Complete genome sequence of Indonesian probiotic strain Lactiplantibacillus plantarum subsp. plantarum Dad-13

ABSTRACT Previous studies have investigated the probiotic properties of L. plantarum subsp. plantarum Dad-13. Nevertheless, genomic sequence data from previous studies were not yet available to support each probiotic characteristic. This study focused on the complete genome sequence of the strain to validate its role in specific probiotic properties.

otic strain discovered by isolating it from dadih, a fermented milk product made from buffalo milk that originated in Minangkabau, Indonesia, in 1997.After 16s rRNA sequencing, the strain was identified as Lactobacillus plantarum (1), and its taxological name was changed to Lactiplantibacillus plantarum subsp.plantarum.Preceding studies have reported the survival ability in the gastrointestinal tract (2)(3)(4), safety (5), and physiological benefits (6-9) of the strain.Clinical studies have reported positive health effects of L. plantarum Dad-13 (10)(11)(12).The strain has been used in numerous probi otic foods such as fermented milk, yogurt, and chocolate.Although previous genomic analysis have been conducted (6,7), the genome sequence analysis in this study further analyzes the strain using Nanopore to assemble a complete genome.
The sequencing process used default parameters for all software unless otherwise specified.The study used the first passage from the freeze-dried collection of the strain (FNCC0461) which was obtained from the Food and Nutrition Culture Collection.The strain was inoculated in de Man Rogosa Sharpe (MRS) broth media (Merck, Darmstadt, Germany; catalog number: 110661) and incubated at 37°C for 24 h.Then, one single colony was taken for sequencing.
The DNA of the strain was isolated using Zymo Research Quick-DNA HMW Mag Bead Kit (D6060 version) with ligation of sequencing adapters.DNA concentration was determined using both NanoDrop spectrophotometers and Qubit fluorometers.Library preparations, including DNA shearing, used Oxford Nanopore Technology kits.GridION sequencing was operated by MinKNOW software version 20.06.9.Base calling was performed using Guppy version 4.0.11with high accuracy mode (13).All FASTQ files were filtered using Flitlong software (Ryan R. Wick, https://github.com/rrwick/Filtlong),and the quality was visualized using NanoPlot (14).De novo assembly and the circularization of contigs were conducted using Flye software version 2.8.1 (15).Then, the data were polished with Medaka software (ONT Research, https://github.com/nanoporetech/medaka).The assembled contig was aligned to the reference genome using Mauve version 2.4.0 (15) to detect any misassemblies or overlaps between contigs.Annotation was conducted using DFAST software (16) and RAST webserver (https://rast.nmpdr.org/).The assembled genomes and annotation were assessed using Busco (17) and CheckM (18) software.The annotated protein sequence was used for BLAST database (BLAST program from https://blast.ncbi.nlm.nih.gov/) to identify matches in the database for at least 90% identity and 90% length.
It must be noted that the genome assembly in this study was only performed using Nanopore reads where sequencing and assembly errors can occur.After the analysis, there was a total genome size of 3,404,719 bp, 44.3% G + C content, N50 value of 3,184,937 bp, 331 subsystems, 3,186 coding sequences, 2 contigs (with protein-encoding genes), and RNAs.Specific probiotic characteristic genes, according to FAO/WHO (19), are shown in Table 1.

TABLE 1
List of putative genes present in L. plantarum Dad-13 that are responsible for probiotic characteristics