A Revamped Amplification Method that Enables Nearly Complete Single-Cell Genomes

With a new methodology, scientists are now getting more complete genomic results from single cells.

The Science

Studying the genomes of microorganisms is incredibly useful for understanding the microbiome of the earth, but most species do not grow easily in the lab. To access the genomes of uncultured microbes, scientists usually employ two different methods; shotgun metagenomic sequencing and single-cell sequencing. For years, extracting single cells and then amplifying their DNA for genome sequencing was no easy task. The traditional way of collecting them is through a method called Multiple Displacement Amplification, and most genomes that were acquired through it were usually only around 30% complete. Described in ISME Communications, a method called Primary Template-Directed Amplification (PTA) yields a much higher completion, above 80% in most cases.

The Impact

PTA is a refined capability that is desirable in the microbiome field due to its high recovery of single-cell genomes. This is key to better understanding all the organisms that live in our immediate microbiome. The U.S. Department of Energy (DOE) Joint Genome Institute (JGI), a DOE Office of Science User Facility located at Lawrence Berkeley National Laboratory, is heavily invested in being the bridge to help scientists and researchers achieve their analysis goals. Offering this tool establishes the JGI as a leader in single-cell genomic amplification at a large scale.

Summary

The two most common methods for collecting genomes from microorganisms are shotgun (group) metagenomic sequencing and single-cell (individual) sequencing. Shotgun metagenomic sequencing takes much less time than its single-cell counterpart. In this approach, DNA from all the different species is mixed together and shredded up before sequencing, then computer algorithms stitch the pieces together and attempt to put them back into the original genomes. These metagenome-assembled genomes (MAGs) that came from shotgun sequencing are tricky because it’s difficult to link the connections to them from the genetic elements that scientists have found, e.g. plasmids and other mobile elements. Moreover, the MAGs are composites of high similar genomes, and no cell in the microbiome exactly matches the MAG. This is where single-cell sequencing comes in.

Single-cell genome sequencing involves taking a single cell and isolating it for analysis. Once it’s alone, the cell breaks apart and its DNA is copied millions of times using a method named Multiple Displacement Amplification to generate enough DNA to put on a sequencer. As previously mentioned, it’s a method that’s been used for years but in most cases, the majority of the cell’s genome is missing from the final assembly. So while researchers are confident all the DNA sequences came from one cell, including components like plasmids, the picture is far from complete. 

In 2021, scientists at St. Jude Children’s Research Hospital developed a new method of genome amplification called Primary Template-Directed Amplification (PTA) that yielded more complete and evenly covered genomes when tested in human cells. To see if this method could also improve genome recovery in microbiome studies, these scientists (now at Stanford University) collaborated with the JGI. Working together, they found most bacterial genomes from water and soil communities were at least 80% complete when studied with PTA, compared to <30% complete when studied with MDA.

With the introduction of PTA, JGI users can get more complete single-cell genomes more consistently. JGI users can request this capability when submitting a proposal.