Scientists at the Qingdao Institute of Bioenergy and Bioprocess Technology (QIBEBT) of the Chinese Academy of Sciences (CAS) have achieved a breakthrough in the field of bacterial single-cell whole genome sequencing. As detailed in their study, published in Frontiers in Bioengineering and Biotechnology, the team managed to considerably reduce bias in the gene amplification stage, which is crucial for the precision of the genome sequencing process.
Traditional gene sequencing methods rely on processing millions of cells simultaneously. This, while beneficial for larger scale studies, results in loss of individual cell information and can obscure research that relies on single-cell consideration. These issues are particularly prominent with bacteria, as the small size of bacterial cells results in significantly lower DNA content compared to human or animal cells.
Gene amplification, a procedure sometimes referred to as "molecular photocopying," is a fundamental step in gene sequencing. It produces millions, sometimes billions, of copies of DNA segments. However, in bacterial single-cell DNA amplification, the process can generate biases that distort the representation of different genome regions, due to the limited amount of DNA available for copying.
Aiming to reduce such bias, lead author ZHANG Jia and his team have introduced a process termed Improved Single-cell Genome Amplification (iSGA). The iSGA process improves the efficiency and robustness of the phi29 DNA polymerase—a protein widely used in single-cell whole genome amplification—through protein engineering and process engineering. However, the traditional use of this protein has been plagued by low genome coverage and efficiency issues.
Through a method known as compartmentalized self-replication (CSR), the team enhanced the structure of the phi29 DNA polymerase, optimizing its activity, specificity, and stability. Alongside modifying the amplification buffer, the group also devised a more efficient decontamination process to further decrease contamination during amplification. The newly developed HotJa Phi29 DNA polymerase shows considerable improvement in genome coverage at higher temperatures compared to existing commercial options, according to Prof. XU Jian, a senior author on the study.
With an eye towards improving their method and making it more cost-effective, the researchers are now focused on the development of new instruments and reagents. The goal is to empower microbiologists to sort and sequence any individual microbial cells on Earth while maintaining high data quality and reducing reagent costs.
Traditional gene sequencing methods rely on processing millions of cells simultaneously. This, while beneficial for larger scale studies, results in loss of individual cell information and can obscure research that relies on single-cell consideration. These issues are particularly prominent with bacteria, as the small size of bacterial cells results in significantly lower DNA content compared to human or animal cells.
Gene amplification, a procedure sometimes referred to as "molecular photocopying," is a fundamental step in gene sequencing. It produces millions, sometimes billions, of copies of DNA segments. However, in bacterial single-cell DNA amplification, the process can generate biases that distort the representation of different genome regions, due to the limited amount of DNA available for copying.
Aiming to reduce such bias, lead author ZHANG Jia and his team have introduced a process termed Improved Single-cell Genome Amplification (iSGA). The iSGA process improves the efficiency and robustness of the phi29 DNA polymerase—a protein widely used in single-cell whole genome amplification—through protein engineering and process engineering. However, the traditional use of this protein has been plagued by low genome coverage and efficiency issues.
Through a method known as compartmentalized self-replication (CSR), the team enhanced the structure of the phi29 DNA polymerase, optimizing its activity, specificity, and stability. Alongside modifying the amplification buffer, the group also devised a more efficient decontamination process to further decrease contamination during amplification. The newly developed HotJa Phi29 DNA polymerase shows considerable improvement in genome coverage at higher temperatures compared to existing commercial options, according to Prof. XU Jian, a senior author on the study.
With an eye towards improving their method and making it more cost-effective, the researchers are now focused on the development of new instruments and reagents. The goal is to empower microbiologists to sort and sequence any individual microbial cells on Earth while maintaining high data quality and reducing reagent costs.