Supplementary Figure 8: Identification of FACS-enriched in situ transconjugants by 16S sequencing. | Nature Methods

Supplementary Figure 8: Identification of FACS-enriched in situ transconjugants by 16S sequencing.

From: Metagenomic engineering of the mammalian gut microbiome in situ

Supplementary Figure 8

(a) Implementation of MAGIC in a mouse model with fecal bacterial analysis by FACS, antibiotic selection, and sequencing. (b) FACS dot plots of in situ conjugations using EcGT2 donors with vector libraries pGT-L1, L2, and L3. Green boxes define the sorted GFP+mCherry transconjugant populations. Each plot shows fluorescence expression of bacteria from the combined fecal samples of 3 cohoused mice. The experiment was run 3 independent times, with similar results. (c) 16S taxonomic classification of FACS-enriched transconjugants from in situ mouse experiments using vector libraries pGT-L1, L2, and L3 at 6 h post-gavage. Relative abundance of each OTU in the unsorted population is shown in the grayscale heat map, and fold enrichment for transconjugants of each OTU is shown in the orange heat map, with annotated taxonomic identities. Bracketed values indicate confidence of taxonomic assignment by RDP Classifier. Each column represents data from a separately housed cohort of 3 mice whose fecal samples were combined for analysis. Genera with successfully cultivated isolates are denoted by stars. (d) The pGT-L3 transconjugant population from (b) was further analyzed by comparison of Q4 enriched OTUs against Q3 OTUs, which represent a sample of the GFP native bacteria population, and by enrichment analysis of Q4 samples that were sorted again for Q4. Enriched GFP+ transconjugants were robust whether compared against the total fecal population or against Q3. 7 out of 11 OTUs enriched in Q4 were present in the double-sorted Q4 population, indicating that Q4 sorting is robust. The OTUs lost upon double-sorting were obligate anaerobes and likely sensitive to prolonged aerobic conditions during double-sorting.

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