Hiroyoshi Aoki’s research while affiliated with Advanced Photonics and other places

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Publications (3)


Schematic view of an agarose gel microcapsule (AGM) and its preparation process. (a) Schematic diagram of the AGM developed in this study for single-cell isolation and multiple displacement amplification (MDA). The AGM consists of an agarose hydrogel shell and an alginate sol core. The alginate core provides a picolitre-scale reaction chamber for MDA, while the agarose shell acts both as a permeable envelope for enzymes and small molecules and as a protective wall against larger particles and molecules such as bacterial cells and genomic/amplified DNA. (b) Preparation scheme for AGMs containing Escherichia coli cell(s). The alginate cores and agarose shells are gelated with Ca²⁺ from CaCO3 and cooled in an emulsion, respectively. Polyglyceryl-6 octacaprylate (PGO) can suppress the aggregation of AGMs during agarose gelation by cooling. Finally, the alginate gel cores are solated with EDTA by chelating Ca²⁺. ISA isostearyl alcohol.
Yield enhancement of multiple displacement amplification (MDA) by alginate core solation. The effect of alginate core solation on the productivity of MDA within agarose gel microcapsules (AGMs) was evaluated. The genomic DNA of Escherichia coli encapsulated within AGMs was amplified by MDA with or without alginate solation using EDTA. The amplified DNA was detected with SYBR Green I. A phase contrast image (red) and epifluorescent image (green, SYBR Green I) are overlaid. MDA in agarose gel beads (without an alginate core), which was prepared using gelation of agarose droplets containing E. coli in oil emulsion, was also conducted as a control experiment. Arrows indicate E. coli cells stained with SYBR Green I. Bar = 100 µm.
Comparison of genome completeness and amplification bias of single-cell genomes obtained by MDA-in-AGM and FACS-MDA. (a) Completeness and number of contigs of SAGs during the sequencing process. Box-and-violin plots (blue: MDA-in-AGM; red: FACS-MDA) are shown for Escherichia coli and two bacterial species as examples from the mock community of human gut bacteria (Supplementary Tables S2, S5 and S6). Datapoints (purple circles) and their arithmetic means (rhombuses) are shown on the plots. In the E. coli graphs, the results for DNA prepared from cultured E. coli cells without MDA are also shown (green circles). In the left panels, significant differences are indicated with asterisks (P < 0.05) or double asterisks (P < 0.01). (b) The number of sequence reads mapped against genome regions is shown as an indicator of amplification bias caused during MDA. The numbers of reads corresponding to different coverages used for de novo assembly (shown as ‘Cov.’) and genome completeness (%) (‘Compl.’) estimated using CheckM are shown.
Relationships between genome completeness and GC contents of single-cell genomes in the mock community of human gut bacteria. Single-cell amplified genomes (SAGs) from the mock community comprising 15 strains of human gut bacteria are plotted with their GC contents against their genome completeness. The SAGs were assembled from 0.3 M randomly chosen read pairs, while MS485-1-51 of MDA-in-AGM (0.26 M read pairs) and CS1–94 of FACS-MDA (0.29 M read pairs) used all read pairs since they were fewer than 0.3 M. The bacterial species comprising the mock community and detailed results are shown in Supplementary Tables S4–S6. Blue and red indicate MDA-in-AGM and FACS-MDA, respectively. Squares, circles and rhombuses indicate gram-positive, gram-negative and gram-variable bacteria, respectively.
Agarose gel microcapsules enable easy-to-prepare, picolitre-scale, single-cell genomics, yielding high-coverage genome sequences
  • Article
  • Full-text available

October 2022

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110 Reads

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6 Citations

Hiroyoshi Aoki

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Michiru Shimizu

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Yutaka Yamagata

A novel type of agarose gel microcapsule (AGM), consisting of an alginate picolitre sol core and an agarose gel shell, was developed to obtain high-quality, single-cell, amplified genomic DNA of bacteria. The AGM is easy to prepare in a stable emulsion with oil of water-equivalent density, which prevents AGM aggregation, with only standard laboratory equipment. Single cells from a pure culture of Escherichia coli, a mock community comprising 15 strains of human gut bacteria, and a termite gut bacterial community were encapsulated within AGMs, and their genomic DNA samples were prepared with massively parallel amplifications in a tube. The genome sequencing did not need second-round amplification and showed an average genome completeness that was much higher than that obtained using a conventional amplification method on the microlitre scale, regardless of the genomic guanine–cytosine content. Our novel method using AGM will allow many researchers to perform single-cell genomics easily and effectively, and can accelerate genomic analysis of yet-uncultured microorganisms.

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Agarose gel microcapsules enable easy-to-prepare, picolitre-scale single-cell genomics, yielding near-complete genome sequences

January 2021

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190 Reads

A novel type of agarose gel microcapsule (AGM), consisting of an alginate picolitre sol core and an agarose gel shell, was developed to obtain high-quality single-cell amplified genomic DNA of bacteria. The AGM is easy to prepare in a stable emulsion with oil of water-equivalent density which prevents AGM aggregation, with only standard laboratory equipment. Single cells from a pure culture of Escherichia coli , a mock community comprising 15 strains of human gut bacteria, and a termite gut bacterial community were encapsulated within AGMs, and their genomic DNAs were obtained with massively parallel amplifications in a tube. The genome sequencing did not need second-round amplification, and showed an average genome completeness that was much higher than that obtained by the conventional amplification method in microlitre scale, regardless of the genomic guanine-cytosine contents. Our novel method using AGMs allows many researchers to perform single-cell genomics easily and effectively, and can enhance the genome analysis of yet-uncultured microorganisms.


Fig. 4
Agarose Gel Microcapsules Enable Easy-to-prepare, Picolitre-scale Single-cell Genomics, Yielding Near-complete Genome Sequences

January 2021

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13 Reads

A novel type of agarose gel microcapsule (AGM), consisting of an alginate picolitre sol core and an agarose gel shell, was developed to obtain high-quality single-cell amplified genomic DNA of bacteria. The AGM is easy to prepare in a stable emulsion with oil of water-equivalent density which prevents AGM aggregation, with only standard laboratory equipment. Single cells from a pure culture of Escherichia coli , a mock community comprising 15 strains of human gut bacteria, and a termite gut bacterial community were encapsulated within AGMs, and their genomic DNA samples were prepared with massively parallel amplifications in a tube. The genome sequencing did not need second-round amplification, and showed an average genome completeness that was much higher than that obtained using a conventional amplification method in microlitre scale, regardless of the genomic guanine-cytosine contents. Our novel method using the AGM allows many researchers to perform single-cell genomics easily and effectively, and can accelerate the genome analysis of yet-uncultured microorganisms.