7% α-La2 2 CARAGRGTSYYGMDVW 142822 11.9% 3 CARVGDGYNYAFDIW 34320 2.9% 4 PKC412 order CAVAGTGYAFDIW 17429 1.4% 5 CARAGGGTSYYGMDVW 11394 0.9% 6 CAKLRGGPTKGDWYFDVW 9688 0.8% 7 CATGDAFDMW 9287 0.8% α-La3 8 CARGHYGMDVW 7675 0.6% 9 CARDEGNAFDIW 7303 0.6%
10 CARGSLGAFDIW 5761 0.5% α-La4 11 CAKLRGPTLPRYSFDYW 5601 0.5% 12 CARDPLGKLGPEEYYYGMDVW 4598 0.4% 13 CARDSMWVVAAKRKLHNCFDPW 4939 0.4% 14 CARDRGYGVDYW 3331 0.3% 15 CARDLGAGMDVW 3256 0.3% 16 CARQQLAAFDIW 3037 0.3% 17 CARDKGHEAFDIW 2589 0.2% 18 CARDGGDAFDIW 2029 0.2% 19 CARDYGEAFDIW 1585 0.1% 20 CARIGGGKRRSHFDYW 1438 0.1% *Total number of quality reads from the Ion Torrent sequencing run = 1,203,589. Discussion The expanding field of metagenomics continues to search for robust ways to obtain high-quality genomes from under-represented or rare species in a given sample. Improvements in sequencing throughput will enable access to lower abundance populations, but a “pre-enrichment/pre-clearing” step before the analysis can provide complementary and significant results. We describe a novel and adaptable approach for sequencing
low abundance genomes from microbial communities, with potential improvements in the genomic coverage of low abundance species where standard single cell approaches result in incomplete genomes or may have missed the organism AZD8931 mouse altogether. We demonstrate the use of phage display to select antibodies against a bacterial species with exquisite specificity. The use of in vitro display potentially Nutlin-3a price allows the method buy DAPT to be adapted
to any organism or microbiome, does not rely on commercially available antibodies, and generates antibodies that are highly renewable and amenable to further engineering to modify affinity or specificity . To demonstrate the feasibility of the approach, we first targeted Lactobacillus acidophilus, a bacteria naturally found in environmental samples from food to feces and is a principal commensal bacterium of the human gut. The tested α-La1 scFv proved to be extremely specific and did not recognize other common gut microflora (such as Bifidumbacterium and E. coli). While it is practically impossible to prove that this scFv does not recognize any other bacteria, when tested on other Lactobacilli such as L. helveticus, which is highly similar to L. acidophilus, we did not observe binding, providing strong evidence that the scFv is species-specific. The target protein recognized by our scFv was identified as the Surface layer protein A (SlpA). S-layer proteins are highly abundant and ubiquitous crystalline surface structures [41, 42] that have been implicated as a principal component for the organism’s probiotic functions [52, 53]. Other Lactobacilli tested in this study produce S-layer proteins that are highly similar (73% identical for L. helveticus) (Figure 2B), but which can nevertheless be distinguished by our α-La1 scFv, demonstrating the high degree of specificity achievable.