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Improved coverage of cDNA-AFLP by sequential digestion of immobilized cDNA.

Weiberg A, Pöhler D, Morgenstern B, Karlovsky P - BMC Genomics (2008)

Bottom Line: cDNA-AFLP is a transcriptomics technique which does not require prior sequence information and can therefore be used as a gene discovery tool.Some transcripts are represented by more than one fragment while other escape detection, causing redundancy and reducing the coverage of the analysis, respectively.For A. thaliana, human and mice transcriptome, the use of two marking enzymes and three sequentially applied releasing enzymes for each of the marking enzymes is recommended.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Phytopathology and Mycotoxin Research Division, University of Goettingen, Goettingen, Germany. aweiber1@gwdg.de

ABSTRACT

Background: cDNA-AFLP is a transcriptomics technique which does not require prior sequence information and can therefore be used as a gene discovery tool. The method is based on selective amplification of cDNA fragments generated by restriction endonucleases, electrophoretic separation of the products and comparison of the band patterns between treated samples and controls. Unequal distribution of restriction sites used to generate cDNA fragments negatively affects the performance of cDNA-AFLP. Some transcripts are represented by more than one fragment while other escape detection, causing redundancy and reducing the coverage of the analysis, respectively.

Results: With the goal of improving the coverage of cDNA-AFLP without increasing its redundancy, we designed a modified cDNA-AFLP protocol. Immobilized cDNA is sequentially digested with several restriction endonucleases and the released DNA fragments are collected in mutually exclusive pools. To investigate the performance of the protocol, software tool MECS (Multiple Enzyme cDNA-AFLP Simulation) was written in Perl. cDNA-AFLP protocols described in the literature and the new sequential digestion protocol were simulated on sets of cDNA sequences from mouse, human and Arabidopsis thaliana. The redundancy and coverage, the total number of PCR reactions, and the average fragment length were calculated for each protocol and cDNA set.

Conclusion: Simulation revealed that sequential digestion of immobilized cDNA followed by the partitioning of released fragments into mutually exclusive pools outperformed other cDNA-AFLP protocols in terms of coverage, redundancy, fragment length, and the total number of PCRs. Primers generating 30 to 70 amplicons per PCR provided the highest fraction of electrophoretically distinguishable fragments suitable for normalization. For A. thaliana, human and mice transcriptome, the use of two marking enzymes and three sequentially applied releasing enzymes for each of the marking enzymes is recommended.

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Effect of sequence quality on coverage. cDNA-AFLP were simulated on RefSeq and UniGene sequences using the sequential digestion protocol with two marking and three releasing enzymes. Enzyme combinations leading to the highest coverage were selected from the set listed in Tab. 2. Black bar: UniGene; white bar: RefSeq.
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Figure 5: Effect of sequence quality on coverage. cDNA-AFLP were simulated on RefSeq and UniGene sequences using the sequential digestion protocol with two marking and three releasing enzymes. Enzyme combinations leading to the highest coverage were selected from the set listed in Tab. 2. Black bar: UniGene; white bar: RefSeq.

Mentions: While comparing cDNA-AFLP protocols, we noticed that the quality of sequence data used for the simulations affected the coverage. Comparing the results obtained with RefSeq and UniGene cDNA sets confirmed this observation in that coverage was lower for the UniGene set (Fig. 5). RefSeq are subsets of high-quality sequences from UniGene (see Tab. 1 for details). The RefSeq and UniGene data sets for A. thaliana differ mainly in the higher number of ambiguous bases in UniGene data while the average sequence lengths are similar. Mouse and human data differ much more in the average sequence length but the difference in the proportion of ambiguous nucleotides is comparable to A. thaliana data.


Improved coverage of cDNA-AFLP by sequential digestion of immobilized cDNA.

Weiberg A, Pöhler D, Morgenstern B, Karlovsky P - BMC Genomics (2008)

Effect of sequence quality on coverage. cDNA-AFLP were simulated on RefSeq and UniGene sequences using the sequential digestion protocol with two marking and three releasing enzymes. Enzyme combinations leading to the highest coverage were selected from the set listed in Tab. 2. Black bar: UniGene; white bar: RefSeq.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2577664&req=5

Figure 5: Effect of sequence quality on coverage. cDNA-AFLP were simulated on RefSeq and UniGene sequences using the sequential digestion protocol with two marking and three releasing enzymes. Enzyme combinations leading to the highest coverage were selected from the set listed in Tab. 2. Black bar: UniGene; white bar: RefSeq.
Mentions: While comparing cDNA-AFLP protocols, we noticed that the quality of sequence data used for the simulations affected the coverage. Comparing the results obtained with RefSeq and UniGene cDNA sets confirmed this observation in that coverage was lower for the UniGene set (Fig. 5). RefSeq are subsets of high-quality sequences from UniGene (see Tab. 1 for details). The RefSeq and UniGene data sets for A. thaliana differ mainly in the higher number of ambiguous bases in UniGene data while the average sequence lengths are similar. Mouse and human data differ much more in the average sequence length but the difference in the proportion of ambiguous nucleotides is comparable to A. thaliana data.

Bottom Line: cDNA-AFLP is a transcriptomics technique which does not require prior sequence information and can therefore be used as a gene discovery tool.Some transcripts are represented by more than one fragment while other escape detection, causing redundancy and reducing the coverage of the analysis, respectively.For A. thaliana, human and mice transcriptome, the use of two marking enzymes and three sequentially applied releasing enzymes for each of the marking enzymes is recommended.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Phytopathology and Mycotoxin Research Division, University of Goettingen, Goettingen, Germany. aweiber1@gwdg.de

ABSTRACT

Background: cDNA-AFLP is a transcriptomics technique which does not require prior sequence information and can therefore be used as a gene discovery tool. The method is based on selective amplification of cDNA fragments generated by restriction endonucleases, electrophoretic separation of the products and comparison of the band patterns between treated samples and controls. Unequal distribution of restriction sites used to generate cDNA fragments negatively affects the performance of cDNA-AFLP. Some transcripts are represented by more than one fragment while other escape detection, causing redundancy and reducing the coverage of the analysis, respectively.

Results: With the goal of improving the coverage of cDNA-AFLP without increasing its redundancy, we designed a modified cDNA-AFLP protocol. Immobilized cDNA is sequentially digested with several restriction endonucleases and the released DNA fragments are collected in mutually exclusive pools. To investigate the performance of the protocol, software tool MECS (Multiple Enzyme cDNA-AFLP Simulation) was written in Perl. cDNA-AFLP protocols described in the literature and the new sequential digestion protocol were simulated on sets of cDNA sequences from mouse, human and Arabidopsis thaliana. The redundancy and coverage, the total number of PCR reactions, and the average fragment length were calculated for each protocol and cDNA set.

Conclusion: Simulation revealed that sequential digestion of immobilized cDNA followed by the partitioning of released fragments into mutually exclusive pools outperformed other cDNA-AFLP protocols in terms of coverage, redundancy, fragment length, and the total number of PCRs. Primers generating 30 to 70 amplicons per PCR provided the highest fraction of electrophoretically distinguishable fragments suitable for normalization. For A. thaliana, human and mice transcriptome, the use of two marking enzymes and three sequentially applied releasing enzymes for each of the marking enzymes is recommended.

Show MeSH
Related in: MedlinePlus