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Inducing Ploidy Increases Genome Coverage, Reduces Bias of Single-Cell Sequencing

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By Monica Heger

This article was originally published March 19.

Increasing chromosome content in single bacterial cells can boost genome coverage and reduce amplification biases for single-cell sequencing techniques, according to researchers from Los Alamos National Laboratory.

Presenting earlier this month at Cambridge Healthtech Institute's XGen Congress in San Diego, Calif., Armand Dichosa, a postdoctoral research associate at Los Alamos National Laboratory, described a technique to induce chromosome replication in cells while inhibiting cell division, prior to sequencing. His team is now working on developing and commercializing a universal assay that would enable the technique to be used on a broad range of cells.

Dichosa's talk was one of several at the conference that addressed new single-cell sequencing techniques to improve assembly, increase genome coverage, and reduce amplification bias — the major challenges of single-cell sequencing (IS 3/13/2012).

Dichosa described to In Sequence LANL's approach as essentially using a drug to prevent cell division after chromosomal duplication within a cell. That increases the amount of genomic content available when subjecting the DNA to single-cell sequencing, which increases coverage and reduces amplification bias.

More specifically, the researchers hypothesized that treating bacterial cells with a compound called PC19 would halt cytokinesis after chromosomal duplication but before cell division by inhibiting FtsZ, a protein involved in cytokinesis of bacteria and archaeal cells.

To test this, they treated Bacillus subtilis cells with PC19, let the cells grow for between 40 to 60 minutes, and then used flow cytometry to sort out single cells. Cells treated with PC19 were nearly double the size of untreated cells, "a good indicator" that the chromosomes had replicated, but the cell had not divided, said Dichosa.

To further confirm that the larger cell size corresponded to increased genomic content, the team used qPCR to test for markers of replication and termination and found that the treated cells had about twice the number of replication and termination sites — indicating that the chromosomes had doubled, Dichosa explained. They also found that maximum DNA content was achieved after letting treated cells grow for about 50 minutes.

Next, the team used multiple displacement amplification, a common amplification method for single-cell sequencing that uses the enzyme phi29 to amplify DNA in a linear fashion, and sequenced the whole genomes of four cells — two treated with PC19 and two controls — on the Illumina Genome Analyzer.

After sequencing, the team compared the genome coverage and bias between the treated and untreated cells. They used a method called locus bias score, or LBS, which uses qPCR to quantify genomic amplification and generate a variance score. A lower score indicates less bias.

"We found a four-fold decrease in amplification bias by inducing ploidy," Dichosa said. Untreated cells had a bias score between 7.9 and 8.62, while treated cells had bias scores between 2.0 and 2.64.

Additionally, inducing ploidy increased genome coverage. Mapping the reads to a reference genome the researchers found that polyploid cells had about 10 percent more mapped reads, and coverage was further improved when comparing de novo assembly between treated and untreated cells.

"This is where you see the advantage of inducing ploidy," Dichosa said. Genome recovery for the control cells was around 50 percent, he said, but inducing ploidy boosted recovery to around 70 percent.

"It made the assembly easier and more accurate," he added.

Dichosa said the next step is to make the method broadly applicable to a wide range of cells by identifying other compounds that could be used to inhibit cell division and induce ploidy.

PC19 will likely work on some other bacterial cells, since most of them use the protein FtsZ in cytokinesis. However, the cytokinesis mechanism can be slightly different depending on the species, so inhibiting FtsZ will not yield the same results for all bacteria. For instance, when the team tested the compound on the gram negative bacteria Escherichia coli, there was "little to no response," Dichosa said.

The team is now trying to expand the applicability of the technique by finding a "drug that will work on a broader range of bacteria," he said.

The researchers have made strides on developing a "universal cocktail," which they will likely patent and potentially commercialize it if they are able to demonstrate it on metagenomic samples from the marine environment or soil, said Dichosa. But, the method is "currently in its infancy," he said, adding that "we're definitely not there yet."

Dichosa said that at Los Alamos National Laboratory, he would like to use the method for applications like pathogen detection and metagenomic analysis, especially for looking at rare species in a sample.

Single-cell sequencing is preferable to metagenomic sequencing for these types of studies, he said, because metagenomic sequencing is "biased for the more abundant members of the community." Additionally, metagenomic sequencing often yields only pieces of the single organism's genome, rather than the whole genome. It offers a snapshot of the most abundant members of the community, rather than a detailed look at a single, potentially rare and pathogenic species.

"With this method, you have the power to target single cells," he said, which brings "you much closer to identifying the organism at the genomic level."


Have topics you'd like to see covered by In Sequence? Contact the editor at mheger [at] genomeweb [.] com.

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