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Efficient targeted transcript discovery via array-based normalization of RACE libraries.

Djebali S, Kapranov P, Foissac S, Lagarde J, Reymond A, Ucla C, Wyss C, Drenkow J, Dumais E, Murray RR, Lin C, Szeto D, Denoeud F, Calvo M, Frankish A, Harrow J, Makrythanasis P, Vidal M, Salehi-Ashtiani K, Antonarakis SE, Gingeras TR, Guigó R - Nat. Methods (2008)

Bottom Line: Random clone selection from the RACE mixture, however, is an ineffective sampling strategy if the dynamic range of transcript abundances is large.This approach, RACEarray, is superior to direct cloning and sequencing of RACE products because it specifically targets new transcripts and often results in overall normalization of transcript abundance.We show theoretically and experimentally that this strategy leads indeed to efficient sampling of new transcripts, and we investigated multiplexing the strategy by pooling RACE reactions from multiple interrogated loci before hybridization.

View Article: PubMed Central - PubMed

Affiliation: Grup de Recerca en Informàtica Biomèdica, Institut Municipal d'Investigació Mèdica/Universitat Pompeu Fabra, Dr. Aiguader 88, 08003 Barcelona, Spain.

ABSTRACT
Rapid amplification of cDNA ends (RACE) is a widely used approach for transcript identification. Random clone selection from the RACE mixture, however, is an ineffective sampling strategy if the dynamic range of transcript abundances is large. To improve sampling efficiency of human transcripts, we hybridized the products of the RACE reaction onto tiling arrays and used the detected exons to delineate a series of reverse-transcriptase (RT)-PCRs, through which the original RACE transcript population was segregated into simpler transcript populations. We independently cloned the products and sequenced randomly selected clones. This approach, RACEarray, is superior to direct cloning and sequencing of RACE products because it specifically targets new transcripts and often results in overall normalization of transcript abundance. We show theoretically and experimentally that this strategy leads indeed to efficient sampling of new transcripts, and we investigated multiplexing the strategy by pooling RACE reactions from multiple interrogated loci before hybridization.

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absolute number and cumulative proportion of all (a) and novel (b) projected RACEfrags originating from index exonsA projected RACEfrag is a maximal set of RACEfrags that transitively overlap (see Supplementary Methods and Supplementary Figure 3). In the X-axis, index exons are ordered from 3' to 5' of the gene. For instance, the most 3’ exon generates 22% of all projected RACEfrags. The two most 3’ exons generate 40 % of them, and so on. Adding the 5’ most index exon generates 10% of all projected RACEfrags.
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Figure 4: absolute number and cumulative proportion of all (a) and novel (b) projected RACEfrags originating from index exonsA projected RACEfrag is a maximal set of RACEfrags that transitively overlap (see Supplementary Methods and Supplementary Figure 3). In the X-axis, index exons are ordered from 3' to 5' of the gene. For instance, the most 3’ exon generates 22% of all projected RACEfrags. The two most 3’ exons generate 40 % of them, and so on. Adding the 5’ most index exon generates 10% of all projected RACEfrags.

Mentions: We carried out, 5’ RACE on 10 exons, evenly distributed 3’ to 5’, of 44 genes, each gene mapping to a different ENCODE region20,21 (Table 1). We used polyA+ RNA from 12 human tissues, and the RACE reactions were pooled before being hybridized to the ENCODE arrays16,17. Each pool contained 44 RACE reactions, each one originating from one exon from a different gene, and thus from a different ENCODE region. Detailed results are presented in Supplementary Results. Figure 4 displays the proportion of all RACEfrags that originate from primers in exons from the 3’ to 5’. The cumulative distribution, although inconclusive, suggest and optimal interrogation strategy, in which RACE of the most 5’ and 3’ exons is likely to give rise to a larger number of novel RACEfrags, compared with RACE of internal exons (see Supplementary Figure 3).


Efficient targeted transcript discovery via array-based normalization of RACE libraries.

Djebali S, Kapranov P, Foissac S, Lagarde J, Reymond A, Ucla C, Wyss C, Drenkow J, Dumais E, Murray RR, Lin C, Szeto D, Denoeud F, Calvo M, Frankish A, Harrow J, Makrythanasis P, Vidal M, Salehi-Ashtiani K, Antonarakis SE, Gingeras TR, Guigó R - Nat. Methods (2008)

absolute number and cumulative proportion of all (a) and novel (b) projected RACEfrags originating from index exonsA projected RACEfrag is a maximal set of RACEfrags that transitively overlap (see Supplementary Methods and Supplementary Figure 3). In the X-axis, index exons are ordered from 3' to 5' of the gene. For instance, the most 3’ exon generates 22% of all projected RACEfrags. The two most 3’ exons generate 40 % of them, and so on. Adding the 5’ most index exon generates 10% of all projected RACEfrags.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2713501&req=5

Figure 4: absolute number and cumulative proportion of all (a) and novel (b) projected RACEfrags originating from index exonsA projected RACEfrag is a maximal set of RACEfrags that transitively overlap (see Supplementary Methods and Supplementary Figure 3). In the X-axis, index exons are ordered from 3' to 5' of the gene. For instance, the most 3’ exon generates 22% of all projected RACEfrags. The two most 3’ exons generate 40 % of them, and so on. Adding the 5’ most index exon generates 10% of all projected RACEfrags.
Mentions: We carried out, 5’ RACE on 10 exons, evenly distributed 3’ to 5’, of 44 genes, each gene mapping to a different ENCODE region20,21 (Table 1). We used polyA+ RNA from 12 human tissues, and the RACE reactions were pooled before being hybridized to the ENCODE arrays16,17. Each pool contained 44 RACE reactions, each one originating from one exon from a different gene, and thus from a different ENCODE region. Detailed results are presented in Supplementary Results. Figure 4 displays the proportion of all RACEfrags that originate from primers in exons from the 3’ to 5’. The cumulative distribution, although inconclusive, suggest and optimal interrogation strategy, in which RACE of the most 5’ and 3’ exons is likely to give rise to a larger number of novel RACEfrags, compared with RACE of internal exons (see Supplementary Figure 3).

Bottom Line: Random clone selection from the RACE mixture, however, is an ineffective sampling strategy if the dynamic range of transcript abundances is large.This approach, RACEarray, is superior to direct cloning and sequencing of RACE products because it specifically targets new transcripts and often results in overall normalization of transcript abundance.We show theoretically and experimentally that this strategy leads indeed to efficient sampling of new transcripts, and we investigated multiplexing the strategy by pooling RACE reactions from multiple interrogated loci before hybridization.

View Article: PubMed Central - PubMed

Affiliation: Grup de Recerca en Informàtica Biomèdica, Institut Municipal d'Investigació Mèdica/Universitat Pompeu Fabra, Dr. Aiguader 88, 08003 Barcelona, Spain.

ABSTRACT
Rapid amplification of cDNA ends (RACE) is a widely used approach for transcript identification. Random clone selection from the RACE mixture, however, is an ineffective sampling strategy if the dynamic range of transcript abundances is large. To improve sampling efficiency of human transcripts, we hybridized the products of the RACE reaction onto tiling arrays and used the detected exons to delineate a series of reverse-transcriptase (RT)-PCRs, through which the original RACE transcript population was segregated into simpler transcript populations. We independently cloned the products and sequenced randomly selected clones. This approach, RACEarray, is superior to direct cloning and sequencing of RACE products because it specifically targets new transcripts and often results in overall normalization of transcript abundance. We show theoretically and experimentally that this strategy leads indeed to efficient sampling of new transcripts, and we investigated multiplexing the strategy by pooling RACE reactions from multiple interrogated loci before hybridization.

Show MeSH