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PhyloPrimer: a taxon-specific oligonucleotide design platform
Gilda Varliero1, Jared Wray1, Cédric Malandain2
1School of Biological Sciences, University of Bristol, Bristol, UK.
Peerj
|May 14, 2021
Summary
PhyloPrimer is a new platform that semi-automates the design of specific DNA primers for microbial detection. It improves accuracy by comparing gene sequences and validating primers computationally, aiding biomonitoring studies.
Area of Science:
- Microbiology
- Bioinformatics
- Molecular Biology
Background:
- Environmental and biomedical studies often require detecting specific organisms or gene functions in microbiome samples.
- Targeted PCR (polymerase chain reaction) is suitable for hypotheses involving a small number of genes, but primer design in complex microbial communities is challenging and can yield non-specific results.
Purpose of the Study:
- To introduce PhyloPrimer, a user-friendly platform for semi-automating the design of taxon-specific oligonucleotide (oligo) primers for genes of interest.
- To enhance primer specificity by employing a positive/negative consensus approach, maximizing differences between targeted and non-targeted gene sequences.
Main Methods:
- PhyloPrimer retrieves and aligns GenBank gene sequences, visualizing relationships via a dynamic tree.
- It designs oligos based on selected sequences and uses non-selected sequences to maximize specificity.
- Oligos are checked for secondary structures using nearest-neighbor calculations and for off-target matches via in silico PCR, including degenerate bases.
Main Results:
- The platform was validated using microbial mock communities to design genus- and species-specific primers for detecting Streptococcus species.
- PhyloPrimer demonstrated effective performance in designing specific primers for microbial detection in mock communities.
- The software also supports designing non-taxonomically specific oligos, designing from preselected sequences, and checking predesigned oligos.
Conclusions:
- PhyloPrimer is the first platform to semi-automate taxon-specific oligo design, significantly improving primer specificity for microbial detection.
- The tool addresses the challenge of primer design in complex microbial communities, offering validation through computational methods.
- PhyloPrimer is a valuable resource for biomonitoring and detection studies, enhancing the accuracy of PCR-based microbial analysis.
Keywords:
Bioinformatics pipelineConsensus sequenceEnvironmental sampleOligo designPCRPrimersTaxon specificity
