Bacterial single-cell, whole-genome sequencing using engineered polymerases

Genome sequencing of single bacterial cells has long been technically difficult due to irreversible biases in the gene amplification step of the process, making it difficult to produce a high-coverage genome sequence from exactly one bacterial cell.

A new process developed by researchers at the Chinese Academy of Sciences’ (CAS) Qingdao Institute of Bioenergy and Bioprocess Technology (QIBEBT) Single-Cell Center greatly reduces that bias and opens new vistas of single-cell research. Their study was published Frontiers in Bioengineering and Biotechnology On June 29.

This is common when using traditional gene sequencing methods that use millions of cells at a time. But such ordering of cells inevitably blurs the differences between cells, making it difficult to explore relationships between different cell lineages or to conduct any other type of research that relies on the consideration of one cell at a time. Such technical challenges are particularly acute for bacteria, because a bacterial cell is so small that its DNA content is several orders of magnitude less than that of a human or animal.

In order to sequence genes or entire genomes, the DNA must first be “amplified.” Gene amplification, sometimes called “molecular photocopying,” can make millions or even billions of copies of DNA segments.

Amplification poses few problems for bulk gene sequencing. However, in bacterial single-cell DNA amplification, especially of whole genomes, the amount of DNA available for replication is very limited, and the amplification process can introduce biases such as over- or under-representation of different regions of the genome.

“Reducing amplification bias is a holy grail for single-cell genome amplification researchers,” said Zhang Jia, a biotechnologist at QIBEBT and lead author of the paper. “We think we have now found a way to solve this problem.”

The researchers developed a process called Improved Single Cell Genome Amplification (ISGA). This primarily involves protein engineering and process engineering into the amplification capacity of Fi29 DNA polymerase. Fi29 DNA polymerase is commonly used in single-cell whole genome amplification, but suffers from problems of low genome coverage and low efficiency.

“The iSGA phi29 DNA polymerase we developed is more than twice as efficient and powerful as conventional phi29 DNA polymerase,” Zhang Jia said. “It’s about eleven times cheaper than the main commercial version that’s commonly available.”

The researchers achieved this by engineering the polymerase to increase its amplification ability. Using a method called compartmentalized self-replication (CSR) to develop Fi29 DNA polymerase, they modified its structure to increase its activity, specificity, and stability. “The newly developed DNA polymerase, which we call HotJa Phi29 DNA polymerase, shows better genome coverage (99.75%) at higher temperatures than existing commercial polymerases,” said senior author Prof. Xu Jian said.

They optimized the amplification reaction conditions and modified the amplification buffer (a solution that provides a suitable chemical environment for DNA polymerase) to improve the stability and activity of Fi29 DNA polymerase. In addition, the researchers developed a more efficient decontamination method to minimize contamination during the amplification process.

The researchers now plan to optimize their iSGA method and HotJa Phi29 DNA polymerase for various applications and sample types and further reduce costs. The team has developed a series of tools including RACS-Seq, FlowRACS and EasySort for functional sorting of microbial single cells. Their goal, by developing these tools and reagents, is to enable microbiologists to classify and sequence any individual microbial cell on Earth without worrying about data quality or reagent cost.