小川 雅人 (コガワ マサト)

写真a

所属

研究院(研究機関) ナノ・ライフ創新研究機構

職名

次席研究員(研究院講師)

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  • 修士

 

研究分野 【 表示 / 非表示

  • 分子生物学

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

    Rieka Chijiiwa, Masahito Hosokawa, Masato Kogawa, Yohei Nishikawa, Keigo Ide, Chikako Sakanashi, Kai Takahashi, Haruko Takeyama

    Microbiome   8 ( 1 ) 5 - 5  2020年01月  [査読有り]  [国際誌]

     概要を見る

    BACKGROUND: The 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 single-cell 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. RESULTS: Inulin-feeding changed the mouse fecal microbiome composition to increase Bacteroides spp., resulting in the production of abundant succinate 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 2 phyla, 4 classes, 7 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. CONCLUSIONS: Our single-cell genomics approach generated a massive amount of SAGs, enabling a functional analysis of uncultured bacteria in the intestinal microbiome. This enabled us to estimate metabolic lineages involved in the bacterial 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. Video abstract.

    DOI PubMed

  • High-throughput identification of peptide agonists against GPCRs by co-culture of mammalian reporter cells and peptide-secreting yeast cells using droplet microfluidics.

    Yaginuma K, Aoki W, Miura N, Ohtani Y, Aburaya S, Kogawa M, Nishikawa Y, Hosokawa M, Takeyama H, Ueda M

    Scientific reports   9 ( 1 ) 10920  2019年07月  [査読有り]

    DOI PubMed

  • Obtaining high-quality draft genomes from uncultured microbes by cleaning and co-assembly of single-cell amplified genomes.

    Masato Kogawa, Masahito Hosokawa, Yohei Nishikawa, Kazuki Mori, Haruko Takeyama

    Scientific reports   8 ( 1 ) 2059 - 2059  2018年02月  [査読有り]  [国際誌]

     概要を見る

    Single-cell genomics is a straightforward approach to obtain genomes from uncultured microbes. However, sequence reads from a single-cell amplified genome (SAG) contain significant bias and chimeric sequences. Here, we describe Cleaning and Co-assembly of a Single-Cell Amplified Genome (ccSAG), a novel analytical workflow to obtain composite single-cell genomes with elimination of sequence errors. By the integration of ccSAG with a massively parallel single-cell genome amplification platform based on droplet microfluidics, we can generate multiple SAGs and effectively integrate them into the composite genomes quality equivalent to the data obtained from bulk DNA. We obtained two novel draft genomes from single gut microbial cells with high completeness (>96.6%) and extremely low contamination (<1.25%). Moreover, we revealed the presence of single nucleotide polymorphisms in the specific gene by sequence comparison at the single-cell level. Thus, the workflow yields near-complete genomes from uncultured microbes, and enables analyses of genetic heterogeneity within identical strains.

    DOI PubMed

  • Massively parallel whole genome amplification for single-cell sequencing using droplet microfluidics

    Masahito Hosokawa, Yohei Nishikawa, Masato Kogawa, Haruko Takeyama

    Scientific Reports   7 ( 1 ) 5199  2017年12月  [査読有り]

     概要を見る

    Massively parallel single-cell genome sequencing is required to further understand genetic diversities in complex biological systems. Whole genome amplification (WGA) is the first step for single-cell sequencing, but its throughput and accuracy are insufficient in conventional reaction platforms. Here, we introduce single droplet multiple displacement amplification (sd-MDA), a method that enables massively parallel amplification of single cell genomes while maintaining sequence accuracy and specificity. Tens of thousands of single cells are compartmentalized in millions of picoliter droplets and then subjected to lysis and WGA by passive droplet fusion in microfluidic channels. Because single cells are isolated in compartments, their genomes are amplified to saturation without contamination. This enables the high-throughput acquisition of contamination-free and cell specific sequence reads from single cells (21,000 single-cells/h), resulting in enhancement of the sequence data quality compared to conventional methods. This method allowed WGA of both single bacterial cells and human cancer cells. The obtained sequencing coverage rivals those of conventional techniques with superior sequence quality. In addition, we also demonstrate de novo assembly of uncultured soil bacteria and obtain draft genomes from single cell sequencing. This sd-MDA is promising for flexible and scalable use in single-cell sequencing.

    DOI PubMed

  • Analysis of environmental bacteria at single-cell level

    Masahito Hosokawa, Yohei Nishikawa, Masato Kogawa, Haruko Takeyama

    TRANSDUCERS 2017 - 19th International Conference on Solid-State Sensors, Actuators and Microsystems     634 - 637  2017年07月  [査読有り]

     概要を見る

    Single-cell genomics has enabled the exploration of cellular diversity in environmental microbes. However, current genome sequencing techniques, which utilizes next-generation sequencing (NGS), typically require nanogram to microgram levels of input DNA sample. Since single bacterial cells contain only a few femtograms of DNA, we have to amplify their genomes to adequate amount for sequencing. We aimed to develop a novel system for precise and high throughput single-cell genomics, to elucidate environmental microbial diversity. In this study, we have developed droplet-based microfluidic system to produce the compartmentalized reaction vessels for single-cell genome sequencing.

    DOI

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