinded validation of the protocol with 384 separate patient DNAs. All heterozygous variants were detected with the optimised assays.This is the first HRM approach to screen the entire coding region of the BRCA1 and BRCA2 genes using one set of reaction conditions in a multi plate 384 well format using specifically designed primers. The parallel screening of a relatively large number of samples enables better detection of sequence variants. HRM has the advantages of decreasing the necessary sequencing by more than 90%. This markedly reduced cost of sequencing will result in BRCA1 and BRCA2 mutation testing becoming accessible to individuals who currently do not undergo mutation testing because of the significant costs involved.Inactivating germline mutations in the BRCA1 and BRCA2 tumour suppressor genes dramatically escalates the risk of developing breast and/or ovarian cancer by up to 20 fold [1-4]. Due to the highly penetrant nature of germline mutations within BRCA1 and BRCA2, it is of importance to identify a woman as being a carrier of a mutation as early intervention measures including breast screening and prophylactic bilateral salphingo-oophorectomy or mastectomy can be offered [5]. More recently, it has been recognised that BRCA1 or BRCA2 mutant tumours are sensitive to PARP inhibitors an
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