Comparison among three methods for obtaining chloroplast genome sequences from the conifer Pinus massoniana

The chloroplast genome (CPG) is a powerful tool for phylogenetic studies. Many CPGs have been determined using NGS. However, the large nuclear-genome and difficult CPG-DNA separation in conifers limit their application in related research. In this study, three methods (PCR + Sanger, PCR + HiSeq, cpDNA+HiSeq) for obtaining the CPGs of Pinus massoniana were compared for sequence accuracy, time and cost. PCR + Sanger obtained the most accurate CPGs with advantages in cost (3.08$/kb) and time (2-3 days); PCR + HiSeq generated some DNA fragments with low depth, and the SNPs false-positive-rate (0.44) and sequencing error-rate (0.0265) of this method were higher than those of the cpDNA+HiSeq. Moreover, the cost (~6.17$/kb) and time (4-5 weeks) would significantly increase when HiSeq sequencing were outsourced to sequencing service company. Thus, for the study of intraspecific and interspecies variation in CPGs, CPG sequences can be obtained by comprehensive methods to bridge the method shortcomings. Scuh as sequence accuracy, cost and time.