Bacterial genomic dna extraction kit

Genomic DNA was extracted with a bacterial genomic DNA extraction kit (Omega Bio‐tek, Inc.), according to the manufacturer's instructions, and the sequence was determined by the Illumina HiSeq 2000. The reads from the sequencing were assembled de novo using the Velvet 1.2.10 program. The genomes of the type strains that were similar to Y74^T were retrieved from GenBank. The average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) were used to assess the degree of similarity of each pair. The ANI was calculated with the JSpeciesWS (Richter, Rossello‐Mora, Glockner, & Peplies, 2016). The ANI could be divided into ANIb and ANIm, depending on the BLASTN (Basic Local Alignment Search Tool) algorithm or the MUMMER ultra‐rapid aligning tool. The dDDH was computed by an online tool, GGDC 2.0: the results of this computation were obtained using the recommended formula 2 (Meier‐Kolthoff, Auch, Klenk, & Goker, 2013). The genome of strain Y74^T was annotated using IMG Annotation Pipeline v.5.0.3 (Chen et al., 2019). The G + C content of the DNA of strain Y74^T was deduced from the genomic data. Y74^T horizontal gene transfer analysis by the method of Bertelli, Laird, and Williams (2017).

Bacterial genomic DNA extraction kit (No. D6942-01) was purchased from OMEGA Biotek Company. Bacterial genomic total RNA extraction kit (No. DP430) was purchased from Tiangen (Beijing) Company. DNA marker, restriction enzymes, reverse transcription reagent, Taq DNA polymerase, and other corresponding reagents were all purchased from TaKaRa Bao Bioengineering (Dalian) Co., Ltd. Competent cell E. coli DH5α was provided by Beijing ComWin Biotech Co., Ltd. T vector was purchased from TransGen Biotech (Beijing) Company. PCR amplified primers for bacteria nifH gene in high-throughput sequencing and nifH gene primers for qRT-PCR analysis were all used universal primers for bacterial nifH gene, and the primers were synthesized by Beijing Allwegene Technology Co., Ltd.
The main equipment used in this study was PCR amplification apparatus (Model number: ABI-2720), Real-Time PCR apparatus (Model number: ABI 7500), Biological analyzer (Model number: Agilent2100), GEL imaging system (Model number: Agilent2100), Ultraviolet spectrophotometer (NANODROP 2000) and Electrophoresis apparatus (Model number: DYY-6C).

The plaque and saliva mixtures were thawed in the centrifugal tube. After thawing, the saliva samples were centrifuged at 12,000 rpm for 10 min and the diposit was used for DNA extraction. A bacterial genomic DNA extraction kit (Omega Bio-Tek, Inc. USA) was used to extract the bacterial genomic DNA from the samples. The extraction procedure was carried out according to the instructions of the kit.