An automated approach to generating expressed sequence catalogues

Meier‐Ewert, Sebastian; Maier, Elmar; Ahmadi, Ali; Curtis, Jon; Lehrach, Hans

doi:10.1038/361375a0

Cited by 102 publications

(31 citation statements)

References 10 publications

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“…The method of hybridization of short synthetic oligonucleotide probes to cloned cDNA sequences under high stringency conditions to extract genetic information has been demonstrated by a number of research groups in recent years (Lehrach et al 1990; Lennon and Lehrach 1991;Meier-Ewert et al 1993;Drmanac et al 1996;Milosavljevic et al 1996). Oligonucleotide fingerprinting is an efficient and fast approach to extract parallel gene expression information about all genes that are represented in a cDNA library from a specific tissue under analysis.…”

mentioning

confidence: 99%

Large-Scale Clustering of cDNA-Fingerprinting Data

Herwig

Poustka²,

Müller³

et al. 1999

Genome Res.

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194

120

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Clustering is one of the main mathematical challenges in large-scale gene expression analysis. We describe a clustering procedure based on a sequential k-means algorithm with additional refinements that is able to handle high-throughput data in the order of hundreds of thousands of data items measured on hundreds of variables. The practical motivation for our algorithm is oligonucleotide fingerprinting-a method for simultaneous determination of expression level for every active gene of a specific tissue-although the algorithm can be applied as well to other large-scale projects like EST clustering and qualitative clustering of DNA-chip data. As a pairwise similarity measure between two p-dimensional data points, x and y, we introduce mutual information that can be interpreted as the amount of information about x in y, and vice versa. We show that for our purposes this measure is superior to commonly used metric distances, for example, Euclidean distance. We also introduce a modified version of mutual information as a novel method for validating clustering results when the true clustering is known. The performance of our algorithm with respect to experimental noise is shown by extensive simulation studies. The algorithm is tested on a subset of 2029 cDNA clones coming from 15 different genes from a cDNA library derived from human dendritic cells. Furthermore, the clustering of these 2029 cDNA clones is demonstrated when the entire set of 76,032 cDNA clones is processed.

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confidence: 99%

Large-Scale Clustering of cDNA-Fingerprinting Data

Herwig

Poustka²,

Müller³

et al. 1999

Genome Res.

Self Cite

194

120

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“…However, its full potential could not be unleashed because identifying all different clones present in an enriched library would have required a major sequencing effort of cDNA inserts. Instead, we have employed robotic-based devices [21], as developed for automated library handling in genomics [22,23], for picking and high-density gridding of phage clones to rapidly catalogue redundant cDNA inserts. This efficient strategy enabled us to find rare clones in large, selectively enriched phage populations, and hence to define a vast variety of structurally different IgE-binding proteins form the mould Aspergillus fumigatus.…”

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confidence: 99%

Rapid Identification of Allergen-Encoding cDNA Clones by Phage Display and High-Density Arrays

Kodzius¹,

Rhyner²,

Konthur

et al. 2003

CCHTS

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Abstract:We describe a high-throughput, quantitative technology for fast identification of all different clones present in selectively enriched phage surface-displayed cDNA libraries. The strategy is based on a combination of phage display and high-density arrays. To demonstrate the utility of the method cDNAs of Aspergillus fumigatus cloned into phagemid pJuFo were expressed on the tip of filamentous M13 phage and affinityselected on solid phase-immobilized serum IgE from allergic patients. Enriched phagemid libraries were amplified in bacteria, plated and arrayed into 384-well microtitre plates by robotic colony picking. cDNA inserts were amplified by high-throughput PCR and gridded onto high-density filter membranes. Filters were iteratively probed with randomly-sequenced inserts until all clones were identified. Eighty-one different sequences encoding IgE-binding proteins likely to cover a large part of the allergen repertoire of the mould were found. This approach represents a widely applicable method for rapid high-throughput identification of all individual cDNAs present in selectively enriched libraries.

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“…ICRFp524), and 20,000 cDNA clones from the adult-liver library (see Resource Availability). Fifty-five thousand clones from the ICRFp524 and 20,000 clones from the ICRFp532 libraries were selected for OFP analysis (Meier-Ewert et al 1993Drmanac et al 1996;Milosavljevic et al 1996;Clark et al 1999). cDNA inserts first were PCR-amplified and spotted onto filters.…”

Section: Resultsmentioning

confidence: 99%

“…Typically, one representative clone is rearrayed for further analysis, such as tag sequencing. OFP technology has been used previously in the mapping of the herpes simplex virus genome (Craig et al 1990), the selection of clones for genomic shotgun sequencing (Radelof et al 1998), and for the characterization and normalization of cDNA libraries (Meier-Ewert et al 1993Drmanac et al 1996;Milosavljevic et al 1996;Poustka et al 1999). Here, we have used OFP to characterize 75,000 cDNAs from zebrafish embryonic and adult liver cDNA libraries, and provide the most extensive validation of OFP to date.…”

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confidence: 99%

An Oligonucleotide Fingerprint Normalized and Expressed Sequence Tag Characterized Zebrafish cDNA Library

Clark

Hennig²,

Herwig³

et al. 2001

Genome Res.

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The zebrafish is a powerful system for understanding the vertebrate genome, allowing the combination of genetic, molecular, and embryological analysis. Expressed sequence tags (ESTs) provide a rapid means of identifying an organism's genes for further analysis, but any EST project is limited by the availability of suitable libraries. Such cDNA libraries must be of high quality and provide a high rate of gene discovery. However, commonly used normalization and subtraction procedures tend to select for shorter, truncated, and internally primed inserts, seriously affecting library quality. An alternative procedure is to use oligonucleotide fingerprinting (OFP) to precluster clones before EST sequencing, thereby reducing the re-sequencing of common transcripts. Here, we describe the use of OFP to normalize and subtract 75,000 clones from two cDNA libraries, to a minimal set of 25,102 clones. We generated 25,788 ESTs (11,380 3Ј and 14,408 5Ј) from over 16,000 of these clones. Clustering of 10,654 high-quality 3Ј ESTs from this set identified 7232 clusters (likely genes), corresponding to a 68% gene diversity rate, comparable to what has been reported for the best normalized human cDNA libraries, and indicating that the complete set of 25,102 clones contains as many as 17,000 genes. Yet, the library quality remains high. The complete set of 25,102 clones is available for researchers as glycerol stocks, filters sets, and as individual EST clones. These resources have been used for radiation hybrid, genetic, and physical mapping of the zebrafish genome, as well as positional cloning and candidate gene identification, molecular marker, and microarray development.

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An automated approach to generating expressed sequence catalogues

Cited by 102 publications

References 10 publications

Large-Scale Clustering of cDNA-Fingerprinting Data

Large-Scale Clustering of cDNA-Fingerprinting Data

Rapid Identification of Allergen-Encoding cDNA Clones by Phage Display and High-Density Arrays

An Oligonucleotide Fingerprint Normalized and Expressed Sequence Tag Characterized Zebrafish cDNA Library

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