Virtual microfluidics for digital quantification and single-cell sequencing

Xu, Li; Brito, Ilana L.; Alm, Eric J.; Blainey, Paul C.

doi:10.1038/nmeth.3955

Cited by 86 publications

(90 citation statements)

References 38 publications

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“…According to the definition used in Almeida et al [16], 40% of the draft genomes of quality higher than medium were qualified as near-complete (> 90% completeness and < 5% contamination, Additional file 1: Table S5). Hence, SAG-gel platform can provide increased numbers of draft genomes from single microbial cells, with 14 higher completeness than other single-cell genome sequencing techniques [22,23,[29][30][31][32].…”

Section: Microbiome Composition Monitoring Demonstrated Rapid Responsmentioning

confidence: 99%

Single-cell genomics of uncultured bacteria reveals dietary fiber responders in the mouse gut microbiota

Chijiiwa

Hosokawa

Kogawa

et al. 2019

Preprint

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BackgroundThe gut microbiota can have dramatic effects on host metabolism; however, current genomic strategies for uncultured bacteria have several limitations that hinder their ability to identify responders to metabolic changes in the microbiota. In this study, we describe a novel singlecell genomic sequencing technique that can identify metabolic responders at the species level without the need for reference genomes, and apply this method to identify bacterial responders to an inulin-based diet in the mouse gut microbiota. ResultsInulin feeding changed the mouse fecal microbiome composition to increase Bacteroides spp., and increased microbial metabolic function resulted in increased succinate concentrations in the mouse intestine. Using our massively parallel single-cell genome sequencing technique, named SAG-gel platform, we obtained 346 single-amplified genomes (SAGs) from mouse gut microbes before and after dietary inulin supplementation. After quality control, the SAGs were classified as 267 bacteria, spanning two phyla, four classes, seven orders, and 14 families, and 31 different strains of SAGs were graded as high-and medium-quality draft genomes. From these, we have successfully obtained the genomes of the dominant inulin responders, Bacteroides spp., and identified their polysaccharide utilization loci and their specific metabolic pathways for succinate production. ConclusionsOur single-cell genomics approach generated a massive amount of SAGs, enabling functional analysis of uncultured bacteria in the intestinal microbiome and consideration of host responses. This enabled us to estimate metabolic lineages involved in the bacterial 2 fermentation of dietary fiber and metabolic outcomes such as short-chain fatty acid production in the intestinal environment based on the fibers ingested. The technique allows the in-depth isolation and characterization of uncultured bacteria with specific functions in the microbiota, and could be exploited to improve human and animal health. BackgroundThe gut microbiota plays a crucial role in the control of host physiology and metabolism through the release and transformation of metabolites like short-chain fatty acids (SCFAs) [1,2]. Many studies have demonstrated that dietary fiber supplementation can modulate gut microbiota composition and promote SCFA production [3][4][5]. This dietary fiber metabolism is particularly important for non-digestible inulin-type fructans classified as prebiotics, which are substrates that promote the growth of beneficial microorganisms in the gut [6][7][8][9][10][11][12]. Thus, to enhance the benefits of dietary fiber to the host, it is important to identify inulin responders in the complex microbiome community of the host intestine, and characterize their mechanisms of inulin fermentation and potential for SCFA production.Recently developed metagenomic approaches can provide comprehensive microbial profiling and putative functions using 16S rRNA gene amplicon sequencing and shotgun metagenome sequencing. These methods have enabled the ...

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Section: Microbiome Composition Monitoring Demonstrated Rapid Responsmentioning

confidence: 99%

Single-cell genomics of uncultured bacteria reveals dietary fiber responders in the mouse gut microbiota

Chijiiwa

Hosokawa

Kogawa

et al. 2019

Preprint

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“…Following an additional sample rinse with ddH 2 O and aspiration, these two distinct components were mixed by pipetting and immediately added in 20 ul volumes (as a combined mixture) to the sample to allow for a 10.8% w/v hydrogel to polymerize for 1 hour at room temperature. This hydrogel would slow diffusion during the amplification reaction (18).…”

Section: In Situ Preparationmentioning

confidence: 99%

DNA microscopy: Optics-free spatio-genetic imaging by a stand-alone chemical reaction

Weinstein

Regev

Zhang

2018

Preprint

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Analyzing the spatial organization of molecules in cells and tissues is a cornerstone of biological research and clinical practice. However, despite enormous progress in profiling the molecular constituents of cells, spatially mapping these constituents remains a disjointed and machinery-intensive process, relying on either light microscopy or direct physical registration and capture. Here, we demonstrate DNA microscopy, a new imaging modality for scalable, optics-free mapping of relative biomolecule positions. In DNA microscopy of transcripts, transcript molecules are tagged in situ with randomized nucleotides, labeling each molecule uniquely. A second in situ reaction then amplifies the tagged molecules, concatenates the resulting copies, and adds new randomized nucleotides to uniquely label each concatenation event. An algorithm decodes molecular proximities from these concatenated sequences, and infers physical images of the original transcripts at cellular resolution. Because its imaging power derives entirely from diffusive molecular dynamics, DNA microscopy constitutes a chemically encoded microscopy system.

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“…Xu et al . developed a hydrogel-based virtual microfluidics for MDA 53 . It relied on hydrogel-limited diffusion to compartmentalize templates and reaction products.…”

Section: Genomicsmentioning

confidence: 99%

“…It relied on hydrogel-limited diffusion to compartmentalize templates and reaction products. It is an alternative to the complicated microfluidic system for compartmentalization 53 . They covalently crosslinked poly(ethylene glycol) (PEG) hydrogels under mild conditions which did not damage templates or inhibit subsequent reactions.…”

Section: Genomicsmentioning

confidence: 99%

Microfluidics for genome-wide studies involving next generation sequencing

Murphy

2017

Biomicrofluidics

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Next-generation sequencing (NGS) has revolutionized how molecular biology studies are conducted. Its decreasing cost and increasing throughput permit profiling of genomic, transcriptomic, and epigenomic features for a wide range of applications. Microfluidics has been proven to be highly complementary to NGS technology with its unique capabilities for handling small volumes of samples and providing platforms for automation, integration, and multiplexing. In this article, we review recent progress on applying microfluidics to facilitate genome-wide studies. We emphasize on several technical aspects of NGS and how they benefit from coupling with microfluidic technology. We also summarize recent efforts on developing microfluidic technology for genomic, transcriptomic, and epigenomic studies, with emphasis on single cell analysis. We envision rapid growth in these directions, driven by the needs for testing scarce primary cell samples from patients in the context of precision medicine.

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Virtual microfluidics for digital quantification and single-cell sequencing

Cited by 86 publications

References 38 publications

Single-cell genomics of uncultured bacteria reveals dietary fiber responders in the mouse gut microbiota

Single-cell genomics of uncultured bacteria reveals dietary fiber responders in the mouse gut microbiota

DNA microscopy: Optics-free spatio-genetic imaging by a stand-alone chemical reaction

Microfluidics for genome-wide studies involving next generation sequencing

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