Labor- and cost-effective long-read amplicon sequencing using a plasmid analysis service: Application to transposon-inserted alleles in the Japanese morning glory

Sequencing PCR fragments amplified from specific regions of genomes is a fundamental technique in molecular genetics. Sanger sequencing is commonly used for the analysis; however, amplicon sequencing utilizing next-generation sequencing has also become widespread. Additionally, long-read amplicon sequencing, using Nanopore or PacBio sequencers to analyze long PCR fragments, has emerged, although it often remains more expensive than Sanger sequencing. Recently, low-cost commercial services for full-length plasmid DNA sequencing using Nanopore sequencers have been launched in several countries, including Japan. This study explored the potential of these services for sequencing long PCR fragments without the need for cloning into plasmid DNA. PCR fragments of 4-11 kb, amplified from the DFR-B gene involved in anthocyanin biosynthesis, with or without Tpn1 transposons in Japanese morning glory, were circularized using T4 ligase and analyzed as templates. Although some inaccuracies in the length of homopolymer stretches were observed, the remaining sequences were obtained without significant errors. This method could potentially reduce the labor and costs associated with cloning, primer synthesis, and sequence assembly, making it a viable option for the analysis of long PCR fragment sequences.