Coupling the specificity of phage-selected α-La1 scFv with FACS allowed precise manipulation of a population on a per-cell basis, making possible the sufficient enrichment of L. acidophilus for >99.8% genome coverage using both reference mapping and de novo assembly. While it is common to observe this level of coverage for de novo assembly when the target organism is cultured prior to sequencing in the laboratory, Selleckchem YH25448 the level of coverage reported here for a bacteria extracted from an environmental sample is exceptional. For sequencing, we easily and rapidly sorted 50 L. acidophilus cells from an environmental sample (yogurt) where L. acidophilus comprised
~0.2% of the population and were able to rapidly detect and quantify L. acidophilus at ~0.1% in a mock community comprising nine other species. Although we only tested compositions as low as ~0.1%, we are confident that L. acidophilus could be identified from mixtures where it is even lower in relative abundance with detection limited solely by the total number of cells available in a mixture and time available for sorting. While detection and enrichment PX-478 of rare species is an obvious use of these antibodies, depletion of common species may be equally important, as bias towards high abundance species is a well-known issue
when performing shotgun metagenomics [54–57] and, potentially, non-targeted single cell genomics. Our single cell analysis shows that L. acidophilus is completely depleted from the sample in the negative sort gate (P2; Figure 4), demonstrating the feasibility of both depletion and enrichment. until Separation methods, namely immunoprecipitation, micromanipulation, and flow cytometry have been described to improve genome sequencing, and the approach described here may also be applicable to other microbes
found in microbiomes without being limited to organisms with innate fluorescence , distinct morphology and/or high genome copy number . In this study we generated a scFv against an organism that can be cultured in the lab as a demonstration that recombinant antibodies can be raised against a specific organism and used to dissect, phylotype, and recover complete genomes for organisms from microbial communities. We used an organism with a reference genome in order to selleck accurately assess genome coverage. Future studies will involve selecting antibodies directly against uncultivable organisms within complex microbiomes. We provide proof of principle, using selection against a mock community, that such an approach is potentially feasible: HCDR3 sequences of three of the antibodies selected against the pure culture were identical to those of antibodies selected against the mock community.