Draft Genome Sequence of Bacillus safensis Strain Sami, Isolated from Leaf Veins of Ficus religiosa.

Here, I report the draft genome sequence of a novel Bacillus safensis strain, Sami, isolated from leaf veins of Ficus religiosa F. religiosa is a large tree native to the Indian subcontinent and Indochina. The draft genome of B. safensis is 3.67 Mb.

ANNOUNCEMENT

Bacillus safensis is normally considered a rhizobacterium and is found in root nodules of members of the Fabaceae family of plants (1). B. safensis is considered a plant growth promoter (1). Here, I report the draft genome sequence of a novel B. safensis strain, Sami, which was isolated from Ficus religiosa leaf veins. This is a new habitat of B. safensis (2).

To isolate the organism, the first step was to remove all kinds of contamination on the outer surface of the leaves, as B. safensis resides inside the leaf veins. Leaves of F. religiosa were washed with ethanol and sodium hypochlorite. After the leaves were washed, they were dried with flame. Leaf veins were cut with a blade and streaked onto nutrient agar at 37°C for 24 h. To extract genomic DNA for whole-genome sequencing, bacterial culture was grown in tryptic soy broth at 37°C. DNA extraction was performed using Qiagen’s DNeasy UltraClean microbial kit, and DNA integrity was checked by using gel electrophoresis. Library construction was performed using Illumina Nextera XT DNA library prep kits. The final library was quantified by quantitative PCR (qPCR) and an Agilent Bioanalyzer. The insert size for libraries was 350 bp.

The Illumina NextSeq 500 platform was used for sequencing the paired-end library. A total of 8,980,391 reads were generated after sequencing was performed. The read length was 2 × 75 bp, and the mean quality (Q) score for the sequence reads was 30. MiSeq Reporter v3.0 software with default parameters was used for adapter trimming. Those sequences which had >90% sequence similarity to adapter sequences were trimmed. The mean length of sequence reads after quality trimming was 72 bp. These high-quality paired-end reads were assembled by using the Unicycler assembler v0.4.6.0 with default parameters (3). Contigs having less than 0.25 of the chromosomal depth were filtered out. For SPAdes assembly, the lowest k-mer size was 0.2, and the highest was 0.95 (expressed as fractions of read length). Contigs shorter than 100 bp were excluded from the FASTA file.

Unicycler generated 30 contigs with a total length of 3,672,548 bp and an N50 value of 528,055. The mean sequence coverage depth of the final genome assembly was 30×. A total of 3,692 protein-coding regions were predicted using the NCBI Prokaryotic Genome Annotation Pipeline (4). One possible CRISPR locus was also predicted using CRISPRfinder (5; see also http://crispr.i2bc.paris-saclay.fr/). The GC content is 41.6% (6). After a genome BLAST search of the NCBI database, comparison with closely matching species was drawn using average nucleotide identity (ANI) values (7; see also http://enve-omics.ce.gatech.edu/ani). During ANI calculation, a window size of 1,000 bp and a step size of 200 bp were selected for fragment options. ANI values determined that B. safensis FO-36b (GenBank accession number CP010405), with an ANI value of 97.5%, is the closest species whose genome is available.

Data availability.

This sequence was submitted to NCBI GenBank under accession number CP032830. Raw sequence reads were also deposited in the NCBI Sequence Read Archive under accession number SRR8181849.