ThermoAlign

ThermoAlign encodes DNA hybridization thermodynamics and evaluates full-length oligonucleotide-template alignments genome-wide to automate the design of specific primers for targeted resequencing.

Key Features:

• Thermodynamic encoding: Encodes DNA hybridization thermodynamics for oligonucleotide binding prediction.
• Thermoalignment: Evaluates thermodynamic properties of full-length oligonucleotide-template alignments across the genome to predict primer specificity.
• Automated oligonucleotide design: Generates primer and oligonucleotide candidates for targeted resequencing applications.
• Repeat-genome handling: Identifies target-specific oligonucleotides in complex, high-repeat genomes, demonstrated with maize (~85% repeat content).
• PCR and resequencing support: Predicts primers compatible with standard PCR, long-range PCR, and amplicon resequencing workflows.
• Directed graph amplicon tiling: Uses directed graph analysis to determine minimum amplicon tiling paths when single primer-pair amplification is not possible.

Scientific Applications:

• Targeted resequencing: Design of primers and oligonucleotides for focused resequencing of genomic regions.
• Primer specificity assessment: Prediction and selection of primers with minimized off-target binding in genome-wide contexts.
• Resequencing in repetitive genomes: Enabling amplification and resequencing of regions within highly repetitive genomes such as maize.
• Amplicon tiling for large regions: Planning minimum-coverage amplicon tiling paths for contiguous region resequencing when single amplicons cannot span targets.

Methodology:

Encodes DNA hybridization thermodynamics; performs thermoalignment by evaluating thermodynamic properties of full-length oligonucleotide-template alignments across the genome; applies directed graph analysis to compute minimum amplicon tiling paths; automates oligonucleotide/primer candidate generation for targeted resequencing.