ThermoAlign: a genome-aware primer design tool for tiled amplicon resequencing
Date
2017-03-16
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Nature Publishing Group
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 amplificationbased
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.
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Citation
Francis, F. et al. ThermoAlign: a genome-aware primer design tool for tiled amplicon resequencing. Sci. Rep. 7, 44437; doi: 10.1038/srep44437 (2017)