Reducing bias in cDNA sequence representation by molecular selection

Coche, Th.; Dewez, Monique

doi:10.1093/nar/22.21.4545

Cited by 11 publications

(5 citation statements)

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“…Second, we reduced the complexity of the transcriptome by cDNA normalization prior to sequencing. cDNA normalization lead to a significant increase in transcriptome sequencing efficiency by equalizing the representation of high, medium and rarely expressed transcripts in the cDNA population [ 50 - 52 ]. Since many transcripts are temporally and/or spatially expressed during plant development, RNA pooled from different tissues at different developmental stages ensured the coverage of temporal- and spatial-specific transcripts.…”

Section: Discussionmentioning

confidence: 99%

From RNA-seq to large-scale genotyping - genomics resources for rye (Secale cereale L.)

et al. 2011

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BackgroundThe improvement of agricultural crops with regard to yield, resistance and environmental adaptation is a perpetual challenge for both breeding and research. Exploration of the genetic potential and implementation of genome-based breeding strategies for efficient rye (Secale cereale L.) cultivar improvement have been hampered by the lack of genome sequence information. To overcome this limitation we sequenced the transcriptomes of five winter rye inbred lines using Roche/454 GS FLX technology.ResultsMore than 2.5 million reads were assembled into 115,400 contigs representing a comprehensive rye expressed sequence tag (EST) resource. From sequence comparisons 5,234 single nucleotide polymorphisms (SNPs) were identified to develop the Rye5K high-throughput SNP genotyping array. Performance of the Rye5K SNP array was investigated by genotyping 59 rye inbred lines including the five lines used for sequencing, and five barley, three wheat, and two triticale accessions. A balanced distribution of allele frequencies ranging from 0.1 to 0.9 was observed. Residual heterozygosity of the rye inbred lines varied from 4.0 to 20.4% with higher average heterozygosity in the pollen compared to the seed parent pool.ConclusionsThe established sequence and molecular marker resources will improve and promote genetic and genomic research as well as genome-based breeding in rye.

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Section: Discussionmentioning

confidence: 99%

From RNA-seq to large-scale genotyping - genomics resources for rye (Secale cereale L.)

et al. 2011

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“…After the hybridization cycle, the cDNA population is divided into two fractions: ds cDNA containing the majority of the initial cDNA (except for very rare genes), and single-stranded (ss) cDNA comprising initial transcripts at largely comparable concentrations. This ss cDNA fraction is then isolated using a technique such as selective amplification, hydroxyapatite columns, magnetic beads, or enzymatic hydrolysis of ds cDNA (Coche and Dewez, 1994;Soares et al, 1994;Luk'ianov et al, 1996;Carninci et al, 2000;Zhulidov et al, 2004), and used for the construction of a normalized cDNA library.…”

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confidence: 99%

Normalizing cDNA Libraries

Богданова

Shagina²,

Barsova

et al. 2010

CP Molecular Biology

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The characterization of rare messages in cDNA libraries is complicated by the substantial variations that exist in the abundance levels of different transcripts in cells and tissues. The equalization (normalization) of cDNA is a helpful approach for decreasing the prevalence of abundant transcripts, thereby facilitating the assessment of rare transcripts. This unit provides a method for duplex-specific nuclease (DSN)-based normalization, which allows for the fast and reliable equalization of cDNA, thereby facilitating the generation of normalized, full-length-enriched cDNA libraries, and enabling efficient RNA analyses.

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“…(4) Cloning of the normalized cDNA library [2][3][4][5][6][7][8][9][10][11]. The main differences between the available normalization methods consist in the procedure of separation of the normalized ss cDNA fraction.…”

Section: Introductionmentioning

confidence: 99%

“…The main differences between the available normalization methods consist in the procedure of separation of the normalized ss cDNA fraction. The separation is accomplished using several approaches, such as the physical separation of ss-and ds-fractions by the chromatography on hydroxylapatite [2,3,8] or by means of paramagnetic beads [7,11], the cleavage of the ds-fraction by restriction endonucleases [5], and the amplification of the ssfraction using the PCR suppression effect [10]. Unfortunately, most of the available approaches are unsuitable for the normalization of cDNA samples enriched with full-length sequences.…”

Section: Introductionmentioning

confidence: 99%

A method for the preparation of normalized cDNA libraries enriched with full-length sequences

et al. 2005

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We developed a new method for the preparation of normalized cDNA libraries enriched with full-length sequences. It is based on the properties of the recently characterized duplex-specific nuclease from the hepatopancreas of the Kamchatka crab. The duplex-specific nuclease is thermostable, it effectively cleaves double-stranded DNA and is inactive toward single-stranded DNA (Shagin et al., Genome Res., 2002, vol. 12, pp. 1935-1942). Our method enables the normalization of cDNA samples enriched with full-length sequences without use of laborious and ineffective stages of physical separation. The efficiency of the method was demonstrated in model experiments using cDNA samples from several human tissues.

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Reducing bias in cDNA sequence representation by molecular selection

Cited by 11 publications

References 4 publications

From RNA-seq to large-scale genotyping - genomics resources for rye (Secale cereale L.)

From RNA-seq to large-scale genotyping - genomics resources for rye (Secale cereale L.)

Normalizing cDNA Libraries

A method for the preparation of normalized cDNA libraries enriched with full-length sequences

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