Computer-Based Methods for the Mouse Full-Length cDNA Encyclopedia: Real-Time Sequence Clustering for Construction of a Nonredundant cDNA Library

Konno, Hiroyuki

doi:10.1101/gr.gr-1457r

Cited by 28 publications

(17 citation statements)

References 33 publications

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“…Microtiter plates of clones can be copied and frozen, allowing the picked library to be stored indefinitely. Accurate tracking of the plates of clones throughout the picking and archiving process is an essential step (Konno et al, 2001), since these plates will be the source for the next steps of PCR amplification, arraying and sequencing.…”

Section: Constructing Microarrays For Non-model Organismsmentioning

confidence: 99%

Interpreting physiological responses to environmental change through gene expression profiling

Gracey

2007

Journal of Experimental Biology

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SUMMARY Identification of differentially expressed genes in response to environmental change offers insights into the roles of the transcriptome in the regulation of physiological responses. A variety of methods are now available to implement large-scale gene expression screens, and each method has specific advantages and disadvantages. Construction of custom cDNA microarrays remains the most popular route to implement expression screens in the non-model organisms favored by comparative physiologists, and we highlight some factors that should be considered when embarking along this path. Using a carp cDNA microarray, we have undertaken a broad, system-wide gene expression screen to investigate the physiological mechanisms underlying cold and hypoxia acclimation. This dataset provides a starting point from which to explore a range of specific mechanistic hypotheses at all levels of organization, from individual biochemical pathways to the level of the whole organism. We demonstrate the utility of two data analysis methods, Gene Ontology profiling and rank-based statistical methods, to summarize the probable physiological function of acclimation-induced gene expression changes, and to prioritize specific genes as candidates for further study.

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Section: Constructing Microarrays For Non-model Organismsmentioning

confidence: 99%

Interpreting physiological responses to environmental change through gene expression profiling

Gracey

2007

Journal of Experimental Biology

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“…1,442,236 sequences were grouped into 171,144 3 0 -end clusters based on sequence similarity (Supplementary Information section 2) [14][15][16] . Of these, 159,789 had no significant BLAST hit to known mouse genes, and were considered potentially novel.…”

Section: The Fantom2 Clone Setmentioning

confidence: 99%

Analysis of the mouse transcriptome based on functional annotation of 60,770 full-length cDNAs

Okazaki¹,

Furuno²,

Kasukawa³

et al. 2002

Nature

1,466

406

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Only a small proportion of the mouse genome is transcribed into mature messenger RNA transcripts. There is an international collaborative effort to identify all full-length mRNA transcripts from the mouse, and to ensure that each is represented in a physical collection of clones. Here we report the manual annotation of 60,770 full-length mouse complementary DNA sequences. These are clustered into 33,409 'transcriptional units', contributing 90.1% of a newly established mouse transcriptome database. Of these transcriptional units, 4,258 are new protein-coding and 11,665 are new non-coding messages, indicating that non-coding RNA is a major component of the transcriptome. 41% of all transcriptional units showed evidence of alternative splicing. In protein-coding transcripts, 79% of splice variations altered the protein product. Whole-transcriptome analyses resulted in the identification of 2,431 sense-antisense pairs. The present work, completely supported by physical clones, provides the most comprehensive survey of a mammalian transcriptome so far, and is a valuable resource for functional genomics.

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“…If not otherwise described, the clustering was made following Konno et al (2001), and other computer analysis, if not otherwise specified, following Okazaki et al (2002), and references therein. For clustering, the linkers and vector sequences were removed from sequences, then the first 200-bp regions were selected and used as TAG sequences.…”

Section: Sequence Analysismentioning

confidence: 99%

Targeting a Complex Transcriptome: The Construction of the Mouse Full-Length cDNA Encyclopedia

Carninci¹,

Waki²,

Shiraki³

et al. 2003

Genome Res.

156

132

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We report the construction of the mouse full-length cDNA encyclopedia, the most extensive view of a complex transcriptome, on the basis of preparing and sequencing 246 libraries. Before cloning, cDNAs were enriched in full-length by Cap-Trapper, and in most cases, aggressively subtracted/normalized. We have produced 1,442,236 successful 3Ј-end sequences clustered into 171,144 groups, from which 60,770 clones were fully sequenced cDNAs Cold Spring Harbor Laboratory Press on May 10, 2018 -Published by genome.cshlp.org Downloaded from annotated in the FANTOM-2 annotation. We have also produced 547,149 5Ј end reads, which clustered into 124,258 groups. Altogether, these cDNAs were further grouped in 70,000 transcriptional units (TU), which represent the best coverage of a transcriptome so far. By monitoring the extent of normalization/subtraction, we define the tentative equivalent coverage (TEC), which was estimated to be equivalent to >12,000,000 ESTs derived from standard libraries. High coverage explains discrepancies between the very large numbers of clusters (and TUs) of this project, which also include non-protein-coding RNAs, and the lower gene number estimation of genome annotations. Altogether, 5Ј-end clusters identify regions that are potential promoters for 8637 known genes and 5Ј-end clusters suggest the presence of almost 63,000 transcriptional starting points. An estimate of the frequency of polyadenylation signals suggests that at least half of the singletons in the EST set represent real mRNAs. Clones accounting for about half of the predicted TUs await further sequencing. The continued high-discovery rate suggests that the task of transcriptome discovery is not yet complete.[Supplemental material available online at www.genome.org.]One of the primary goals of genome sequencing projects is to identify the genome sequences that are transcribed into functional mRNAs, so that full-length cDNAs can be isolated to allow further downstream biology, and functional and structural genomics. The limitations of a priori genome annotation dictate that the transcriptome needs to be identified experimentally via cDNA cloning and sequencing. Although expressed sequence tags (ESTs) (Adams et al. 1991(Adams et al. , 1995Hillier et al. 1996;Marra et al. 1999;Kargul et al. 2001) and ORESTES (Camargo et al. 2001) have been extremely valuable for new gene discovery, these approaches have not allowed highthroughput recovering of full-length cDNA clones nor definition of protein sequence derived from actual cDNA clones. To overcome such problems, we undertook from the year 1995, a strategic project aimed at the comprehensive collection of at least one full-length cDNA derived from each mouse gene, a strategy that is recently becoming useful in similar projects to collect full-length gene collections (Stapleton et al. 2002;Strausberg et al. 2002).Because of the limited processivity of reverse transcriptase and other limitations, standard cDNA libraries generally contain a majority of truncated transcripts. The introductio...

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Computer-Based Methods for the Mouse Full-Length cDNA Encyclopedia: Real-Time Sequence Clustering for Construction of a Nonredundant cDNA Library

Cited by 28 publications

References 33 publications

Interpreting physiological responses to environmental change through gene expression profiling

Interpreting physiological responses to environmental change through gene expression profiling

Analysis of the mouse transcriptome based on functional annotation of 60,770 full-length cDNAs

Targeting a Complex Transcriptome: The Construction of the Mouse Full-Length cDNA Encyclopedia

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