Browsing by Author "Wisser, RJ"
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Item High-Throughput Resequencing of Maize Landraces at Genomic Regions Associated with Flowering Time(Public Library of Science, 2017-01-03) Jamann, Tiffany M; Sood, Shilpa; Wisser, Randall J; Holland, James B; Tiffany M. Jamann; Shilpa Sood; Randall J. Wisser; James B. Holland; Wisser, RJDespite the reduction in the price of sequencing, it remains expensive to sequence and assemble whole, complex genomes of multiple samples for population studies, particularly for large genomes like those of many crop species. Enrichment of target genome regions coupled with next generation sequencing is a cost-effective strategy to obtain sequence information for loci of interest across many individuals, providing a less expensive approach to evaluating sequence variation at the population scale. Here we evaluate amplicon-based enrichment coupled with semiconductor sequencing on a validation set consisting of three maize inbred lines, two hybrids and 19 landrace accessions. We report the use of a multiplexed panel of 319 PCR assays that target 20 candidate loci associated with photoperiod sensitivity in maize while requiring 25 ng or less of starting DNA per sample. Enriched regions had an average on-target sequence read depth of 105 with 98% of the sequence data mapping to the maize ‘B73’ reference and 80% of the reads mapping to the target interval. Sequence reads were aligned to B73 and 1,486 and 1,244 variants were called using SAMtools and GATK, respectively. Of the variants called by both SAMtools and GATK, 30% were not previously reported in maize. Due to the high sequence read depth, heterozygote genotypes could be called with at least 92.5% accuracy in hybrid materials using GATK. The genetic data are congruent with previous reports of high total genetic diversity and substantial population differentiation among maize landraces. In conclusion, semiconductor sequencing of highly multiplexed PCR reactions is a cost-effective strategy for resequencing targeted genomic loci in diverse maize materials.Item ThermoAlign: a genome-aware primer design tool for tiled amplicon resequencing(Nature Publishing Group, 2017-03-16) Francis, Felix; Dumas, Michael D.; Wisser, RJ; Felix Francis, Michael D. Dumas and Randall J.Wisser; Wisser, Randall JeromeIsolating 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.