AUTHOR=Zhang Jia , Su Xiaolu , Wang Yefei , Wang Xiaohang , Zhou Shiqi , Jia Hui , Jing Xiaoyan , Gong Yanhai , Wang Jichao , Xu Jian TITLE=Improved single-cell genome amplification by a high-efficiency phi29 DNA polymerase JOURNAL=Frontiers in Bioengineering and Biotechnology VOLUME=11 YEAR=2023 URL=https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2023.1233856 DOI=10.3389/fbioe.2023.1233856 ISSN=2296-4185 ABSTRACT=

Single-cell genomic whole genome amplification (WGA) is a crucial step in single-cell sequencing, yet its low amplification efficiency, incomplete and uneven genome amplification still hinder the throughput and efficiency of single-cell sequencing workflows. Here we introduce a process called Improved Single-cell Genome Amplification (iSGA), in which the whole single-cell sequencing cycle is completed in a high-efficient and high-coverage manner, through phi29 DNA polymerase engineering and process engineering. By establishing a disulfide bond of F137C-A377C, the amplification ability of the enzyme was improved to that of single-cell. By further protein engineering and process engineering, a supreme enzyme named HotJa Phi29 DNA Polymerase was developed and showed significantly better coverage (99.75%) at a higher temperature (40°C). High single-cell genome amplification ability and high coverage (93.59%) were also achieved for commercial probiotic samples. iSGA is more efficient and robust than the wild-type phi29 DNA polymerase, and it is 2.03-fold more efficient and 10.89-fold cheaper than the commercial Thermo Scientific EquiPhi29 DNA Polymerase. These advantages promise its broad applications in large-scale single-cell sequencing.