A New Method and Application of PacBio Sequencing for Low Copy and Difficulty Preparation Plasmids

Author(s): Xiaoshu Ma, Yusha Wang, Ruikai Jia, Hua Ye.

The Single Molecule Real Time (SMRT) system developed by Pacific Biosciences applies the principle of synthesis while sequencing and uses the SMRT chip as the sequencing carrier. The high starting amount and good integrity of DNA required by PacBio library construction has always been a headache. Generally, the total loading volume of PacBio sequencing is 10μg, and the concentration is not less than 200ng/μL, which is a huge challenge for low-copy plasmids. Tight plasmid means that when the bacterial chromosome replicates once, the plasmid replicates once, and each bacterium only contains 1 to 2 plasmids. The replicon of the plasmid determines the copy number of the plasmid. Low copy plasmids should produce 0.2-1 μg DNA per ml of LB culture. Low-copy plasmids play a key role in gene synthesis. When vectors are used for expression or other purposes, low-copy plasmids are used to reduce resource consumption caused by plasmid expansion. Low-copy plasmids are also used when plasmids have lethal gene clones. In order to solve the problem of low copy plasmid library database construction in PacBio, we used Phi29 polymerase to perform multiple substitution amplification of low copy plasmid, so as to obtain a large number of high molecular weight DNA to meet the computer requirements of PacBio. In addition, this study also established a PacBio sequencing method for bacterial fluids without the need for plasmid extraction steps, thereby reducing time and money costs.