High-Accuracy de novo Assembly and SNP Detection of Chloroplast Genomes using a SMRT Circular Consensus Sequencing Strategy

A circular consensus sequencing (CCS) strategy involving single molecule, real-time (SMRT) DNA sequencing was applied to de novo assembly and SNP detection of chloroplast genomes.

Chloroplast DNA was extracted and sheared from enriched chloroplasts of pooled individuals to construct a short-insert shotgun library for each target species. The sequencing reactions were performed on a PacBio RS platform. Circular consensus sub-reads were generated from long polymerase reads that passed the dumbbell-shaped DNA templates (SMRTbell) with ligated hairpin adaptors multiple times. The complete sequence of the chloroplast genome was generated by mapping all reads to the draft sequence constructed in a step-by-step manner.The chloroplast genome was completely assembled using the data generated from one SMRT Cell without requiring a closely related reference genome. Comparisons of the three assembled 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 sequencing approach yields high-quality chloroplast genomes for accurate prediction of SNP 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.