title: ThermoAlign: a genome-aware primer design tool for tiled amplicon resequencing.
abstract: Isolating and sequencing specific regions in a genome is a cornerstone of molecular biology. This has been facilitated by computationally encoding the thermodynamics of DNA hybridization for automated design of hybridization and priming oligonucleotides. However, the repetitive composition of genomes challenges the identification of target-specific oligonucleotides, which limits genetics and genomics research on many species. Here, a tool called ThermoAlign was developed that ensures the design of target-specific primer pairs for DNA amplification. This is achieved by evaluating the thermodynamics of hybridization for full-length oligonucleotide-template alignments - thermoalignments - across the genome to identify primers predicted to bind specifically to the target site. For amplification-based resequencing of regions that cannot be amplified by a single primer pair, a directed graph analysis method is used to identify minimum amplicon tiling paths. Laboratory validation by standard and long-range polymerase chain reaction and amplicon resequencing with maize, one of the most repetitive genomes sequenced to date (≈85% repeat content), demonstrated the specificity-by-design functionality of ThermoAlign. ThermoAlign is released under an open source license and bundled in a dependency-free container for wide distribution. It is anticipated that this tool will facilitate multiple applications in genetics and genomics and be useful in the workflow of high-throughput targeted resequencing studies.
authors.affiliation: Center for Bioinformatics and Computational Biology, University of Delaware, Newark, DE 19714, USA.|Department of Plant and Soil Sciences, University of Delaware, Newark, DE 19716-1304, USA.
authors.forename: Felix
authors.initials: F
authors.lastname: Francis
authors.affiliation: Department of Plant and Soil Sciences, University of Delaware, Newark, DE 19716-1304, USA.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Dumas
authors.affiliation: Department of Plant and Soil Sciences, University of Delaware, Newark, DE 19716-1304, USA.
authors.forename: Randall J
authors.initials: RJ
authors.lastname: Wisser
mesh_terms: D001483:Base Sequence; D017931:DNA Primers / Q000138:chemical synthesis / Q000378:metabolism*; D018745:Genome, Plant; D059014:High-Throughput Nucleotide Sequencing; D018895:Microsatellite Repeats; D009693:Nucleic Acid Hybridization / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA / Q000379:methods*; D012984:Software; D013816:Thermodynamics; D003313:Zea mays / Q000235:genetics*
chemical_list: D017931:DNA Primers