A circular consensus sequencing (CCS) strategy involving single molecule, real-time (SMRT) DNA sequencing technology was applied to de novo assembly and single nucleotide polymorphism (SNP) detection of chloroplast genomes. Chloroplast DNA was purified from enriched chloroplasts of pooled individuals to construct a shotgun library for each species. The sequencing reactions were performed on a PacBio RS platform. CCS sub-reads were generated from polymerase reads that passed the native dumbbell-shaped DNA templates multiple times. The complete chloroplast genome sequence was generated by mapping all reads to the draft sequence constructed in a step-by-step manner. The full-chain, PCR-free approach eliminates the possible context-specific biases in library construction and sequencing reaction. The chloroplast genome was easily and completely assembled using the data generated from one SMRT Cell without requiring a reference genome. Comparisons of the three assembled Fritillaria genomes to 34.1 kb of validation Sanger sequences revealed 100% concordance, and the detected intraspecies SNPs at a minimum variant frequency of 15% were all confirmed. This simple approach with potential for parallel sequencing yields high-quality chloroplast genomes for sensitive SNP detection and comparative analyses. We recommend this approach for its powerful applicability for evolutionary genetics and genomics studies in plants based on the sequences of chloroplast genomes.
Keywords: Fritillaria; chloroplast genome; circular consensus sequencing (CCS); single molecular real-time (SMRT) sequencing; single nucleotide polymorphism (SNP).
© 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.