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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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distribution of distances of RACEfrags to assigned index exonsThe blue bars reflect the frequency of extension lengths among different length classes. The solid yellow line shows the cumulative frequency of extensions of that length or greater. Most of the RACEfrags map within 1MB of the index primer. The pick at 10MB could reflect RACEfrags 3’ to the 5’ index primer (originated by circularization of the cDNA); note that the average distance between pooled primers is about 10MB.
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Figure 5: distribution of distances of RACEfrags to assigned index exonsThe blue bars reflect the frequency of extension lengths among different length classes. The solid yellow line shows the cumulative frequency of extensions of that length or greater. Most of the RACEfrags map within 1MB of the index primer. The pick at 10MB could reflect RACEfrags 3’ to the 5’ index primer (originated by circularization of the cDNA); note that the average distance between pooled primers is about 10MB.

Mentions: We conducted 5’ RACE on 96 genes in human chromosomes 21 and 22 (Table 1). Reactions were carried out individually on polyA+ RNA from 12 different tissues and subsequently pooled. RACE reactions from different tissues originating from genes each separated by 10 Mb were pooled in groups of 6 on the same chip. Results (Supplementary Results and Figure 5) show that transcripts may span very large genomic space, with about 50% of the RACEfrags more than 3MB away from the index gene. These results need further validation, but they could potentially challenge our current understanding of the structure and organization of transcripts encoded in the human genome, suggesting that distal regions may be connected into individual transcripts more often than previously expected. They also make very challenging the delineation of an effective pooling strategy since only a very sparse pooling appears to guarantee a robust assignment of RACEfrags to primers.


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)

distribution of distances of RACEfrags to assigned index exonsThe blue bars reflect the frequency of extension lengths among different length classes. The solid yellow line shows the cumulative frequency of extensions of that length or greater. Most of the RACEfrags map within 1MB of the index primer. The pick at 10MB could reflect RACEfrags 3’ to the 5’ index primer (originated by circularization of the cDNA); note that the average distance between pooled primers is about 10MB.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: distribution of distances of RACEfrags to assigned index exonsThe blue bars reflect the frequency of extension lengths among different length classes. The solid yellow line shows the cumulative frequency of extensions of that length or greater. Most of the RACEfrags map within 1MB of the index primer. The pick at 10MB could reflect RACEfrags 3’ to the 5’ index primer (originated by circularization of the cDNA); note that the average distance between pooled primers is about 10MB.
Mentions: We conducted 5’ RACE on 96 genes in human chromosomes 21 and 22 (Table 1). Reactions were carried out individually on polyA+ RNA from 12 different tissues and subsequently pooled. RACE reactions from different tissues originating from genes each separated by 10 Mb were pooled in groups of 6 on the same chip. Results (Supplementary Results and Figure 5) show that transcripts may span very large genomic space, with about 50% of the RACEfrags more than 3MB away from the index gene. These results need further validation, but they could potentially challenge our current understanding of the structure and organization of transcripts encoded in the human genome, suggesting that distal regions may be connected into individual transcripts more often than previously expected. They also make very challenging the delineation of an effective pooling strategy since only a very sparse pooling appears to guarantee a robust assignment of RACEfrags to primers.

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