M20 Genomics

VITA GutMicrobiome: Revolutionizing Gut Microbiome Research with High-Throughput Single-Bacterium Transcriptomics

发布时间:2024-03  /  浏览次数:128 次

In March 2022, we proudly unveiled our innovative high-throughput single-bacterium RNA sequencing technology, MscRNA Seq (incooperated in M20 Seq®) alongside the VITApilote® High-Throughput Single-Bacterium Transcriptome Kit. Our groundbreaking advancement sparked a transformative shift in microbiological research towards single-bacterium transcriptomics.

As scientific exploration in microbiology ventures into unknown territories with increased complexity and precision, particularly within the intricate realm of gut microbiota, we have committed to confronting emerging challenges and facilitating a deeper understanding of this vital microbial community. We are thrilled to present the latest addition to our VITA single-cell transcriptome product family - VITA GutMicrobiome (Figure 1). This product stands as the world's first high-throughput single-bacterium transcriptomic solution designed for gut microbiome studies, empowering researchers to gain comprehensive insights into the intricate world of gut microbiota.

Figure 1. VITA GutMicrobiome product

The introduction of VITA GutMicrobiome marks the addition of a powerful tool available to researchers studying the functionality of gut microbiota. By expanding the scope of analysis to the single-cell level, this breakthrough holds the potential to significantly advance comprehensive investigations in various fields, including gut microbiota functions, live biotherapeutic products, fecal microbiota transplantation, microbiota-drug interactions, and beyond.

 

The Current Status of Gut Microbiota Research

The gut microbiota can be considered a major "hidden organ" of the human body, with approximately 30 trillion human cells and an estimated 100 trillion microorganisms in the gut. These microorganisms play crucial roles in nutrient absorption, drug metabolism, immune regulation, disease development, and even post-COVID sequela[1-3].

In recent years, the number of published articles on gut microbiota has exponentially increased (Figure 2). However, the primary research tools used at the nucleic acid level continue to be the metagenomics and metatranscriptomics techniques introduced nearly two decades ago.

Figure 2. Number of research articles published on the gut microbiota/microbiome over the past 23 years on PubMed

While these conventional methods are mature due to their long-term use, they have inherent limitations, especially in precisely analyzing the functional heterogeneity of microbial populations. For instance, the clinical use of broad-spectrum antibiotics may disrupt the balance of gut microbiota leading to the emergence of antibiotic-resistant bacteria and potentially severe consequences like Clostridioides difficile infection. Scientists have recognized that antibiotic resistance in microbial populations is a highly heterogeneous process. Even strains with identical genetic backgrounds may develop into subpopulations with different functional phenotypes under the pressure of environmental antibiotics. This heterogeneity can rapidly change with environmental variations, and current clinical detection and analysis methods often lack sensitivity to detect such antibiotic heteroresistance[4-6] (Figure 3).

Figure 3. Conventional detection methods for bacterial resistance fail to identify heteroresistant bacterial populations.

Many bacterial species in the gut microbiota are challenging to culture in vitro, making functional studies post-pure cultivation impractical. Among the currently used techniques, metagenomics, as a tool at the genomic level, struggles to capture rapid changes in functional heterogeneity. While techniques such as metatranscriptomics and microarray can analyze functional phenotypes at the transcriptome level, they only provide averaged information for the population, thereby preventing the aquisition of information on the heterogeneity of piovital subgroups. Flow cytometry can analyze functional heterogeneity at the protein level but is confined to a limited number of known targets and faces challenges in providing comprehensive analysis and exploration. Existing single-bacterium transcriptomics technologies on other platforms (estbalished techniques lacking commercial products as of now) are solely applicable for the analysis of cultured bacteria and have not been applied to complex microbial communities [7-9].

Hence, there is an urgent need for innovative technologies tailored for precise analysis of functional heterogeneity in microbiome research. Such technologies will not only facilitate the investigation of antibiotic heteroresistance in gut microbiota mentioned earlier but will also hold significance for other research directions, such as microbiota-drug interactions, microbiota-host interactions, fecal microbiota transplantation, and the development of live biotherapeutic products.

 

VITA GutMicrobiome – The World's First High-Throughput Single-Bacterium Transcriptome Product for Gut Microbiota

In response to the evolving needs in microbiome research, we proudly present the world's first high-throughput single-bacterium transcriptomie product for gut microbiota - VITA GutMicrobiome. Our product comprises the VITApilote® GutMicrobiome Single-Bacterium Transcriptome Kit and Chip, VITAcruizer® Single-Cell Partitioning System, and the VITAseer® Bioinformatics Software. All components are designed to seamlessly integrate, delivering an end-to-end solution for single-bacterium transcriptome sequencing of gut microbiota.

Notably, users of VITA products, can forego the necessity of acquiring additional instruments. Any installed VITAcruizer® instrument can facilitate experiments on the single-bacterium transcriptome of microbiota samples. Such convenience enables high-throughput single-cell transcriptome experiments, covering a spectrum of samples from eukaryotic FFPE sections/blocks to microbiota, all conducted on the same instrument and realizes a comprehensive approach for high-throughput single-cell transcriptomics.

Our VITA GutMicrobiome High-Throughput Single-Bacterium Transcriptome Kit is specifically designed for direct application to gut microbiota samples, eliminating  the need for in vitro cultivation. In contrast to other existing technologies, VITA GutMicrobiome offers several additional advantages:

1. Analyzing the functional and phenotypic heterogeneity of gut microbiota

VITA GutMicrobiome transcends mere identification of different bacterial species in the gut microbiota and reveals distinct transcriptional states of individual cells from the same species. This capability proves especially effective in analyzing alterations in functional and phenotypic heterogeneity of gut microbiota under specific or dynamic environmental conditions.

2. Enhancing the precision of the transcriptome analysis for gut microbiome

In the analysis of metagenomics and metatranscriptomics, one of the major challenges lies in determining the species origin of each sequencing read. Distinguishing between reads from closely related species with high homogeneity can be challenging. However, VITA GutMicrobiome operates at the single-bacterium level, allowing researchers to intricately identify the cellular origin of each sequencing read and thereby determine its species origin. This advancement facilitates precise analysis of the gut microbiota transcriptome, empowering researchers to explore the complex network of microbial communities in detail.

3. Conducting precise correlation analysis between bacteria and bacteriophages

VITA GutMicrobiome can accurately elucidate the correlation between gene expression of individual bacteria and the expression of their internal bacteriophages. This capability exposes the heterogeneity of phage invasion in the gut microbiota and its impact on bacterial functions and phenotypes.

4. Investigating interaction patterns between bacteria and between bacteria and hosts

VITA GutMicrobiome enables to precisely profile the expression of ligands and receptors for various signaling pathways at the single-bacterium level. Therefore, it can facilitate the analysis of interactions within bacterial communities at the single-bacterium level. Additionally, its seamless combination with our VITApilote® High-Throughput Eukaryotic Single-Cell Transcriptome kit empowers the investigation of interactions between gut bacteria and hosts.

 

Elevating Insights: Performance and Applications of VITA GutMicrobiome

Our assessment of VITA GutMicrobiome, using human gut microbiota (feces) samples, demonstrated its outstanding performance and ability to reveal alterations in the functionality of human gut microbiota.

VITA GutMicrobiome: Exceptional performance

In a human gut microbiota sample with a captured cell count of 10,000 VITA GutMicrobiome achieved a median Unique Molecular Identifier (UMI) count of 350 and a median gene count of 113 (Figure 4). These metrics align with the elevated levels of single-bacterium transcriptome data from pure cultures reported in existing literature[7-9].

Figure 4. Violin plots of the UMI count (left) and the gene count (right) of a human gut microbiota sample

Furthermore, the analysis of the obtained data identified a total of 67 different bacterial species in the sample (Figure 5). The list included renowned core bacterial genera in the gut, such as Prevotella and Megasphaera, as well as newly discovered human gut bacterial species like Neobittarella massillensis.

Figure 5. Species composition of the sample (species ranked below tenth in abundance are labeled in white)

VITA GutMicrobiome: Revealing the functional dynamics of the gut microbiota

To further assess the application of VITA GutMicrobiome in human gut microbiota research, we collected gut microbiota samples from the same healthy individual at 9 AM and 6 PM. We utilized our VITA GutMicrobiome High-Throughput Single-Bacterium Transcriptome product to compare changes in the incidence of highly abundand bacterial species in the two samples.

Interestingly, the abundance of species such as Megamonas funiformis, Prevotellamassilia timonensis, and Prevotella hominis decreased from 9 AM to 6 PM, while Prevotella copri_A and Prevotella sp900557255 were detected at increased frequencies at 6 PM (Figure 6).

Figure 6. Species composition of gut microbiota at 9 AM (left) and 6 PM (right) for the same healthy individual (only species with abundance >1% are displayed)

For a more in-depth exploration of the functional mechanisms underlying the changes in the gut microbiota composition, we focused on the most abundant bacterial species, Megamonas funiformis, for further subclustering. Megamonas funiformis was isolated and identified from the gut microbiota of Japanese subjects in 2008[10] and has since been found in populations of various ethnic groups. It is a common species in the gut microbiota of Asian individuals and is closely associated with nutritional metabolism[11].

UMAP analysis unveiled the presence of three distinct subpopulations within Megamonas funiformis across both samples (Figure 7, left). Subpopulation 1 exhibited consistent occurrence at both time points, while subpopulation 0 displayed higher abundance in the 9 AM sample (Figure 7, right, red arrow). Notably, subpopulation 2 was solely detectable in the 6 PM sample (Figure 7, right, blue arrow).

 

Figure 7. Subpopulations of Megamonas funiformis

Differential gene expression analysis results indicated that Megamonas funiformis subpopulation 2, uniquely present in the 6 PM sample, showed elevated expression levels of genes associated with carbohydrate and protein metabolism and transport  (Figure 8). This suggests that the variation in Megamonas funiformis at different time points may be related to the dietary rhythm and nutritional metabolism of the subject.

Figure 8. Differential gene expression heatmap of the 3 subpopulations of Megamonas funiformis (Gene names are concealed here to protect unpublished data.)

Moreover, as VITA GutMicrobiome can provide precise insights into gene expression within individual bacterial cells, it facilitates a detailed analysis of bacteriophage gene expression within each bacterial species in the gut microbiota. This capability allows for the investigation of specific mechanisms by which bacteriophages influence the functionality of the gut microbiota and human health.

The analytical approach demonstrated above can be applied to analyze bacterial species relevant to a broad spectrum of research objectives in the gut microbiota. This approach can provide a detailed functional atlas of the gut microbiome associated with particular biological phenomena such as dietary patterns, pathologies, pharmaceutical interventions and more. Furthermore, it can be utilized to uncover bacterial subpopulations and functional pathways within the gut microbiota that play essential roles in the onset or progression of specific diseases, as well as the effectiveness of drugs.

 

VITA GutMicrobiome: A New Era in Gut Microbiome Research

Our VITA GutMicrobiome High-Throughput Single-Bacterium Transcriptome Kit succsesfully overcomes the limitations that previously hindered the application of high-throughput single-bacterium transcriptome technologies to microbiota samples. This breakthrough signifies a paradigm shift in gut microbiome research, transcending the conventional boundaries of functional genomics and species identification, entering a new era of in-depth and precise exploration of functional and phenotyopic diverstiy.

Having undergone assessment on over 500 microbiota samples already, the VITA GutMicrobiome Kit significantly expands the horizons of gut microbiome studies, paving the way for breakthroughs in clinical research and physiology studies, as well as drug development. We are confident that our innovative solution will empower researchers to achieve unparalleled outcomes in their endeavors.

Figure 9. VITA product family

 References:

[1] Lee KA, et al. The gut microbiome: what the oncologist ought to know. Br J Cancer. 2021; 125(9): 1197-1209.

[2] de Vos WM, et al. Gut microbiome and health: mechanistic insights. Gut. 2022; 71(5): 1020-1032.

[3] Liu Q, et al. Gut microbiota dynamics in a prospective cohort of patients with post-acute COVID-19 syndrome. Gut. 2022; 71(3): 544-552.

[4] Andersson, D.I., Nicoloff, H. & Hjort, K. Mechanisms and clinical relevance of bacterial heteroresistance. Nat Rev Microbiol. 2019; 17, 479–496.

[5] Xu Z, et al. Droplet-based high-throughput single microbe RNA sequencing by smRandom-seq. Nat Commun. 2023; 14(1): 5130.

[6] Martins BM & Locke JC. Microbial individuality: how single-cell heterogeneity enables population level strategies. Curr Opin Microbiol. 2015; 24: 104-12.

[7] Blattman, S.B., et al. Prokaryotic single-cell RNA sequencing by in situ combinatorial indexing.  Nat Microbiol. 2020; 5(10): 1192-1201.

[8] Kuchina, A., et al. Microbial single-cell RNA sequencing by split-pool barcoding. Science. 2021; 371(6531): eaba5257.

[9] Ma P, et al. Bacterial droplet-based single-cell RNA-seq reveals antibiotic-associated heterogeneous cellular states. Cell. 2023; 186(4): 877-891.

[10] Sakon H, et al. Sutterella parvirubra sp. nov. and Megamonas funiformis sp. nov., isolated from human faeces. Int J Syst Evol Microbiol. 2008; 58(Pt 4): 970-975.

[11] Lee, G, et al. Distinct signatures of gut microbiome and metabolites associated with significant fibrosis in non-obese NAFLD. Nat Commun. 2020; 11: 4982.

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