Massively parallel single-cell sequencing of genetic loci in diverse microbial populations

Abstract

Single cell genetic heterogeneity is ubiquitous in microbial populations and an important aspect of microbial biology. However, we lack a broadly applicable and accessible method to study this heterogeneity at the single cell level. Here, we introduce a simple, robust, and generalizable platform for quantitative and massively parallel single cell sequencing of target genetic loci in microbes using ultrahigh-throughput droplet microfluidics (Droplet Targeted Amplicon Sequencing or DoTA-seq). Using DoTA-seq, we elucidate the highly diverse single cell ON/OFF states of the phase-variable capsule synthesis operons in the prevalent human gut species Bacteroides fragilis. In addition, we quantify the shifts in antibiotic resistance gene abundances in different species in a 25 member human gut microbial community in response to antibiotics. By sequencing tens of thousands of single-cells derived from a human fecal sample, we identify links between plasmid replicons and the taxonomic lineages of their associated hosts. In sum, DoTA-seq is an accessible and broadly applicable tool for profiling single-cell genetic variation in microbiomes.

Shaping human gut community assembly and butyrate production by controlling the arginine dihydrolase pathway

“…Bottom-up assembly enables a deeper understanding of the contributions of monoculture growth and inter-species interactions in response to environmental factors. While we focused on a single metabolic pathway, higherthroughput genetic approaches could also be used to discover and rank the influence of diverse molecular pathways on target community functions [59][60][61] . Our strategy could be applied to the pathway hits identified via high-throughput genetic pipelines to understand their quantitative effects and develop the capability to tune these pathways to precisely control community behaviors.…”

“…By integrating mechanistic computational modeling, synthetic biology and bottom-up community assembly, we revealed the extent to which a single mechanism of interaction contributed to community assembly. While we focused on a single metabolic pathway, high-throughput genetic manipulation approaches could also be used to discover and rank the influence of diverse molecular pathways on target community functions 64–66 . Our strategy to quantitatively determine the contribution of metabolic pathways could be applied to the pathway hits to identify novel control knobs for steering microbiomes to desired states.…”

Single-cell analysis of multiple invertible promoters reveals differential inversion rates as a strong determinant of bacterial population heterogeneity

“…To investigate B. fragilis population diversification at the single-cell level, we used an ultrahigh-throughput, single-cell sequencing method referred to as droplet targeted amplification sequencing (DoTA-seq) (24). DoTA-seq can analyze combinations of genes in diverse Gram-positive and Gram-negative bacteria at the single-cell level.…”

“…This workflow is a simplified version of a single-cell sequencing workflow suitable for many types of microbes (24). Please refer to the manuscript of the original workflow for a detailed step-by-step protocol and additional guidelines.…”

High-throughput single-cell sequencing of multiple invertible promoters reveals a strong determinant of bacterial population heterogeneity

“…To investigate B. fragilis population diversification at the single-cell level, we used an ultrahigh-throughput, single-cell sequencing method referred to as Droplet Targeted Amplification sequencing (DoTA-seq) (19). DoTA-seq can analyze combinations of genes in diverse gram-positive and gram-negative bacteria at the single-cell level.…”