PCR-activated cell sorting for cultivation-free enrichment and sequencing of rare microbes

Abstract

Microbial systems often exhibit staggering diversity, making the study of rare, interesting species challenging. For example, metagenomic analyses of mixed-cell populations are often dominated by the sequences of the most abundant organisms, while those of rare microbes are detected only at low levels, if at all. To overcome this, selective cultivation or fluorescence-activated cell sorting (FACS) can be used to enrich for the target species prior to sequence analysis; however, since most microbes cannot be grown in the lab, cultivation strategies often fail, while cell sorting requires techniques to uniquely label the cell type of interest, which is often not possible with uncultivable microbes. Here, we introduce a culture-independent strategy for sorting microbial cells based on genomic content, which we term PCR-activated cell sorting (PACS). This technology, which utilizes the power of droplet-based microfluidics, is similar to FACS in that it uses a fluorescent signal to uniquely identify and sort target species. However, PACS differs importantly from FACS in that the signal is generated by performing PCR assays on the cells in microfluidic droplets, allowing target cells to be identified with high specificity with suitable design of PCR primers and TaqMan probes. The PACS assay is general, requires minimal optimization and, unlike antibody methods, can be developed without access to microbial antigens. Compared to non-specific methods in which cells are sorted based on size, granularity, or the ability to take up dye, PACS enables genetic sequence-specific sorting and recovery of the cell genomes. In addition to sorting microbes, PACS can be applied to eukaryotic cells, viruses, and naked nucleic acids.

Fig 1. The PACS workflow applied to a model microbial system.

A microbial sample consisting of K-12 E coli harboring wild type TolA and a spike-in variant (ΔTolA) is created from growth cultures (Fig. 1A). This sample is then encapsulated together with PCR reagent to form a single emulsion (Fig. 1B). This emulsion is then collected and thermocycled, with PCR-positive droplets experiencing an increase in Taqman fluorescence (Fig. 1C). This emulsion is then DEP sorted for bright drops (Fig. 1D), and these drops are ruptured to release genomic content which is sequenced to verify sorting efficacy (Fig. 1E).

Fig 2. Taqman PCR detection of TolA gene on E. coli bacteria.

E coli bacteria are encapsulated with PCR reagents in droplets and are thermocycled. Fig. 2A, upper, Drops containing bacteria with the TolA gene are bright, whereas this is absent in Fig. 2A, lower, which has E coli without this gene. Fig. 2B shows the dependency of the fraction of loading drops which are bright versus the poisson loading ratio. The different curves represent different calculated curves if the E. coli lysis factor k was varied.

Fig 3. DEP droplet sorting device.

Fig. 3, upper, shows the device layout, with the salt “moat” insulating the drops from any stray electric fields potentially originating from the salt electrode. This device consists of the reinjection junction, Fig. 3, left, at which the reinjected emulsion is spaced out, as well as the sorting junction, Fig. 3, middle, which is where detection and sorting occurs. Fig. 3, right, shows positive and negative droplet sorting events.

Fig 4. Droplet detection and sorted drops.

Fig. 4, left, is the PMT timetrace of recorded signals from the optical droplet detection setup. There is a clear peak at 32.5 ms, which corresponds to a bright drop that is sorted. Fig. 4, right, are the fluorescence images of thermocycled drops before and after DEP sorting. Scale bars are 100 μm.

Fig 5. Sequencing verification and genome enrichment.

Fig. 5A is a representative electropherogram of the LpoA gene and its mutant counterpart after Sanger sequencing LpoA from sorted bacterial genomes. There are clear base calls on the double nucleotide mutation. Fig. 5B shows the sequencing results, with clear enrichments for the TolA/ΔLpoA bacterial strain for both spike-in ratios.