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Long-fragment targeted capture for long-read sequencing of plastomes

Abstract:

Premise: Third-generation sequencing methods generate significantly longer reads than those produced using alternative sequencing methods. This provides increased possibilities for the study of biodiversity, phylogeography, and population genetics. We developed a protocol for in-solution enrichment hybridization capture of long DNA fragments applicable to complete plastid genomes. Methods and Results: The protocol uses cost-effective in-house probes developed via long-range PCR and was used in six non-model monocot species (Poaceae: African rice, pearl millet, fonio; and three palm species). DNA was extracted from fresh and silica gel–dried leaves. Our protocol successfully captured long-read plastome fragments (3151 bp median on average), with an enrichment rate ranging from 15% to 98%. DNA extracted from silica gel–dried leaves led to low-quality plastome assemblies when compared to DNA extracted from fresh tissue. Conclusions: Our protocol could also be generalized to capture long sequences from specific nuclear fragments.