Balanced-Size and Long-Size Cloning of Full-Length, Cap-Trapped cDNAs into Vectors of the Novel λ-FLC Family Allows Enhanced Gene Discovery Rate and Functional Analysis

Carninci, Piero; Shibata, Yûkô; Hayatsu, Norihito; Itoh, Masayoshi; Shiraki, Toshiyuki; Hirozane, Tomoko; Watahiki, Akira; Shibata, Kazuhiro; Konno, Hiroyuki; Muramatsu, Masami; Hayashizaki, Yoshihide

doi:10.1006/geno.2001.6601

Cited by 71 publications

(65 citation statements)

References 29 publications

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“…Alternatively, owing to technical limitations in cDNA library constructions, cDNAs deriving from very long mRNAs are not cloned as efficiently as others. Few full-length cDNA clone inserts are longer than 6 kb in practice, and this should include both UTRs and the coding sequence (Carninci et al 2001). We may therefore expect very long transcripts to escape sequencing more frequently than others.…”

Section: Discussionmentioning

confidence: 99%

The disparate nature of “intergenic” polyadenylation sites

Lopez

Granjeaud²,

Ara³

et al. 2006

RNA

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The termination of mature eukaryotic mRNAs occurs at specific polyadenylation sites located downstream from stop codons in the 39-untranslated region (UTR). An accurate delineation of these sites is essential for the study of 39-UTR-based gene regulation and for the design of pertinent probes for transcriptome analysis. Although typical poly(A) sites are located between 0 and 2 kb from the stop codon, EST sequence analyses have identified sites located at unexpectedly long ranges (5-10 kb) in a number of genes. Here we perform a complete mapping of EST and full-length cDNA sequences on the mouse and human genome to observe putative poly(A) sites extending beyond annotated 39-ends and into the intergenic regions. We introduce several quality parameters for poly(A) site prediction and train a classification tree to associate P-values to predicted sites. We observe a higher than background level of high-scoring sites up to 12-15 kb past the stop codon, both in human and mouse. This leads to an estimate of about 5000 human genes having unreported 39-end extensions and about 3500 novel polyadenylated transcripts lying in present ''intergenic'' regions. These high-scoring, long-range poly(A) sites corresponding to novel transcripts and gene extensions should be incorporated into current human and mouse gene repositories.

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Section: Discussionmentioning

confidence: 99%

The disparate nature of “intergenic” polyadenylation sites

Lopez

Granjeaud²,

Ara³

et al. 2006

RNA

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“…The cDNA was synthesized by using trehalose-thermoactivated reverse transcriptase (24), and full-length cDNA was recovered by using the biotinylated CAP trapper method (25,26). The -full-length cDNA vector (27) and the single-strand linker ligation method (28) were used in the construction of the cDNA libraries. One round of normalization (29) was performed to construct the NAA-and BA-treated cDNA libraries to reduce the frequency of highly expressed mRNAs in the libraries.…”

Section: Methodsmentioning

confidence: 99%

Comparative genomics ofPhyscomitrella patensgametophytic transcriptome andArabidopsis thaliana: Implication for land plant evolution

Nishiyama

Fujita

Shin-I

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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323

266

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The mosses and flowering plants diverged >400 million years ago. The mosses have haploid-dominant life cycles, whereas the flowering plants are diploid-dominant. The common ancestors of land plants have been inferred to be haploid-dominant, suggesting that genes used in the diploid body of flowering plants were recruited from the genes used in the haploid body of the ancestors during the evolution of land plants. To assess this evolutionary hypothesis, we constructed an EST library of the moss Physcomitrella patens, and compared the moss transcriptome to the genome of Arabidopsis thaliana. We constructed full-length enriched cDNA libraries from auxin-treated, cytokinin-treated, and untreated gametophytes of P. patens, and sequenced both ends of >40,000 clones. These data, together with the mRNA sequences in the public databases, were assembled into 15,883 putative transcripts. Sequence comparisons of A. thaliana and P. patens showed that at least 66% of the A. thaliana genes had homologues in P. patens. Comparison of the P. patens putative transcripts with all known proteins, revealed 9,907 putative transcripts with high levels of similarity to vascular plant genes, and 850 putative transcripts with high levels of similarity to other organisms. The haploid transcriptome of P. patens appears to be quite similar to the A. thaliana genome, supporting the evolutionary hypothesis. Our study also revealed that a number of genes are moss specific and were lost in the flowering plant lineage.

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“…In particular, Archaeorhizomycetes stand out as a group with exceptionally short ITS1 and ITS2 barcodes. Because of the very strong size selection in certain cloning methods and PCR (Kanagawa 2003;Carninci et al 2011), the unprecedented dominance of Archaeorhizomycetes in molecular studies based on cloning of long DNA fragments (Schadt et al 2003;Castro et al 2010) may represent an extreme example of a cloning bias.…”

Section: Analytical Biasesmentioning

confidence: 99%

Shotgun metagenomes and multiple primer pair-barcode combinations of amplicons reveal biases in metabarcoding analyses of fungi

Tedersoo¹,

Anslan²,

Bahram³

et al. 2015

429

399

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Rapid development of high-throughput (HTS) molecular identification methods has revolutionized our knowledge about taxonomic diversity and ecology of fungi. However, PCR-based methods exhibit multiple technical shortcomings that may bias our understanding of the fungal kingdom. This study was initiated to quantify potential biases in fungal community ecology by comparing the relative performance of amplicon-free shotgun metagenomics and amplicons of nine primer pairs over seven nuclear ribosomal DNA (rDNA) regions often used in metabarcoding analyses. The internal transcribed spacer (ITS) barcodes ITS1 and ITS2 provided greater taxonomic and functional resolution and richness of operational taxonomic units (OTUs) at the 97% similarity threshold compared to barcodes located within the ribosomal small subunit (SSU) and large subunit (LSU) genes. All barcode-primer pair combinations provided consistent results in ranking taxonomic richness and recovering the importance of floristic variables in driving fungal community composition in soils of Papua New Guinea. The choice of forward primer explained up to 2.0% of the variation in OTU-level analysis of the ITS1 and ITS2 barcode data sets. Across the whole data set, barcode-primer pair combination explained 37.6-38.1% of the variation, which surpassed any environmental signal. Overall, the metagenomics data set recovered a similar taxonomic overview, but resulted in much lower fungal rDNA sequencing depth, inability to infer OTUs, and high RESEARCH ARTICLELeho Tedersoo et al. / MycoKeys 10: 1-43 (2015) 2 uncertainty in identification. We recommend the use of ITS2 or the whole ITS region for metabarcoding and we advocate careful choice of primer pairs in consideration of the relative proportion of fungal DNA and expected dominant groups.

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Balanced-Size and Long-Size Cloning of Full-Length, Cap-Trapped cDNAs into Vectors of the Novel λ-FLC Family Allows Enhanced Gene Discovery Rate and Functional Analysis

Cited by 71 publications

References 29 publications

The disparate nature of “intergenic” polyadenylation sites

The disparate nature of “intergenic” polyadenylation sites

Comparative genomics ofPhyscomitrella patensgametophytic transcriptome andArabidopsis thaliana: Implication for land plant evolution

Shotgun metagenomes and multiple primer pair-barcode combinations of amplicons reveal biases in metabarcoding analyses of fungi

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