Nov 14, 2025
Isolation and hybrid genome assembly of Bacillus subtilis SNUY1
- Gwon Mo Yang1,
- Na Yun Kim1,
- Donghwa Chung1,2,
- Hyo Jin Kim1,2
- 1Graduate School of International Agricultural Technology, Seoul National University, Pyeongchang 25354, Republic of Korea;
- 2Institutes of Green Bio Science and Technology, Seoul National University, Pyeongchang 25354, Republic of Korea
- Seoul National University
Protocol Citation: Gwon Mo Yang, Na Yun Kim, Donghwa Chung, Hyo Jin Kim 2025. Isolation and hybrid genome assembly of Bacillus subtilis SNUY1. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkyjmdg5r/v1
Manuscript citation:
Isolation and hybrid genome assembly of Bacillus subtilis SNUY1. Protocols.io. DOI: [insert DOI here]
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 14, 2025
Last Modified: November 14, 2025
Protocol Integer ID: 232401
Keywords: antifungal activity, Bacillus subtilis SNUY1, edible coating, hybrid sequencing, hybrid genome assembly of bacillus subtilis snuy1, bacillus subtilis snuy1 from doenjang, bacillus subtilis snuy1, hybrid genome assembly, sequencing taxonomic identification, genome sequencing, taxonomic identification
Funders Acknowledgements:
Ministry of Trade, Industry and Energy (MOTIE, Korea)
Grant ID: A100-20250023
Disclaimer
This protocol was developed and optimized specifically for the isolation and genome sequencing of Bacillus subtilis SNUY1 from a Doenjang sample. The described materials and methods are provided as a reference framework for similar studies but are not intended as universal procedures. Laboratories are encouraged to adjust parameters such as media composition, incubation temperature, and DNA quantification settings according to their strain characteristics and facility protocols.
Abstract
This protocol minimally documents the procedures used to isolate Bacillus subtilis SNUY1 from Doenjang, perform pre-sequencing taxonomic identification (near-full-length 16S rRNA; Sanger), and report DNA concentration and purity measured for whole-genome sequencing. Values are reported as dataset-specific, non-prescriptive information.
Guidelines
1. Quantify extracted DNA using the QuantiFluor dsDNA System (Promega) with a Victor Nivo reader (PerkinElmer).
2. Assess purity with a NanoPhotometer (IMPLEN) to verify the A260/280 ratio.
3. Representative values for the SNUY1 preparation (approximately 439 ng/μL DNA and an A260/280 ratio of ~1.8) are provided for reference only; actual concentrations may vary depending on strain, sample quality, and instrument calibration.
Materials
Agar/broth media: LA/LB, NA/NB, TSA/TSB, MRS
Sterile distilled water (DW)
Glycerol (molecular biology grade)
PrepMan Ultra (Thermo Fisher Scientific)
QuantiFluor dsDNA System (Promega) and Victor Nivo reader (PerkinElmer)
NanoPhotometer (IMPLEN)
Troubleshooting
Section 1. Sampling and strain isolation (used for SNUY1)
Homogenize 1 g of Doenjang in 9 mL sterile distilled water; perform serial 10-fold dilutions.
Spread on LA, NA, TSA, and MRS agar; incubate at 30°C and 37°C until well-isolated colonies appear.
Inoculate picked colonies into 5 mL LB, NB, TSB, or MRS; incubate 24 h at 30°C and/or 37°C (pre-cultures).
Inoculate the corresponding broth at 1% (v/v) and incubate 24 h (main cultures). Prepare glycerol stocks at 25% (v/v) and store at −80°C. For genome DNA preparation, subculture SNUY1 on TSA and grow in TSB at 30°C.
Section 2. Pre-sequencing taxonomic identification (near-full-length 16S rRNA; Sanger)
Extract DNA from a single colony (PrepMan Ultra; per manufacturer).
PCR primers: 27F (5′-AGAGTTTGATCMTGGCTCAG-3′), 1492R (5′-TACGGYTACCTTGTTACGACTT-3′). Cycling: 94°C 5 min; 30×(94°C 30 s, 55°C 30 s, 72°C 90 s); 72°C 10 min.
Sanger sequencing with internal primers 785F (5′-GGATTAGATACCCTGGTA-3′) and 907R (5′-CCGTCAATTCMTTTGAGTTT-3′).
Analyze the assembled sequence using BLASTN for taxonomic identification. Example result (for reference only): The best match for the tested isolate was Bacillus subtilis (post-assembly ANI using pyani v0.2.7: 99.87% ANI, 98.3% alignment coverage vs GCF_045037735.1).
Section 3. DNA concentration and purity (reporting only)
Quantify extracted DNA using the QuantiFluor dsDNA System (Promega) with a Victor Nivo reader (PerkinElmer).
Assess purity with a NanoPhotometer (IMPLEN) to verify the A260/280 ratio.
Representative values for the SNUY1 preparation (approximately 439 ng/μL DNA and an A260/280 ratio of ~1.8) are provided for reference only; actual concentrations may vary depending on strain, sample quality, and instrument calibration.
Protocol references
Genome assembly for B. subtilis SNUY1 available under GenBank accession CP199918.
Acknowledgements
Ministry of Trade, Industry and Energy (MOTIE)
Grand ID: 20018683, Development of Technology for Manufacturing Biomass-based Cellulose Fibers and Commercializing Edible Coating