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1.  Hypergeometric analysis of tiling-array and sequence data: detection and interpretation of peaks 
Probing protein-deoxyribonucleic acid (DNA) is gaining popularity as it sheds light on molecular mechanisms that regulate the expression of genes. Currently, tiling-arrays and next-generation sequencing technology can be used to measure these interactions. Both methods generate a signal over the genome in which contiguous regions of peaks on the genome represent the presence of an interacting molecule. Many methods do exist to identify functional regions of interest (ROIs) on the genome. However the detection of ROIs are often not an end-point in research questions and it therefore requires data dragging between tools to relate the ROIs to information present in databases, such as gene-ontology, pathway information, or enrichment of certain genomic content. We introduce hypergeometric analysis of tiling-array and sequence data (HATSEQ), a powerful tool that accurately identifies functional ROIs on the genome where a genomic signal significantly deviates from the general genome-wide behavior. HATSEQ also includes a number of built-in post-analyses with which biological meaning can be attached to the detected ROIs in terms of gene pathways and de-novo motif analysis, and provides different visualizations and statistical summaries for the detected ROIs. In addition, HATSEQ has an intuitive graphic user interface that lowers the barrier for researchers to analyze their data without the need of scripting languages. We compared the results of HATSEQ against two other popular chromatin immunoprecipitation sequencing (ChIP-Seq) methods and observed overlap in the detected ROIs but HATSEQ is more specific in delineating the peak boundaries. We also discuss the versatility of HATSEQ by using a Signal Transducer and Activator of Transcription 1 (STAT1) ChIP-Seq data-set, and show that the detected ROIs are highly specific for the expected STAT1 binding motif. HATSEQ is freely available at: http://hema13.erasmusmc.nl/index.php/HATSEQ.
doi:10.2147/AABC.S51271
PMCID: PMC3810201  PMID: 24187504
bioinformatics; NGS analysis; ChIP-Seq; peak detection
2.  Comparison and calibration of transcriptome data from RNA-Seq and tiling arrays 
BMC Genomics  2010;11:383.
Background
Tiling arrays have been the tool of choice for probing an organism's transcriptome without prior assumptions about the transcribed regions, but RNA-Seq is becoming a viable alternative as the costs of sequencing continue to decrease. Understanding the relative merits of these technologies will help researchers select the appropriate technology for their needs.
Results
Here, we compare these two platforms using a matched sample of poly(A)-enriched RNA isolated from the second larval stage of C. elegans. We find that the raw signals from these two technologies are reasonably well correlated but that RNA-Seq outperforms tiling arrays in several respects, notably in exon boundary detection and dynamic range of expression. By exploring the accuracy of sequencing as a function of depth of coverage, we found that about 4 million reads are required to match the sensitivity of two tiling array replicates. The effects of cross-hybridization were analyzed using a "nearest neighbor" classifier applied to array probes; we describe a method for determining potential "black list" regions whose signals are unreliable. Finally, we propose a strategy for using RNA-Seq data as a gold standard set to calibrate tiling array data. All tiling array and RNA-Seq data sets have been submitted to the modENCODE Data Coordinating Center.
Conclusions
Tiling arrays effectively detect transcript expression levels at a low cost for many species while RNA-Seq provides greater accuracy in several regards. Researchers will need to carefully select the technology appropriate to the biological investigations they are undertaking. It will also be important to reconsider a comparison such as ours as sequencing technologies continue to evolve.
doi:10.1186/1471-2164-11-383
PMCID: PMC3091629  PMID: 20565764
3.  Detecting transcriptionally active regions using genomic tiling arrays 
Genome Biology  2006;7(7):R59.
A new method for designing and integrating genomic tiling array data is described and applied to Anopheles and human arrays.
We have developed a method for interpreting genomic tiling array data, implemented as the program TranscriptionDetector. Probed loci expressed above background are identified by combining replicates in a way that makes minimal assumptions about the data. We performed medium-resolution Anopheles gambiae tiling array experiments and found extensive transcription of both coding and non-coding regions. Our method also showed improved detection of transcriptional units when applied to high-density tiling array data for ten human chromosomes.
doi:10.1186/gb-2006-7-7-r59
PMCID: PMC1779562  PMID: 16859498
4.  TileProbe: modeling tiling array probe effects using publicly available data 
Bioinformatics  2009;25(18):2369-2375.
Motivation: Individual probes on an Affymetrix tiling array usually behave differently. Modeling and removing these probe effects are critical for detecting signals from the array data. Current data processing techniques either require control samples or use probe sequences to model probe-specific variability, such as with MAT. Although the MAT approach can be applied without control samples, residual probe effects continue to distort the true biological signals.
Results: We propose TileProbe, a new technique that builds upon the MAT algorithm by incorporating publicly available data sets to remove tiling array probe effects. By using a large number of these readily available arrays, TileProbe robustly models the residual probe effects that MAT model cannot explain. When applied to analyzing ChIP-chip data, TileProbe performs consistently better than MAT across a variety of analytical conditions. This shows that TileProbe resolves the issue of probe-specific effects more completely.
Availability: http://www.biostat.jhsph.edu/∼hji/cisgenome/index_files/tileprobe.htm
Contact: hji@jhsph.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btp425
PMCID: PMC2735670  PMID: 19592393
5.  Efficient oligonucleotide probe selection for pan-genomic tiling arrays 
BMC Bioinformatics  2009;10:293.
Background
Array comparative genomic hybridization is a fast and cost-effective method for detecting, genotyping, and comparing the genomic sequence of unknown bacterial isolates. This method, as with all microarray applications, requires adequate coverage of probes targeting the regions of interest. An unbiased tiling of probes across the entire length of the genome is the most flexible design approach. However, such a whole-genome tiling requires that the genome sequence is known in advance. For the accurate analysis of uncharacterized bacteria, an array must query a fully representative set of sequences from the species' pan-genome. Prior microarrays have included only a single strain per array or the conserved sequences of gene families. These arrays omit potentially important genes and sequence variants from the pan-genome.
Results
This paper presents a new probe selection algorithm (PanArray) that can tile multiple whole genomes using a minimal number of probes. Unlike arrays built on clustered gene families, PanArray uses an unbiased, probe-centric approach that does not rely on annotations, gene clustering, or multi-alignments. Instead, probes are evenly tiled across all sequences of the pan-genome at a consistent level of coverage. To minimize the required number of probes, probes conserved across multiple strains in the pan-genome are selected first, and additional probes are used only where necessary to span polymorphic regions of the genome. The viability of the algorithm is demonstrated by array designs for seven different bacterial pan-genomes and, in particular, the design of a 385,000 probe array that fully tiles the genomes of 20 different Listeria monocytogenes strains with overlapping probes at greater than twofold coverage.
Conclusion
PanArray is an oligonucleotide probe selection algorithm for tiling multiple genome sequences using a minimal number of probes. It is capable of fully tiling all genomes of a species on a single microarray chip. These unique pan-genome tiling arrays provide maximum flexibility for the analysis of both known and uncharacterized strains.
doi:10.1186/1471-2105-10-293
PMCID: PMC2753849  PMID: 19758451
6.  A hidden Markov model approach for determining expression from genomic tiling micro arrays 
BMC Bioinformatics  2006;7:239.
Background
Genomic tiling micro arrays have great potential for identifying previously undiscovered coding as well as non-coding transcription. To-date, however, analyses of these data have been performed in an ad hoc fashion.
Results
We present a probabilistic procedure, ExpressHMM, that adaptively models tiling data prior to predicting expression on genomic sequence. A hidden Markov model (HMM) is used to model the distributions of tiling array probe scores in expressed and non-expressed regions. The HMM is trained on sets of probes mapped to regions of annotated expression and non-expression. Subsequently, prediction of transcribed fragments is made on tiled genomic sequence. The prediction is accompanied by an expression probability curve for visual inspection of the supporting evidence. We test ExpressHMM on data from the Cheng et al. (2005) tiling array experiments on ten Human chromosomes [1]. Results can be downloaded and viewed from our web site [2].
Conclusion
The value of adaptive modelling of fluorescence scores prior to categorisation into expressed and non-expressed probes is demonstrated. Our results indicate that our adaptive approach is superior to the previous analysis in terms of nucleotide sensitivity and transfrag specificity.
doi:10.1186/1471-2105-7-239
PMCID: PMC1481622  PMID: 16672042
7.  Geoseq: a tool for dissecting deep-sequencing datasets 
BMC Bioinformatics  2010;11:506.
Background
Datasets generated on deep-sequencing platforms have been deposited in various public repositories such as the Gene Expression Omnibus (GEO), Sequence Read Archive (SRA) hosted by the NCBI, or the DNA Data Bank of Japan (ddbj). Despite being rich data sources, they have not been used much due to the difficulty in locating and analyzing datasets of interest.
Results
Geoseq http://geoseq.mssm.edu provides a new method of analyzing short reads from deep sequencing experiments. Instead of mapping the reads to reference genomes or sequences, Geoseq maps a reference sequence against the sequencing data. It is web-based, and holds pre-computed data from public libraries. The analysis reduces the input sequence to tiles and measures the coverage of each tile in a sequence library through the use of suffix arrays. The user can upload custom target sequences or use gene/miRNA names for the search and get back results as plots and spreadsheet files. Geoseq organizes the public sequencing data using a controlled vocabulary, allowing identification of relevant libraries by organism, tissue and type of experiment.
Conclusions
Analysis of small sets of sequences against deep-sequencing datasets, as well as identification of public datasets of interest, is simplified by Geoseq. We applied Geoseq to, a) identify differential isoform expression in mRNA-seq datasets, b) identify miRNAs (microRNAs) in libraries, and identify mature and star sequences in miRNAS and c) to identify potentially mis-annotated miRNAs. The ease of using Geoseq for these analyses suggests its utility and uniqueness as an analysis tool.
doi:10.1186/1471-2105-11-506
PMCID: PMC2972303  PMID: 20939882
8.  At-TAX: a whole genome tiling array resource for developmental expression analysis and transcript identification in Arabidopsis thaliana 
Genome Biology  2008;9(7):R112.
A developmental expression atlas, At-TAX, based on whole-genome tiling arrays, is presented along with associated analysis methods.
Gene expression maps for model organisms, including Arabidopsis thaliana, have typically been created using gene-centric expression arrays. Here, we describe a comprehensive expression atlas, Arabidopsis thaliana Tiling Array Express (At-TAX), which is based on whole-genome tiling arrays. We demonstrate that tiling arrays are accurate tools for gene expression analysis and identified more than 1,000 unannotated transcribed regions. Visualizations of gene expression estimates, transcribed regions, and tiling probe measurements are accessible online at the At-TAX homepage.
doi:10.1186/gb-2008-9-7-r112
PMCID: PMC2530869  PMID: 18613972
9.  TiArA: A Virtual Appliance for the Analysis of Tiling Array Data 
PLoS ONE  2010;5(4):e9993.
Background
Genomic tiling arrays have been described in the scientific literature since 2003, yet there is a shortage of user-friendly applications available for their analysis.
Methodology/Principal Findings
Tiling Array Analyzer (TiArA) is a software program that provides a user-friendly graphical interface for the background subtraction, normalization, and summarization of data acquired through the Affymetrix tiling array platform. The background signal is empirically measured using a group of nonspecific probes with varying levels of GC content and normalization is performed to enforce a common dynamic range.
Conclusions/Significance
TiArA is implemented as a standalone program for Linux systems and is available as a cross-platform virtual machine that will run under most modern operating systems using virtualization software such as Sun VirtualBox or VMware. The software is available as a Debian package or a virtual appliance at http://purl.org/NET/tiara.
doi:10.1371/journal.pone.0009993
PMCID: PMC2848623  PMID: 20376318
10.  Use of tiling array data and RNA secondary structure predictions to identify noncoding RNA genes 
BMC Genomics  2007;8:244.
Background
Within the last decade a large number of noncoding RNA genes have been identified, but this may only be the tip of the iceberg. Using comparative genomics a large number of sequences that have signals concordant with conserved RNA secondary structures have been discovered in the human genome. Moreover, genome wide transcription profiling with tiling arrays indicate that the majority of the genome is transcribed.
Results
We have combined tiling array data with genome wide structural RNA predictions to search for novel noncoding and structural RNA genes that are expressed in the human neuroblastoma cell line SK-N-AS. Using this strategy, we identify thousands of human candidate RNA genes. To further verify the expression of these genes, we focused on candidate genes that had a stable hairpin structures or a high level of covariance. Using northern blotting, we verify the expression of 2 out of 3 of the hairpin structures and 3 out of 9 high covariance structures in SK-N-AS cells.
Conclusion
Our results demonstrate that many human noncoding, structured and conserved RNA genes remain to be discovered and that tissue specific tiling array data can be used in combination with computational predictions of sequences encoding structural RNAs to improve the search for such genes.
doi:10.1186/1471-2164-8-244
PMCID: PMC1949828  PMID: 17645787
11.  Integrating Prior Knowledge in Multiple Testing under Dependence with Applications to Detecting Differential DNA Methylation 
Biometrics  2012;68(3):774-783.
Summary
DNA methylation has emerged as an important hallmark of epigenetics. Numerous platforms including tiling arrays and next generation sequencing, and experimental protocols are available for profiling DNA methylation. Similar to other tiling array data, DNA methylation data shares the characteristics of inherent correlation structure among nearby probes. However, unlike gene expression or protein DNA binding data, the varying CpG density which gives rise to CpG island, shore and shelf definition provides exogenous information in detecting differential methylation. This paper aims to introduce a robust testing and probe ranking procedure based on a non-homogeneous hidden Markov model that incorporates the above-mentioned features for detecting differential methylation. We revisit the seminal work of Sun and Cai (2009, J. R. Stat. Soc. B. 71, 393-424) and propose modeling the non-null using a non-parametric symmetric distribution in two-sided hypothesis testing. We show that this model improves probe ranking and is robust to model misspecification based on extensive simulation studies. We further illustrate that our proposed framework achieves good operating characteristics as compared to commonly used methods in real DNA methylation data that aims to detect differential methylation sites.
doi:10.1111/j.1541-0420.2011.01730.x
PMCID: PMC3449228  PMID: 22260651
Non-homogeneous Hidden Markov Model; False Discovery Rate; Microarray; CpG Island; Kernel Density Estimation; Semiparametric Model
12.  chipD: a web tool to design oligonucleotide probes for high-density tiling arrays 
Nucleic Acids Research  2010;38(Web Server issue):W321-W325.
chipD is a web server that facilitates design of DNA oligonucleotide probes for high-density tiling arrays, which can be used in a number of genomic applications such as ChIP-chip or gene-expression profiling. The server implements a probe selection algorithm that takes as an input, in addition to the target sequences, a set of parameters that allow probe design to be tailored to specific applications, protocols or the array manufacturer’s requirements. The algorithm optimizes probes to meet three objectives: (i) probes should be specific; (ii) probes should have similar thermodynamic properties; and (iii) the target sequence coverage should be homogeneous and avoid significant gaps. The output provides in a text format, the list of probe sequences with their genomic locations, targeted strands and hybridization characteristics. chipD has been used successfully to design tiling arrays for bacteria and yeast. chipD is available at http://chipd.uwbacter.org/.
doi:10.1093/nar/gkq517
PMCID: PMC2896189  PMID: 20529880
13.  Whole-Genome Tiling Array Analysis of Mycobacterium leprae RNA Reveals High Expression of Pseudogenes and Noncoding Regions▿ †  
Journal of Bacteriology  2009;191(10):3321-3327.
Whole-genome sequence analysis of Mycobacterium leprae has revealed a limited number of protein-coding genes, with half of the genome composed of pseudogenes and noncoding regions. We previously showed that some M. leprae pseudogenes are transcribed at high levels and that their expression levels change following infection. In order to clarify the RNA expression profile of the M. leprae genome, a tiling array in which overlapping 60-mer probes cover the entire 3.3-Mbp genome was designed. The array was hybridized with M. leprae RNA from the SHR/NCrj-rnu nude rat, and the results were compared to results from an open reading frame array and confirmed by reverse transcription-PCR. RNA expression was detected from genes, pseudogenes, and noncoding regions. The signal intensities obtained from noncoding regions were higher than those from pseudogenes. Expressed noncoding regions include the M. leprae unique repetitive sequence RLEP and other sequences without any homology to known functional noncoding RNAs. Although the biological functions of RNA transcribed from M. leprae pseudogenes and noncoding regions are not known, RNA expression analysis will provide insights into the bacteriological significance of the species. In addition, our study suggests that M. leprae will be a useful model organism for the study of the molecular mechanism underlying the creation of pseudogenes and the role of microRNAs derived from noncoding regions.
doi:10.1128/JB.00120-09
PMCID: PMC2687151  PMID: 19286800
14.  OikoBase: a genomics and developmental transcriptomics resource for the urochordate Oikopleura dioica 
Nucleic Acids Research  2012;41(D1):D845-D853.
We report the development of OikoBase (http://oikoarrays.biology.uiowa.edu/Oiko/), a tiling array-based genome browser resource for Oikopleura dioica, a metazoan belonging to the urochordates, the closest extant group to vertebrates. OikoBase facilitates retrieval and mining of a variety of useful genomics information. First, it includes a genome browser which interrogates 1260 genomic sequence scaffolds and features gene, transcript and CDS annotation tracks. Second, we annotated gene models with gene ontology (GO) terms and InterPro domains which are directly accessible in the browser with links to their entries in the GO (http://www.geneontology.org/) and InterPro (http://www.ebi.ac.uk/interpro/) databases, and we provide transcript and peptide links for sequence downloads. Third, we introduce the transcriptomics of a comprehensive set of developmental stages of O. dioica at high resolution and provide downloadable gene expression data for all developmental stages. Fourth, we incorporate a BLAST tool to identify homologs of genes and proteins. Finally, we include a tutorial that describes how to use OikoBase as well as a link to detailed methods, explaining the data generation and analysis pipeline. OikoBase will provide a valuable resource for research in chordate development, genome evolution and plasticity and the molecular ecology of this important marine planktonic organism.
doi:10.1093/nar/gks1159
PMCID: PMC3531137  PMID: 23185044
15.  BioTile, A Perl based tool for the identification of differentially enriched regions in tiling microarray data 
BMC Bioinformatics  2013;14:76.
Background
Genome-wide tiling array experiments are increasingly used for the analysis of DNA methylation. Because DNA methylation patterns are tissue and cell type specific, the detection of differentially methylated regions (DMRs) with small effect size is a necessary feature of tiling microarray ‘peak’ finding algorithms, as cellular heterogeneity within a studied tissue may lead to a dilution of the phenotypically relevant effects. Additionally, the ability to detect short length DMRs is necessary as biologically relevant signal may occur in focused regions throughout the genome.
Results
We present a free open-source Perl application, Binding Intensity Only Tile array analysis or “BioTile”, for the identification of differentially enriched regions (DERs) in tiling array data. The application of BioTile to non-smoothed data allows for the identification of shorter length and smaller effect-size DERs, while correcting for probe specific variation by inversely weighting on probe variance through a permutation corrected meta-analysis procedure employed at identified regions. BioTile exhibits higher power to identify significant DERs of low effect size and across shorter genomic stretches as compared to other peak finding algorithms, while not sacrificing power to detect longer DERs.
Conclusion
BioTile represents an easy to use analysis option applicable to multiple microarray platforms, allowing for its integration into the analysis workflow of array data analysis.
doi:10.1186/1471-2105-14-76
PMCID: PMC3599767  PMID: 23452827
DNA methylation; Differentially methylated region; Tiling microarray; Algorithm; Epigenetic
16.  A continuous-index Bayesian hidden Markov model for prediction of nucleosome positioning in genomic DNA 
Biostatistics (Oxford, England)  2010;12(3):462-477.
Nucleosomes are units of chromatin structure, consisting of DNA sequence wrapped around proteins called “histones.” Nucleosomes occur at variable intervals throughout genomic DNA and prevent transcription factor (TF) binding by blocking TF access to the DNA. A map of nucleosomal locations would enable researchers to detect TF binding sites with greater efficiency. Our objective is to construct an accurate genomic map of nucleosome-free regions (NFRs) based on data from high-throughput genomic tiling arrays in yeast. These high-volume data typically have a complex structure in the form of dependence on neighboring probes as well as underlying DNA sequence, variable-sized gaps, and missing data. We propose a novel continuous-index model appropriate for non-equispaced tiling array data that simultaneously incorporates DNA sequence features relevant to nucleosome formation. Simulation studies and an application to a yeast nucleosomal assay demonstrate the advantages of using the new modeling framework, as well as its robustness to distributional misspecifications. Our results reinforce the previous biological hypothesis that higher-order nucleotide combinations are important in distinguishing nucleosomal regions from NFRs.
doi:10.1093/biostatistics/kxq077
PMCID: PMC3114652  PMID: 21193724
Chromatin structure; Data augmentation; FAIRE; Tiling arrays
17.  SP14 Genome-Wide High-Resolution Mapping and Functional Analysis of DNA Methylation 
Methylation of cytosines in DNA sequences is a major part of epigenetic regulation, resulting in proximal transcriptional silencing and enabling the stable inheritance of a pattern of transcriptional activity. DNA methylation in higher eukaryotes is involved in transposon silencing and regulation of gene expression; however, the full extent to which this mechanism regulates the genome has remained unknown. Tiling arrays representing the entire genome of the flowering plant Arabidopsis thaliana, tiled at 35-bp resolution, provide a platform upon which to analyze the methylated component of the Arabidopsis genome. Hybridization of methylated genomic DNA isolated by 5-methyl-cytosine immunoprecipitation to the whole-genome tiling arrays produced the first comprehensive DNA methylation map of an entire genome, identifying heavy DNA methylation at pericentromeric heterochromatin, repetitive sequences, and regions producing small interfering RNAs. Over one-third of expressed genes contain methylation within transcribed regions, whereas only ~5% of genes show methylation within promoter regions. Genes methylated in transcribed regions are highly expressed and constitutively active, whereas promoter-methylated genes show a greater degree of tissue-specific expression. Whole-genome tiling-array transcriptional profiling of DNA methyltransferase null mutants identified hundreds of genes and intergenic noncoding RNAs with altered expression levels, many of which may be epigenetically controlled by DNA methylation. The approaches developed should assist in the study of DNA methylation in larger and more complex genomes, for which whole-genome tiling arrays are now available.
PMCID: PMC2291889
18.  Mapping the genome landscape using tiling array technology 
Current opinion in plant biology  2007;10(5):534-542.
Summary
With the availability of complete genome sequences for a growing number of organisms, high-throughput methods for gene annotation and analysis of genome dynamics are needed. The application of whole-genome tiling microarrays for studies of global gene expression is providing a more unbiased view of the transcriptional activity within genomes. For example, this approach has led to the identification and isolation of many novel non-protein-coding RNAs (ncRNAs), which have been suggested to comprise a major component of the transcriptome that have novel functions involved in epigenetic regulation of the genome. Additionally, tiling arrays have been recently applied to the study of histone modifications and methylation of cytosine bases (DNA methylation). Surprisingly, recent studies combining the analysis of gene expression (transcriptome) and DNA methylation (methylome) using whole-genome tiling arrays revealed that DNA methylation regulates the expression levels of many ncRNAs. Further capture and integration of additional types of genome-wide data sets will help to illuminate additional hidden features of the dynamic genomic landscape that are regulated by both genetic and epigenetic pathways in plants.
doi:10.1016/j.pbi.2007.07.006
PMCID: PMC2665186  PMID: 17703988
19.  Support vector machines-based identification of alternative splicing in Arabidopsis thaliana from whole-genome tiling arrays 
BMC Bioinformatics  2011;12:55.
Background
Alternative splicing (AS) is a process which generates several distinct mRNA isoforms from the same gene by splicing different portions out of the precursor transcript. Due to the (patho-)physiological importance of AS, a complete inventory of AS is of great interest. While this is in reach for human and mammalian model organisms, our knowledge of AS in plants has remained more incomplete. Experimental approaches for monitoring AS are either based on transcript sequencing or rely on hybridization to DNA microarrays. Among the microarray platforms facilitating the discovery of AS events, tiling arrays are well-suited for identifying intron retention, the most prevalent type of AS in plants. However, analyzing tiling array data is challenging, because of high noise levels and limited probe coverage.
Results
In this work, we present a novel method to detect intron retentions (IR) and exon skips (ES) from tiling arrays. While statistical tests have typically been proposed for this purpose, our method instead utilizes support vector machines (SVMs) which are appreciated for their accuracy and robustness to noise. Existing EST and cDNA sequences served for supervised training and evaluation. Analyzing a large collection of publicly available microarray and sequence data for the model plant A. thaliana, we demonstrated that our method is more accurate than existing approaches. The method was applied in a genome-wide screen which resulted in the discovery of 1,355 IR events. A comparison of these IR events to the TAIR annotation and a large set of short-read RNA-seq data showed that 830 of the predicted IR events are novel and that 525 events (39%) overlap with either the TAIR annotation or the IR events inferred from the RNA-seq data.
Conclusions
The method developed in this work expands the scarce repertoire of analysis tools for the identification of alternative mRNA splicing from whole-genome tiling arrays. Our predictions are highly enriched with known AS events and complement the A. thaliana genome annotation with respect to AS. Since all predicted AS events can be precisely attributed to experimental conditions, our work provides a basis for follow-up studies focused on the elucidation of the regulatory mechanisms underlying tissue-specific and stress-dependent AS in plants.
doi:10.1186/1471-2105-12-55
PMCID: PMC3051901  PMID: 21324185
20.  The incredible shrinking world of DNA microarrays 
Molecular bioSystems  2008;4(7):726-732.
The efficacy of microarrays in examining gene expression, gene and genome structure, protein-DNA interactions, whole-genome similarities and differences, microRNA expression, methylation, (and more), is no longer in question. It is a fast-developing, cutting edge technology that has grown up along with massive sequence databases and is likely to become part of everyday patient care. Many advances have recently expanded the power and utility of microarrays; among them is our development of a new array tiling technique that dramatically increases the scope of coverage of an oligonucleotide tiling array without substantially increasing its cost.
doi:10.1039/b706237k
PMCID: PMC2535915  PMID: 18563246
21.  An efficient pseudomedian filter for tiling microrrays 
BMC Bioinformatics  2007;8:186.
Background
Tiling microarrays are becoming an essential technology in the functional genomics toolbox. They have been applied to the tasks of novel transcript identification, elucidation of transcription factor binding sites, detection of methylated DNA and several other applications in several model organisms. These experiments are being conducted at increasingly finer resolutions as the microarray technology enjoys increasingly greater feature densities. The increased densities naturally lead to increased data analysis requirements. Specifically, the most widely employed algorithm for tiling array analysis involves smoothing observed signals by computing pseudomedians within sliding windows, a O(n2logn) calculation in each window. This poor time complexity is an issue for tiling array analysis and could prove to be a real bottleneck as tiling microarray experiments become grander in scope and finer in resolution.
Results
We therefore implemented Monahan's HLQEST algorithm that reduces the runtime complexity for computing the pseudomedian of n numbers to O(nlogn) from O(n2logn). For a representative tiling microarray dataset, this modification reduced the smoothing procedure's runtime by nearly 90%. We then leveraged the fact that elements within sliding windows remain largely unchanged in overlapping windows (as one slides across genomic space) to further reduce computation by an additional 43%. This was achieved by the application of skip lists to maintaining a sorted list of values from window to window. This sorted list could be maintained with simple O(log n) inserts and deletes. We illustrate the favorable scaling properties of our algorithms with both time complexity analysis and benchmarking on synthetic datasets.
Conclusion
Tiling microarray analyses that rely upon a sliding window pseudomedian calculation can require many hours of computation. We have eased this requirement significantly by implementing efficient algorithms that scale well with genomic feature density. This result not only speeds the current standard analyses, but also makes possible ones where many iterations of the filter may be required, such as might be required in a bootstrap or parameter estimation setting. Source code and executables are available at .
doi:10.1186/1471-2105-8-186
PMCID: PMC1913926  PMID: 17555595
22.  Global Identification and Characterization of Transcriptionally Active Regions in the Rice Genome 
PLoS ONE  2007;2(3):e294.
Genome tiling microarray studies have consistently documented rich transcriptional activity beyond the annotated genes. However, systematic characterization and transcriptional profiling of the putative novel transcripts on the genome scale are still lacking. We report here the identification of 25,352 and 27,744 transcriptionally active regions (TARs) not encoded by annotated exons in the rice (Oryza. sativa) subspecies japonica and indica, respectively. The non-exonic TARs account for approximately two thirds of the total TARs detected by tiling arrays and represent transcripts likely conserved between japonica and indica. Transcription of 21,018 (83%) japonica non-exonic TARs was verified through expression profiling in 10 tissue types using a re-array in which annotated genes and TARs were each represented by five independent probes. Subsequent analyses indicate that about 80% of the japonica TARs that were not assigned to annotated exons can be assigned to various putatively functional or structural elements of the rice genome, including splice variants, uncharacterized portions of incompletely annotated genes, antisense transcripts, duplicated gene fragments, and potential non-coding RNAs. These results provide a systematic characterization of non-exonic transcripts in rice and thus expand the current view of the complexity and dynamics of the rice transcriptome.
doi:10.1371/journal.pone.0000294
PMCID: PMC1808428  PMID: 17372628
23.  HAT: Hypergeometric Analysis of Tiling-arrays with application to promoter-GeneChip data 
BMC Bioinformatics  2010;11:275.
Background
Tiling-arrays are applicable to multiple types of biological research questions. Due to its advantages (high sensitivity, resolution, unbiased), the technology is often employed in genome-wide investigations. A major challenge in the analysis of tiling-array data is to define regions-of-interest, i.e., contiguous probes with increased signal intensity (as a result of hybridization of labeled DNA) in a region. Currently, no standard criteria are available to define these regions-of-interest as there is no single probe intensity cut-off level, different regions-of-interest can contain various numbers of probes, and can vary in genomic width. Furthermore, the chromosomal distance between neighboring probes can vary across the genome among different arrays.
Results
We have developed Hypergeometric Analysis of Tiling-arrays (HAT), and first evaluated its performance for tiling-array datasets from a Chromatin Immunoprecipitation study on chip (ChIP-on-chip) for the identification of genome-wide DNA binding profiles of transcription factor Cebpa (used for method comparison). Using this assay, we can refine the detection of regions-of-interest by illustrating that regions detected by HAT are more highly enriched for expected motifs in comparison with an alternative detection method (MAT). Subsequently, data from a retroviral insertional mutagenesis screen were used to examine the performance of HAT among different applications of tiling-array datasets. In both studies, detected regions-of-interest have been validated with (q)PCR.
Conclusions
We demonstrate that HAT has increased specificity for analysis of tiling-array data in comparison with the alternative method, and that it accurately detects regions-of-interest in two different applications of tiling-arrays. HAT has several advantages over previous methods: i) as there is no single cut-off level for probe-intensity, HAT can detect regions-of-interest at various thresholds, ii) it can detect regions-of-interest of any size, iii) it is independent of probe-resolution across the genome, and across tiling-array platforms and iv) it employs a single user defined parameter: the significance level. Regions-of-interest are detected by computing the hypergeometric-probability, while controlling the Family Wise Error. Furthermore, the method does not require experimental replicates, common regions-of-interest are indicated, a sequence-of-interest can be examined for every detected region-of-interest, and flanking genes can be reported.
doi:10.1186/1471-2105-11-275
PMCID: PMC2892465  PMID: 20492700
24.  Flexible and efficient genome tiling design with penalized uniqueness score 
BMC Bioinformatics  2012;13:323.
Background
As a powerful tool in whole genome analysis, tiling array has been widely used in the answering of many genomic questions. Now it could also serve as a capture device for the library preparation in the popular high throughput sequencing experiments. Thus, a flexible and efficient tiling array design approach is still needed and could assist in various types and scales of transcriptomic experiment.
Results
In this paper, we address issues and challenges in designing probes suitable for tiling array applications and targeted sequencing. In particular, we define the penalized uniqueness score, which serves as a controlling criterion to eliminate potential cross-hybridization, and a flexible tiling array design pipeline. Unlike BLAST or simple suffix array based methods, computing and using our uniqueness measurement can be more efficient for large scale design and require less memory. The parameters provided could assist in various types of genomic tiling task. In addition, using both commercial array data and experiment data we show, unlike previously claimed, that palindromic sequence exhibiting relatively lower uniqueness.
Conclusions
Our proposed penalized uniqueness score could serve as a better indicator for cross hybridization with higher sensitivity and specificity, giving more control of expected array quality. The flexible tiling design algorithm incorporating the penalized uniqueness score was shown to give higher coverage and resolution. The package to calculate the penalized uniqueness score and the described probe selection algorithm are implemented as a Perl program, which is freely available at http://www1.fbn-dummerstorf.de/en/forschung/fbs/fb3/paper/2012-yang-1/OTAD.v1.1.tar.gz.
doi:10.1186/1471-2105-13-323
PMCID: PMC3583072  PMID: 23216884
Tiling array; Targeted sequencing; Probe design; Penalized uniqueness score
25.  Model-based analysis of two-color arrays (MA2C) 
Genome Biology  2007;8(8):R178.
A normalization method based on probe GC content for two-color tiling arrays and an algorithm for detecting peak regions are presented. They are available in a stand-alone Java program.
A novel normalization method based on the GC content of probes is developed for two-color tiling arrays. The proposed method, together with robust estimates of the model parameters, is shown to perform superbly on published data sets. A robust algorithm for detecting peak regions is also formulated and shown to perform well compared to other approaches. The tools have been implemented as a stand-alone Java program called MA2C, which can display various plots of statistical analysis for quality control.
doi:10.1186/gb-2007-8-8-r178
PMCID: PMC2375008  PMID: 17727723

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