Single-cell analysis and spatial resolution of the gut microbiome

Abstract

Over the past decade it has become clear that various aspects of host physiology, metabolism, and immunity are intimately associated with the microbiome and its interactions with the host. Specifically, the gut microbiome composition and function has been shown to play a critical role in the etiology of different intestinal and extra-intestinal diseases. While attempts to identify a common pattern of microbial dysbiosis linked with these diseases have failed, multiple studies show that bacterial communities in the gut are spatially organized and that disrupted spatial organization of the gut microbiome is often a common underlying feature of disease pathogenesis. As a result, focus over the last few years has shifted from analyzing the diversity of gut microbiome by sequencing of the entire microbial community, towards understanding the gut microbiome in spatial context. Defining the composition and spatial heterogeneity of the microbiome is critical to facilitate further understanding of the gut microbiome ecology. Development in single cell genomics approach has advanced our understanding of microbial community structure, however, limitations in approaches exist. Single cell genomics is a very powerful and rapidly growing field, primarily used to identify the genetic composition of microbes. A major challenge is to isolate single cells for genomic analyses. This review summarizes the different approaches to study microbial genomes at single-cell resolution. We will review new techniques for microbial single cell sequencing and summarize how these techniques can be applied broadly to answer many questions related to the microbiome composition and spatial heterogeneity. These methods can be used to fill the gaps in our understanding of microbial communities.

Keywords: genomics; microbiome; sequencing; single cell; spatial resolution.

Figure 1

Single cell sequencing workflow. Single cell isolation: The first step for performing single cell microbial genomics is isolating single cells. Some commonly used single cell isolation techniques include fluorescence-activated cell sorting (FACS), micromanipulation, and microfluidics. (1) FACS involves size- and fluorescence-based separation that separates single cells from a complex microbial community (Rinke et al., 2014). (2) The traditional micromanipulation method includes micro-pipetting in combination with an inverted microscope for the isolation of single cells from a mix of microbial cells (Ishøy et al., 2006). (3) The microfluidics approach combined with droplet encapsulation involves encapsulating individual cells in hydrogel microspheres resulting in isolated single cells in each droplet (Marcy et al., 2007). There have been different modifications of the microfluidics method, based on the core concept of encapsulating single cells in droplets, to yield single cells. Spatial resolution: Various new techniques have been developed in order to obtain spatial genomics information of the gut microbiota, generally either imaging based or sequencing based. (1) High phylogenetic resolution fluorescence in-situ hybridization (HiPR-FISH) employs a binary barcode system based on hybridization of distinct fluorophores (Shi et al., 2020). Spectra are measured using fluorescence microscopy, and spectral barcodes are decoded using machine learning. Identification and spatial visualization of taxa are possible. Tunable expression tools (Whitaker et al., 2017) is a platform for engineering Bacteroides using a novel phage promoter and translation tuning strategy to enable imaging of fluorescent bacteria. Unique fluorescent signals can be used to allow differentiation of species within the gut. (2) Metagenomic plot sampling by sequencing (MaPS-seq) combines genomic and spatial resolution (Sheth et al., 2019). Intact microbiota samples are fractured into particles and are encapsulated in droplets before deep sequencing. This results in the retention of spatial information and can identify species that tend to co-localize in complex samples such as the gut microbiota. Sequencing: (1) The sequencing step of the single cell genomics workflow involves using DNA from the isolated cells to prepare a library. (2) This is followed by high-throughput sequencing which yields (3) critical information identifying the genetic composition microbes and associated gene expression changes in a complex microbial community.