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Improved single-cell genome amplification by a high-efficiency phi29 DNA polymerase

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 Improv...

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Autores principales: Zhang, Jia, Su, Xiaolu, Wang, Yefei, Wang, Xiaohang, Zhou, Shiqi, Jia, Hui, Jing, Xiaoyan, Gong, Yanhai, Wang, Jichao, Xu, Jian
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10347390/
https://www.ncbi.nlm.nih.gov/pubmed/37456715
http://dx.doi.org/10.3389/fbioe.2023.1233856
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author Zhang, Jia
Su, Xiaolu
Wang, Yefei
Wang, Xiaohang
Zhou, Shiqi
Jia, Hui
Jing, Xiaoyan
Gong, Yanhai
Wang, Jichao
Xu, Jian
author_facet Zhang, Jia
Su, Xiaolu
Wang, Yefei
Wang, Xiaohang
Zhou, Shiqi
Jia, Hui
Jing, Xiaoyan
Gong, Yanhai
Wang, Jichao
Xu, Jian
author_sort Zhang, Jia
collection PubMed
description 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.
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spelling pubmed-103473902023-07-15 Improved single-cell genome amplification by a high-efficiency phi29 DNA polymerase Zhang, Jia Su, Xiaolu Wang, Yefei Wang, Xiaohang Zhou, Shiqi Jia, Hui Jing, Xiaoyan Gong, Yanhai Wang, Jichao Xu, Jian Front Bioeng Biotechnol Bioengineering and Biotechnology 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. Frontiers Media S.A. 2023-06-29 /pmc/articles/PMC10347390/ /pubmed/37456715 http://dx.doi.org/10.3389/fbioe.2023.1233856 Text en Copyright © 2023 Zhang, Su, Wang, Wang, Zhou, Jia, Jing, Gong, Wang and Xu. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Bioengineering and Biotechnology
Zhang, Jia
Su, Xiaolu
Wang, Yefei
Wang, Xiaohang
Zhou, Shiqi
Jia, Hui
Jing, Xiaoyan
Gong, Yanhai
Wang, Jichao
Xu, Jian
Improved single-cell genome amplification by a high-efficiency phi29 DNA polymerase
title Improved single-cell genome amplification by a high-efficiency phi29 DNA polymerase
title_full Improved single-cell genome amplification by a high-efficiency phi29 DNA polymerase
title_fullStr Improved single-cell genome amplification by a high-efficiency phi29 DNA polymerase
title_full_unstemmed Improved single-cell genome amplification by a high-efficiency phi29 DNA polymerase
title_short Improved single-cell genome amplification by a high-efficiency phi29 DNA polymerase
title_sort improved single-cell genome amplification by a high-efficiency phi29 dna polymerase
topic Bioengineering and Biotechnology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10347390/
https://www.ncbi.nlm.nih.gov/pubmed/37456715
http://dx.doi.org/10.3389/fbioe.2023.1233856
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