McArthur Lab Citation Tracker

Tuesday, November 17, 2009

CMap 1.01: A comparative mapping application for the Internet.

Bioinformatics. 2009 Jul 30;

Authors: Youens-Clark K, Faga B, Yap IV, Stein L, Ware D

SUMMARY: CMap is a web-based tool for displaying and comparing maps of any type and from any species. A user can compare an unlimited number of maps, view pair-wise comparisons of known correspondences, and search for maps or for features by name, species, type and accession. CMap is freely available, can run on a variety of database engines, and uses only free and open software components. AVAILABILITY: http://www.gmod.org/cmap.

Tuesday, October 6, 2009

Giardia lamblia: a report of drug effects under cell differentiation.

Parasitol Res. 2009 May 12;

Authors: Hausen MA, de Oliveira RP, Gadelha AP, Campanati L, de Carvalho JJ, de Carvalho L, Barbosa HS

The Giardia lamblia life cycle is characterized by two phases during which two major cell differentiation processes take place: encystation and excystation. During encystation, the trophozoites transform into cysts, the resistance form. Once ingested by a susceptible host, the cysts are stimulated to excyst in the stomach, and the excysted trophozoites adhere to the epithelium of the upper small intestine. Our work analyses the effects of four benzimidazole derivatives during Giardia differentiation into cysts and evaluates the excystation efficiency of water resistant cysts. Albendazole (AB) showed the most significant results by inhibiting encystation about 30% and a decreasing rate of excystation efficiency. The ultrastructural organization of the cyst adhesive disk was notably affected by AB treatment. Although other benzimidazoles showed some effect on encystation, they were not able to inhibit the excystation process. It is known that the benzimidazoles affect the cytoskeleton of many organisms but how it interferes in Giardia differentiation processes is our main focus. The importance of studying Giardia's differentiation under drug action is reinforced by the following arguments: (1) Cysts eliminated by hosts undergoing treatment could still be potentially infective; (2) once the host has been treated, it would be desirable that the shedding of cysts into the environment is avoided; (3) the prevention of Giardia dissemination is a question of extreme importance mainly in underdeveloped countries, where poor sanitary conditions are related to high rates of giardiasis. This report concerns the importance of keeping the environment free from infective cysts and on Giardia's drug resistance and differentiating abilities.

DNA Sudoku--harnessing high-throughput sequencing for multiplexed specimen analysis.

Genome Res. 2009 May 15;

Authors: Erlich Y, Chang K, Gordon A, Ronen R, Navon O, Rooks M, Hannon GJ

Next-generation sequencers have sufficient power to analyze simultaneously DNAs from many different specimens, a practice known as multiplexing. Such schemes rely on the ability to associate each sequence read with the specimen from which it was derived. The current practice of appending molecular barcodes prior to pooling is practical for parallel analysis of up to many dozen samples. Here, we report a strategy that permits simultaneous analysis of tens of thousands of specimens. Our approach relies on the use of combinatorial pooling strategies in which pools rather than individual specimens are assigned barcodes. Thus, the identity of each specimen is encoded within the pooling pattern rather than by its association with a particular sequence tag. Decoding the pattern allows the sequence of an original specimen to be inferred with high confidence. We verified the ability of our encoding and decoding strategies to accurately report the sequence of individual samples within a large number of mixed specimens in two ways. First, we simulated data both from a clone library and from a human population in which a sequence variant associated with cystic fibrosis was present. Second, we actually pooled, sequenced, and decoded identities within two sets of 40,000 bacterial clones comprising approximately 20,000 different artificial microRNAs targeting Arabidopsis or human genes. We achieved greater than 97% accuracy in these trials. The strategies reported here can be applied to a wide variety of biological problems, including the determination of genotypic variation within large populations of individuals.

Apollo: A Community Resource for Genome Annotation Editing.

Bioinformatics. 2009 May 13;

Authors: Ed L, Nomi H, Mark G, Raymond C, Suzanna L

SUMMARY: Apollo is a genome annotation-editing tool with an easy to use graphical interface. It is a component of the GMOD project, with ongoing development driven by the community. Recent additions to the software include support for GFF3, support for continuous transcriptome data, a full Chado database interface, integration with remote services for on-the-fly BLAST and Primer BLAST analyses, graphical interfaces for configuring user preferences, and full undo of all edit operations. Apollo's user community continues to grow, including its use as an educational tool for college and high school students. AVAILABILITY: Apollo is a Java application distributed under a free and open source license. Installers for Windows, Linux, Unix, Solaris, and Mac OS X are available at http://apollo.berkeleybop.org, and the source code is available from the SourceForge CVS repository at http://gmod.cvs.sourceforge.net/gmod/apollo.

Zebrafish embryos as models for embryotoxic and teratological effects of chemicals.

Reprod Toxicol. 2009 Apr 27;

Authors: Yang L, Ho NY, Alshut R, Legradi J, Weiss C, Reischl M, Mikut R, Liebel U, Müller F, Strähle U

The experimental virtues of the zebrafish embryo such as small size, development outside of the mother, cheap maintenance of the adult made the zebrafish an excellent model for phenotypic genetic and more recently also chemical screens. The availability of a genome sequence and several thousand mutants and transgenic lines together with gene arrays and a broad spectrum of techniques to manipulate gene functions add further to the experimental strength of this model. Pioneering studies suggest that chemicals can have in many cases very similar toxicological and teratological effects in zebrafish embryos and humans. In certain areas such as cardiotoxicity, the zebrafish appears to outplay the traditional rodent models of toxicity testing. Several pilot projects used zebrafish embryos to identify new chemical entities with specific biological functions. In combination with the establishment of transgenic sensor lines and the further development of existing and new automated imaging systems, the zebrafish embryos could therefore be used as cost effective and ethically acceptable animal models for drug screening as well as toxicity testing.

Altered gene expression in the brain and ovaries of zebrafish exposed to the aromatase inhibitor fadrozole: microarray analysis and hypothesis generation.

Environ Toxicol Chem. 2009 May 7;:1

Authors: Villeneuve DL, Wang RL, Bencic DC, Biales AD, Martinovic D, Lazorchak JM, Toth G, Ankley GT

As part of a research effort examining system-wide responses of the hypothalamic-pituitary-gonadal (HPG) axis in fish to endocrine active chemicals (EACs) with different modes of action, we exposed zebrafish (Danio rerio) to 25 or 100 mug/L of the aromatase inhibitor fadrozole for 24, 48, or 96 h. Global transcriptional response in brain and ovary tissue of fish exposed to 25 mug fadrozole/L was compared to that in control fish using a commercially available 22,000 gene oligonucleotide microarray. Transcripts altered in brain were functionally linked to differentiation, development, DNA replication, and cell cycle. Additionally, multiple genes associated with the one carbon pool by folate pathway (KEGG 00670) were significantly up-regulated. Transcripts altered in ovary were functionally linked to cell-cell adhesion, extracellular matrix, vasculogenesis, and development. Promoter motif analysis identified GATA-binding factor 2, Ikaros 2, alcohol dehydrogenase gene regulator 1, myoblast determining factor, and several heat shock factors as being associated with co-expressed gene clusters that were differentially-expressed following fadrozole exposure. Based on the transcriptional changes observed, we hypothesize that fadrozole elicits neurodegenerative stress in brain tissue and that fish cope with that stress through proliferation of radial glial cells. Additionally, we hypothesize that gene expression changes in the ovary of fadrozole-exposed zebrafish reflect disruption of oocyte maturation and ovulation due to impaired vitellogenesis. These hypotheses and others derived from the microarray results provide a foundation for future studies aimed at understanding responses of the HPG axis to EACs and other chemical stressors.

A novel WRKY-like protein involved in transcriptional activation of cyst wall protein genes in Giardia lamblia.

A novel WRKY-like protein involved in transcriptional activation of cyst wall protein genes in giardia lamblia.

J Biol Chem. 2009 May 7;

Authors: Pan YJ, Cho CC, Kao YY, Sun CH

Synthesis of a protective cyst wall is required for survival outside the host and infection of Giardia lamblia. Little is known of gene regulation of the cyst wall proteins (CWPs) during differentiation into dormant cysts. WRKY homologues constitute a large family of DNA-binding proteins in plants that are involved in several key cellular functions including disease-resistance, stress response, dormancy, and development. A putative wrky gene has been identified in G. lamblia genome. We found that wrky expression levels increased significantly during encystation. The epitope-tagged WRKY was translocated into the nuclei during encystation. Recombinant WRKY specifically bound to the wrky its own promoter and the encystation-induced cwp1 and cwp2 promoters. WRKY contains several key residues for DNA binding and mutation analysis revealed that its binding sequences are similar to those of the known plant WRKY proteins and two of them are positive cis-acting elements of the wrky and cwp2 promoters. Overexpression of WRKY increased the cwp1-2 and myb2 mRNA levels and these gene promoters were bound by WRKY in vivo. Interestingly, the wrky and cwp1-2 genes were up-regulated by the extracellular signal-related kinase 1 (ERK1) overexpression, suggesting that WRKY may be a downstream component of the ERK1 pathway. In addition, a WRKY mutant that can not enter nuclei and an ERK1 mutant lacking the predicted kinase domain showed decreased cwp1-2 gene expression. Our results suggest that the WRKY family has been conserved during evolution and WRKY is an important transactivator of the cwp1-2 genes when G. lamblia differentiation into dormant cysts.

Parallel DNA pyrosequencing unveils new zebrafish microRNAs.

BMC Genomics. 2009 Apr 27;10(1):195

Authors: Soares AR, Pereira PM, Santos B, Egas C, Gomes AC, Arrais J, Oliveira JL, Moura GR, Santos MA

ABSTRACT: BACKGROUND: MicroRNAs (miRNAs) are a new class of small RNAs of approximately 22 nucleotides in length that control eukaryotic gene expression by fine tuning mRNA translation. They regulate a wide variety of biological processes, namely developmental timing, cell differentiation, cell proliferation, immune response and infection. For this reason, their identification is essential to understand eukaryotic biology. Their small size, low abundance and high instability complicated early identification, however cloning/Sanger sequencing and new generation genome sequencing approaches overcame most technical hurdles and are being used for rapid miRNAs identification in many eukaryotes. RESULTS: We have applied 454 DNA pyrosequencing technology to miRNA discovery in zebrafish (Danio rerio). For this, a series of cDNA libraries were prepared from miRNAs isolated at different embryonic time points and from fully developed organs. Each cDNA library was tagged with specific sequences and was sequenced using the Roche FLX genome sequencer. This approach retrieved 90% of the 192 miRNAs previously identified by cloning/Sanger sequencing and bioinformatics. Twenty five novel miRNAs were predicted, 107 miRNA star sequences and also 41 candidate miRNA targets were identified. A miRNA expression profile built on the basis of pyrosequencing read numbers showed high expression of most miRNAs throughout zebrafish development and identified tissue specific miRNAs. CONCLUSIONS: This study increases the number of zebrafish miRNAs from 192 to 217 and demonstrates that a single DNA mini-chip pyrosequencing run is effective in miRNA identification in zebrafish. This methodology also produced sufficient information to elucidate miRNA expression patterns during development and in differentiated organs. Moreover, some zebrafish miRNA star sequences were more abundant than their corresponding miRNAs, suggesting a functional role for the former in gene expression control in this vertebrate model organism.

Wednesday, September 9, 2009

A quick guide to organizing computational biology projects.

PLoS Comput Biol. 2009 Jul;5(7):e1000424

Authors: Noble WS

Tuesday, September 8, 2009

JBrowse: A next-generation genome browser.

Genome Res. 2009 Jul 1;

Authors: Skinner ME, Uzilov AV, Stein LD, Mungall CJ, Holmes IH

We describe an open source, portable, JavaScript-based genome browser, JBrowse, that can be used to navigate genome annotations over the web. JBrowse helps preserve the user's sense of location by avoiding discontinuous transitions, instead offering smoothly-animated panning, zooming, navigation and track selection. Unlike most existing genome browsers, where the genome is rendered into images on the webserver and the role of the client is restricted to displaying those images, JBrowse distributes work between the server and client and therefore uses significantly less server overhead than previous genome browsers. We report benchmark results empirically comparing server- and client-side rendering strategies, review the architecture and design considerations of JBrowse, and describe a simple wiki plugin that allows users to upload and share annotation tracks.

Friday, July 17, 2009

The genome of the blood fluke Schistosoma mansoni.

Nature. 2009 Jul 16;460(7253):352-8

Authors: Berriman M, Haas BJ, Loverde PT, Wilson RA, Dillon GP, Cerqueira GC, Mashiyama ST, Al-Lazikani B, Andrade LF, Ashton PD, Aslett MA, Bartholomeu DC, Blandin G, Caffrey CR, Coghlan A, Coulson R, Day TA, Delcher A, Demarco R, Djikeng A, Eyre T, Gamble JA, Ghedin E, Gu Y, Hertz-Fowler C, Hirai H, Hirai Y, Houston R, Ivens A, Johnston DA, Lacerda D, Macedo CD, McVeigh P, Ning Z, Oliveira G, Overington JP, Parkhill J, Pertea M, Pierce RJ, Protasio AV, Quail MA, Rajandream MA, Rogers J, Sajid M, Salzberg SL, Stanke M, Tivey AR, White O, Williams DL, Wortman J, Wu W, Zamanian M, Zerlotini A, Fraser-Liggett CM, Barrell BG, El-Sayed NM

Schistosoma mansoni is responsible for the neglected tropical disease schistosomiasis that affects 210 million people in 76 countries. Here we present analysis of the 363 megabase nuclear genome of the blood fluke. It encodes at least 11,809 genes, with an unusual intron size distribution, and new families of micro-exon genes that undergo frequent alternative splicing. As the first sequenced flatworm, and a representative of the Lophotrochozoa, it offers insights into early events in the evolution of the animals, including the development of a body pattern with bilateral symmetry, and the development of tissues into organs. Our analysis has been informed by the need to find new drug targets. The deficits in lipid metabolism that make schistosomes dependent on the host are revealed, and the identification of membrane receptors, ion channels and more than 300 proteases provide new insights into the biology of the life cycle and new targets. Bioinformatics approaches have identified metabolic chokepoints, and a chemogenomic screen has pinpointed schistosome proteins for which existing drugs may be active. The information generated provides an invaluable resource for the research community to develop much needed new control tools for the treatment and eradication of this important and neglected disease.

Wednesday, April 29, 2009

Can subtle changes in gene expression be consistently detected with different microarray platforms?

BMC Genomics. 2008;9:124

Authors: Pedotti P, 't Hoen PA, Vreugdenhil E, Schenk GJ, Vossen RH, Ariyurek Y, de Hollander M, Kuiper R, van Ommen GJ, den Dunnen JT, Boer JM, de Menezes RX

BACKGROUND: The comparability of gene expression data generated with different microarray platforms is still a matter of concern. Here we address the performance and the overlap in the detection of differentially expressed genes for five different microarray platforms in a challenging biological context where differences in gene expression are few and subtle. RESULTS: Gene expression profiles in the hippocampus of five wild-type and five transgenic deltaC-doublecortin-like kinase mice were evaluated with five microarray platforms: Applied Biosystems, Affymetrix, Agilent, Illumina, LGTC home-spotted arrays. Using a fixed false discovery rate of 10% we detected surprising differences between the number of differentially expressed genes per platform. Four genes were selected by ABI, 130 by Affymetrix, 3,051 by Agilent, 54 by Illumina, and 13 by LGTC. Two genes were found significantly differentially expressed by all platforms and the four genes identified by the ABI platform were found by at least three other platforms. Quantitative RT-PCR analysis confirmed 20 out of 28 of the genes detected by two or more platforms and 8 out of 15 of the genes detected by Agilent only. We observed improved correlations between platforms when ranking the genes based on the significance level than with a fixed statistical cut-off. We demonstrate significant overlap in the affected gene sets identified by the different platforms, although biological processes were represented by only partially overlapping sets of genes. Aberrances in GABA-ergic signalling in the transgenic mice were consistently found by all platforms. CONCLUSION: The different microarray platforms give partially complementary views on biological processes affected. Our data indicate that when analyzing samples with only subtle differences in gene expression the use of two different platforms might be more attractive than increasing the number of replicates. Commercial two-color platforms seem to have higher power for finding differentially expressed genes between groups with small differences in expression.

Antioxidant responses and NRF2 in synergistic developmental toxicity of PAHs in zebrafish.

Toxicol Sci. 2009 Feb 20;

Authors: Timme-Laragy AR, Van Tiem LA, Linney EA, Di Giulio RT

Early piscine life-stages are sensitive to polycyclic aromatic hydrocarbon (PAH) exposure, which can cause pericardial effusion and craniofacial malformations. We previously reported that certain combinations of PAHs cause synergistic developmental toxicity, as observed with co-exposure to the aryl hydrocarbon receptor (AHR) agonist beta-naphthoflavone (BNF) and cytochrome P4501A inhibitor alpha-naphthoflavone (ANF). Herein, we hypothesized that oxidative stress is a component of this toxicity. We examined induction of antioxidant genes in zebrafish embryos (Danio rerio) exposed to BNF or ANF individually, a BNF+ANF combination, and a pro-oxidant positive control, tert-butylhydroperoxide (tBOOH). We measured total glutathione, and attempted to modulate deformities using the glutathione synthesis inhibitor buthionine sulfoxamine (BSO) and increase glutathione pools with N-acetyl cysteine (NAC). In addition, we used a morpholino to knockdown expression of the antioxidant response element transcription factor NRF2 to determine if this would alter gene expression or increase deformity severity. BNF+ANF co-exposure significantly increased expressions of superoxide dismutase1 and2, glutathione peroxidase 1, pi class glutathione-s-transferase, and glutamate cysteine-ligase to a greater extent than tBOOH, BNF, or ANF alone. BSO pretreatment decreased some glutathione levels, but did not worsen deformities, nor did NAC diminish toxicity. Knockdown of NRF2 increased mortality following tBOOH challenge, prevented significant upregulation of antioxidant genes following both tBOOH and BNF+ANF exposures, and exacerbated BNF+ANF-related deformities. Collectively, these findings demonstrate that antioxidant responses are a component of PAH synergistic developmental toxicity, and that NRF2 is protective against prooxidant and PAH challenges during development.

Gene expression analysis using agilent DNA microarrays.

Methods Mol Biol. 2009;529:133-45

Authors: Stangegaard M

Hybridization of labeled cDNA to microarrays is an intuitively simple and a vastly underestimated process. If it is not performed, optimized, and standardized with the same attention to detail as e.g., RNA amplification, information may be overlooked or even lost. Careful balancing of the amount of labeled cDNA added to each slide reduces dye-bias and slide to slide variation. Efficient mixing of the hybridization solution throughout the hybridization reaction increases signals several fold. The amount of near perfect target-probe hybrids may be reduced by efficient stringency washes of the hybridized microarray slides.

Proteomes and transcriptomes of Apicomplexa - Where's the message?

Int J Parasitol. 2008 Nov 1;

Authors: Wastling JM, Xia D, Sohal A, Chaussepied M, Pain A, Langsley G

The Apicomplexa now have some of the most comprehensive and integrated proteome datasets of all pathogenic micro-organisms. Coverage is currently at a level where these data can be used to help predict the potential biological function of proteins in these parasites, without having to defer to measurement of mRNA levels. Transcriptomic data for the Apicomplexa (microarrays, expressed sequence tag (EST) collections, serial analysis of gene expression (SAGE) and massively parallel signature sequencing (MPSS) tags) are also copious, enabling us to investigate the extent to which global mRNA levels correlate with proteomic data. Here, we present a proteomic and transcriptomic perspective of gene expression in key apicomplexan parasites, including Plasmodium spp., Toxoplasma gondii, Cryptosporidium parvum, Neospora caninum and Theileria spp., and discuss the alternative views of gene expression that they provide. Although proteomic evidence does not detect every protein for every gene, many examples of readily detected proteins whose corresponding genes display little or no detectable transcription, are detected across the Apicomplexa. These examples are not easily explained by the "guilt by association", or "stock and go" hypotheses of gene transcription. With the advent of ultra-high-throughput sequencing technologies there will be a quantum shift in transcriptional analysis which, combined with improving quantitative proteome datasets, will provide a core component of a systems-wide approach to studying the Apicomplexa.

Clustering-based approaches to SAGE data mining.

BioData Min. 2008;1(1):5

Authors: Wang H, Zheng H, Azuaje F

ABSTRACT: Serial analysis of gene expression (SAGE) is one of the most powerful tools for global gene expression profiling. It has led to several biological discoveries and biomedical applications, such as the prediction of new gene functions and the identification of biomarkers in human cancer research. Clustering techniques have become fundamental approaches in these applications. This paper reviews relevant clustering techniques specifically designed for this type of data. It places an emphasis on current limitations and opportunities in this area for supporting biologically-meaningful data mining and visualisation.

A combination of LongSAGE with Solexa sequencing is well suited to explore the depth and the complexity of transcriptome.

BMC Genomics. 2008 Sep 16;9(1):418

Authors: Hanriot L, Keime C, Gay N, Faure C, Dossat C, Wincker P, Scote-Blachon C, Peyron C, Gandrillon O

ABSTRACT: BACKGROUND: "Open" transcriptome analysis methods allow to study gene expression without a priori knowledge of the transcript sequences. As of now, SAGE (Serial Analysis of Gene Expression), LongSAGE and MPSS (Massively Parallel Signature Sequencing) are the mostly used methods for "open" transcriptome analysis. Both LongSAGE and MPSS rely on the isolation of 21 pb tag sequences from each transcript. In contrast to LongSAGE, the high throughput sequencing method used in MPSS enables the rapid sequencing of very large libraries containing several millions of tags, allowing deep transcriptome analysis. However, a bias in the complexity of the transcriptome representation obtained by MPSS was recently uncovered. RESULTS: In order to make a deep analysis of mouse hypothalamus transcriptome avoiding the limitation introduced by MPSS, we combined LongSAGE with the Solexa sequencing technology and obtained a library of more than 11 millions of tags. We then compared it to a LongSAGE library of mouse hypothalamus sequenced with the Sanger method. CONCLUSION: We found that Solexa sequencing technology combined with LongSAGE is perfectly suited for deep transcriptome analysis. In contrast to MPSS, it gives a complex representation of transcriptome as reliable as a LongSAGE library sequenced by the Sanger method.

Comparison of the expression profiles of promastigotes and axenic amastigotes in Leishmania donovani using serial analysis of gene expression.

Parasitol Res. 2008 Jun 22;

Authors: Li Q, Zhao Y, Ni B, Yao C, Zhou Y, Xu W, Wang Z, Qiao Z

In this study, we examined the transcriptome of Leishmania donovani promastigotes and axenic amastigotes to identify differentially regulated mRNAs utilizing the serial analysis of gene expression (SAGE). The axenic culture of amastigotes was initiated from stationary phase promastigotes. Transformation from promastigote to amastigote occurred when cultures in Medium 199 (pH 5.5), supplemented with 20% (v/v) fetal bovine serum, were transferred from 26 degrees C to 37 degrees C. A total of 20,299 and 20,132 tags were generated from promastigote and amastigote libraries, respectively. The containing unique genes identified in these two SAGE libraries were 8,615 and 7,835, respectively. Characteristics of the expressed genes' frequency distribution were remarkably similar in both libraries: the most abundant tags (frequency >/=20), whose levels were equal to or >1.3% of the identified tags, constituted >23% of the total sequenced tags. Correspondingly, 75.72% or 71.65% of the genes accounted for those tags present at low abundance (frequency = 1) contributed only 32.13% or 27.89% of the total tags. A total of 968 genes (11.2% of the total genes in promastigotes and 12.4% in amastigotes) were recorded to have statistically different transcript levels between promastigotes and axenic amastigotes. Of the 968 distinct total genes, there are 326 promastigote-enriched transcripts and 642 amastigote-enriched mRNAs.

Validation of oligoarrays for quantitative exploration of the transcriptome.

BMC Genomics. 2008 May 30;9(1):258

Authors: Nygaard V, Liu F, Holden M, Kuo WP, Trimarchi J, Ohno-Machado L, Cepko CL, Frigessi A, Glad IK, van de Wiel MA, Hovig E, Lyng H

ABSTRACT: BACKGROUND: Oligoarrays have become an accessible technique for exploring the transcriptome, but it is presently unclear how absolute transcript data from this technique compare to the data achieved with tag-based quantitative techniques, such as massively parallel signature sequencing (MPSS) and serial analysis of gene expression (SAGE). By use of the TransCount method we calculated absolute transcript concentrations from spotted oligoarray intensities, enabling direct comparisons with tag counts obtained with MPSS and SAGE. The tag counts were converted to number of transcripts per cell by assuming that the sum of all transcripts in a single cell was 5 * 105. Our aim was to investigate whether the less resource demanding and more widespread oligoarray technique could provide data that were correlated to and had the same absolute scale as those obtained with MPSS and SAGE. RESULTS: A number of 1,777 unique transcripts were detected in common for the three technologies and served as the basis for our analyses. The correlations involving the oligoarray data were not weaker than, but, similar to the correlation between the MPSS and SAGE data, both when the entire concentration range was considered and at high concentrations. The data sets were more strongly correlated at high transcript concentrations than at low concentrations. On an absolute scale, the number of transcripts per cell and gene was generally higher based on oligoarrays than on MPSS and SAGE, and ranged from 1.6 to 9,705 for the 1,777 overlapping genes. The MPSS data were on same scale as the SAGE data, ranging from 0.5 to 3,180 (MPSS) and 9 to1,268 (SAGE) transcripts per cell and gene. The sum of all transcripts per cell for these genes was 3.8 * 105 (oligoarrays), 1.1 * 105 (MPSS) and 7.6 * 104 (SAGE), whereas the corresponding sum for all detected transcripts was 1.1 * 106 (oligoarrays), 2.8 * 105 (MPSS) and 3.8 * 105 (SAGE). CONCLUSION: The oligoarrays and TransCount provide quantitative transcript concentrations that are correlated to MPSS and SAGE data, but, the absolute scale of the measurements differs across the technologies. The discrepancy questions whether the sum of all transcripts within a single cell might be higher than the number of 5 * 105 suggested in the literature and used to convert tag counts to transcripts per cell. If so, this may explain the apparent higher transcript detection efficiency of the oligoarrays, and has to be clarified before absolute transcript concentrations can be interchanged across the technologies. The ability to obtain transcript concentrations from oligoarrays opens up the possibility of efficient generation of universal transcript databases with low resource demands.

Deep cap analysis gene expression (CAGE): genome-wide identification of promoters, quantification of their expression, and network inference.

Biotechniques. 2008 Apr;44(5):627-8, 630, 632

Authors: de Hoon M, Hayashizaki Y

In cap analysis gene expression (CAGE), short ( approximately 20 nucleotide) sequence tags originating from the 5' end of full-length mRNAs are sequenced to identify transcription events on a genome-wide scale. The rapid increase in the throughput of present-day sequencers provides much deeper CAGE tag sequencing, where CAGE tags can be found multiple times for each mRNA in a given experiment. CAGE tag counts can then be used to reliably estimate the cellular concentration of the corresponding mRNA. In contrast to microarray and SAGE expression profiling, CAGE identifies the location of each transcription start site in addition to its expression level. This makes it possible for us to infer a genome-wide network of transcriptional regulation by searching the promoter region surrounding each CAGE-defined transcription start site for potential transcription factor binding sites. Hence, deep CAGE is a unique tool for the construction of a promoter-based network of transcriptional regulation. CAGE-based expression profiling also allows us to identify dynamic promoter usage in time-course experiments and the specific promoter regulated by a given transcription factor in disruption experiments. The sheer size of the short-tag datasets produced by modern sequencers spurs a need for new software development to handle the amount of data generated by next-generation sequencers. In addition, new visualization methods will be needed to represent a promoter-based transcriptional network.