Limits...
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.

Show MeSH
strategy for comprehensive characterization of novel isoforms from annotated genesFirst, RACE (5’, 3’ or both) is performed with primers (black arrows) from one or more annotated exons of known loci. Second, the RACE products are hybridized onto a tiling array, possibly across different cell types and conditions. Third, the detected sites of transcription (RACEfrags, in yellow in the figure) are used to design RT-PCR primers (black arrows). Primers are designed only on RACEfrags corresponding to previously undetected exons. Fourth, one RT-PCR reaction is performed for each primer in a novel RACEfrag, using the original RACE primer as the second primer. Fith, each RT-PCR reaction is cloned separately into a mini-pool. Finally, clones are randomly selected from the RT-PCR mini-pools and sequenced, leading to the identification of novel transcripts.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2713501&req=5

Figure 1: strategy for comprehensive characterization of novel isoforms from annotated genesFirst, RACE (5’, 3’ or both) is performed with primers (black arrows) from one or more annotated exons of known loci. Second, the RACE products are hybridized onto a tiling array, possibly across different cell types and conditions. Third, the detected sites of transcription (RACEfrags, in yellow in the figure) are used to design RT-PCR primers (black arrows). Primers are designed only on RACEfrags corresponding to previously undetected exons. Fourth, one RT-PCR reaction is performed for each primer in a novel RACEfrag, using the original RACE primer as the second primer. Fith, each RT-PCR reaction is cloned separately into a mini-pool. Finally, clones are randomly selected from the RT-PCR mini-pools and sequenced, leading to the identification of novel transcripts.

Mentions: Figure 1 schematizes the RACEarray strategy (Supplementary Figs 1, 2, Supplementary Methods). Given a locus, we first select the exons in which the RACE primers will be designed (Supplementary Fig 3), and carry out the RACE reactions. Second, we hybridize the RACE products onto tiling arrays, and we build the sites of transcription, the so-called RACEfrags (RACE positive fragments)16, from the probe hybridization intensities. A number of filters can be applied to RACEfrags to account for highly expressed genes in the original RNA source, or to identify RACEfrags produced by the amplification of non-targeted loci (Supplementary Fig 4). If RACE reactions from multiple primers have been pooled together before hybridization, complex assignment procedures may need to be employed to assign RACEfrags to interrogated primers. Third, we use the resulting RACEfrags (Supplementary Fig 5) to delineate RT-PCR reactions. One of the primers for each of the reactions is the original RACE primer, and the second primer is designed within each novel RACEfrag. Strategies based on the pattern of co-occurrence of RACEfrags across different assayed conditions can be designed to select the subset of RACEfrags maximizing transcript discovery (Supplementary Fig. 6). Fourth, we clone the products of the resulting RT-PCR into “mini-pools”: each mini-pool contains the amplified transcripts connecting an index exon with a novel RACEfrag. Finally, we randomly select clones from these pools for sequencing.


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)

strategy for comprehensive characterization of novel isoforms from annotated genesFirst, RACE (5’, 3’ or both) is performed with primers (black arrows) from one or more annotated exons of known loci. Second, the RACE products are hybridized onto a tiling array, possibly across different cell types and conditions. Third, the detected sites of transcription (RACEfrags, in yellow in the figure) are used to design RT-PCR primers (black arrows). Primers are designed only on RACEfrags corresponding to previously undetected exons. Fourth, one RT-PCR reaction is performed for each primer in a novel RACEfrag, using the original RACE primer as the second primer. Fith, each RT-PCR reaction is cloned separately into a mini-pool. Finally, clones are randomly selected from the RT-PCR mini-pools and sequenced, leading to the identification of novel transcripts.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: strategy for comprehensive characterization of novel isoforms from annotated genesFirst, RACE (5’, 3’ or both) is performed with primers (black arrows) from one or more annotated exons of known loci. Second, the RACE products are hybridized onto a tiling array, possibly across different cell types and conditions. Third, the detected sites of transcription (RACEfrags, in yellow in the figure) are used to design RT-PCR primers (black arrows). Primers are designed only on RACEfrags corresponding to previously undetected exons. Fourth, one RT-PCR reaction is performed for each primer in a novel RACEfrag, using the original RACE primer as the second primer. Fith, each RT-PCR reaction is cloned separately into a mini-pool. Finally, clones are randomly selected from the RT-PCR mini-pools and sequenced, leading to the identification of novel transcripts.
Mentions: Figure 1 schematizes the RACEarray strategy (Supplementary Figs 1, 2, Supplementary Methods). Given a locus, we first select the exons in which the RACE primers will be designed (Supplementary Fig 3), and carry out the RACE reactions. Second, we hybridize the RACE products onto tiling arrays, and we build the sites of transcription, the so-called RACEfrags (RACE positive fragments)16, from the probe hybridization intensities. A number of filters can be applied to RACEfrags to account for highly expressed genes in the original RNA source, or to identify RACEfrags produced by the amplification of non-targeted loci (Supplementary Fig 4). If RACE reactions from multiple primers have been pooled together before hybridization, complex assignment procedures may need to be employed to assign RACEfrags to interrogated primers. Third, we use the resulting RACEfrags (Supplementary Fig 5) to delineate RT-PCR reactions. One of the primers for each of the reactions is the original RACE primer, and the second primer is designed within each novel RACEfrag. Strategies based on the pattern of co-occurrence of RACEfrags across different assayed conditions can be designed to select the subset of RACEfrags maximizing transcript discovery (Supplementary Fig. 6). Fourth, we clone the products of the resulting RT-PCR into “mini-pools”: each mini-pool contains the amplified transcripts connecting an index exon with a novel RACEfrag. Finally, we randomly select clones from these pools for sequencing.

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