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AlignMiner: a Web-based tool for detection of divergent regions in multiple sequence alignments of conserved sequences.

Guerrero D, Bautista R, Villalobos DP, Cantón FR, Claros MG - Algorithms Mol Biol (2010)

Bottom Line: It accepts alignments (protein or nucleic acid) obtained using any of a variety of algorithms, which does not appear to have a significant impact on the final results.Users do not need to wait until execution is complete and can.even inspect their results on a different computer.In silico and experimental proof-of-concept cases have shown that AlignMiner can be successfully used to designing specific polymerase chain reaction primers as well as potential epitopes for antibodies.

View Article: PubMed Central - HTML - PubMed

Affiliation: Plataforma Andaluza de Bioinformática (Universidad de Málaga), Severo Ochoa, 34, 29590 Málaga, Spain. claros@uma.es.

ABSTRACT

Background: Multiple sequence alignments are used to study gene or protein function, phylogenetic relations, genome evolution hypotheses and even gene polymorphisms. Virtually without exception, all available tools focus on conserved segments or residues. Small divergent regions, however, are biologically important for specific quantitative polymerase chain reaction, genotyping, molecular markers and preparation of specific antibodies, and yet have received little attention. As a consequence, they must be selected empirically by the researcher. AlignMiner has been developed to fill this gap in bioinformatic analyses.

Results: AlignMiner is a Web-based application for detection of conserved and divergent regions in alignments of conserved sequences, focusing particularly on divergence. It accepts alignments (protein or nucleic acid) obtained using any of a variety of algorithms, which does not appear to have a significant impact on the final results. AlignMiner uses different scoring methods for assessing conserved/divergent regions, Entropy being the method that provides the highest number of regions with the greatest length, and Weighted being the most restrictive. Conserved/divergent regions can be generated either with respect to the consensus sequence or to one master sequence. The resulting data are presented in a graphical interface developed in AJAX, which provides remarkable user interaction capabilities. Users do not need to wait until execution is complete and can.even inspect their results on a different computer. Data can be downloaded onto a user disk, in standard formats. In silico and experimental proof-of-concept cases have shown that AlignMiner can be successfully used to designing specific polymerase chain reaction primers as well as potential epitopes for antibodies. Primer design is assisted by a module that deploys several oligonucleotide parameters for designing primers "on the fly".

Conclusions: AlignMiner can be used to reliably detect divergent regions via several scoring methods that provide different levels of selectivity. Its predictions have been verified by experimental means. Hence, it is expected that its usage will save researchers' time and ensure an objective selection of the best-possible divergent region when closely related sequences are analysed. AlignMiner is freely available at http://www.scbi.uma.es/alignminer.

No MeSH data available.


Use of AlignMiner for designing several specific primer pairs for PCR amplification of the different isoforms of the AtGS1 nucleotide sequence (A) The 5' and 3' divergent regions obtained with Entropy that were selected for primer design including the characteristic parameters of each region. (B) Results of the in silico "PCR amplification" with BioPHP [34] using the different primer pairs. Note that the actual 3' primers are complementary to the sequences shown on the right.
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Figure 6: Use of AlignMiner for designing several specific primer pairs for PCR amplification of the different isoforms of the AtGS1 nucleotide sequence (A) The 5' and 3' divergent regions obtained with Entropy that were selected for primer design including the characteristic parameters of each region. (B) Results of the in silico "PCR amplification" with BioPHP [34] using the different primer pairs. Note that the actual 3' primers are complementary to the sequences shown on the right.

Mentions: AlignMiner can be used for selecting specific PCR primers that serve to discriminate among closely-related sequences. As an example, divergent regions were obtained for the five A. thaliana GS1 isoforms (AtGS1 in Table 1). Since all the scoring methods produce similar results for these sequences (Figure 3), the MSA was inspected with DNAW. The resulting divergent regions were sorted by decreasing score and the best regions (scores 0.223 and 0.024) were selected for primer design (Figure 6A and Table 2) with the help of the primer tool. These primers were shown to selectively amplify each isoform of GS1 in silico (Figure 6B), as revealed by "PCR amplification" of the BioPHP suite [35].


AlignMiner: a Web-based tool for detection of divergent regions in multiple sequence alignments of conserved sequences.

Guerrero D, Bautista R, Villalobos DP, Cantón FR, Claros MG - Algorithms Mol Biol (2010)

Use of AlignMiner for designing several specific primer pairs for PCR amplification of the different isoforms of the AtGS1 nucleotide sequence (A) The 5' and 3' divergent regions obtained with Entropy that were selected for primer design including the characteristic parameters of each region. (B) Results of the in silico "PCR amplification" with BioPHP [34] using the different primer pairs. Note that the actual 3' primers are complementary to the sequences shown on the right.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2902484&req=5

Figure 6: Use of AlignMiner for designing several specific primer pairs for PCR amplification of the different isoforms of the AtGS1 nucleotide sequence (A) The 5' and 3' divergent regions obtained with Entropy that were selected for primer design including the characteristic parameters of each region. (B) Results of the in silico "PCR amplification" with BioPHP [34] using the different primer pairs. Note that the actual 3' primers are complementary to the sequences shown on the right.
Mentions: AlignMiner can be used for selecting specific PCR primers that serve to discriminate among closely-related sequences. As an example, divergent regions were obtained for the five A. thaliana GS1 isoforms (AtGS1 in Table 1). Since all the scoring methods produce similar results for these sequences (Figure 3), the MSA was inspected with DNAW. The resulting divergent regions were sorted by decreasing score and the best regions (scores 0.223 and 0.024) were selected for primer design (Figure 6A and Table 2) with the help of the primer tool. These primers were shown to selectively amplify each isoform of GS1 in silico (Figure 6B), as revealed by "PCR amplification" of the BioPHP suite [35].

Bottom Line: It accepts alignments (protein or nucleic acid) obtained using any of a variety of algorithms, which does not appear to have a significant impact on the final results.Users do not need to wait until execution is complete and can.even inspect their results on a different computer.In silico and experimental proof-of-concept cases have shown that AlignMiner can be successfully used to designing specific polymerase chain reaction primers as well as potential epitopes for antibodies.

View Article: PubMed Central - HTML - PubMed

Affiliation: Plataforma Andaluza de Bioinformática (Universidad de Málaga), Severo Ochoa, 34, 29590 Málaga, Spain. claros@uma.es.

ABSTRACT

Background: Multiple sequence alignments are used to study gene or protein function, phylogenetic relations, genome evolution hypotheses and even gene polymorphisms. Virtually without exception, all available tools focus on conserved segments or residues. Small divergent regions, however, are biologically important for specific quantitative polymerase chain reaction, genotyping, molecular markers and preparation of specific antibodies, and yet have received little attention. As a consequence, they must be selected empirically by the researcher. AlignMiner has been developed to fill this gap in bioinformatic analyses.

Results: AlignMiner is a Web-based application for detection of conserved and divergent regions in alignments of conserved sequences, focusing particularly on divergence. It accepts alignments (protein or nucleic acid) obtained using any of a variety of algorithms, which does not appear to have a significant impact on the final results. AlignMiner uses different scoring methods for assessing conserved/divergent regions, Entropy being the method that provides the highest number of regions with the greatest length, and Weighted being the most restrictive. Conserved/divergent regions can be generated either with respect to the consensus sequence or to one master sequence. The resulting data are presented in a graphical interface developed in AJAX, which provides remarkable user interaction capabilities. Users do not need to wait until execution is complete and can.even inspect their results on a different computer. Data can be downloaded onto a user disk, in standard formats. In silico and experimental proof-of-concept cases have shown that AlignMiner can be successfully used to designing specific polymerase chain reaction primers as well as potential epitopes for antibodies. Primer design is assisted by a module that deploys several oligonucleotide parameters for designing primers "on the fly".

Conclusions: AlignMiner can be used to reliably detect divergent regions via several scoring methods that provide different levels of selectivity. Its predictions have been verified by experimental means. Hence, it is expected that its usage will save researchers' time and ensure an objective selection of the best-possible divergent region when closely related sequences are analysed. AlignMiner is freely available at http://www.scbi.uma.es/alignminer.

No MeSH data available.