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.

Keywords

• de novo assembly
• DNA probes
• long‐range PCR
• MinION
• whole plastome sequencing