Complete Genome Sequence of a Gram-Positive Bacterium, Leifsonia sp. Strain PS1209, a Potato Endophyte

ANNOUNCEMENT

In 2019, we isolated a Gram-positive endophyte from apparently healthy tubers of potato cultivar NY166 (potato breeding program, Cornell University). This endophyte persisted in bud tissues following surface sterilization (10% commercial bleach). It was isolated at room temperature after being grown from intact bud tissue using nutrient broth (1) and Richardson's solution (2) after 2 to 3 days of incubation. The sequence of the 16S rRNA gene (3) amplified from genomic DNA showed 99.9% identity to the 16S rRNA gene of Leifsonia lichenia 2Sb^T (4).

A single-colony isolate of Leifsonia sp. PS1209 was stored in 20% glycerol at −80°C and cultured in Luria-Bertani medium (5) at 28°C. High-molecular-weight DNA was extracted from overnight bacterial cultures (after approximately three passages after isolation) using a Wizard genomic DNA purification kit (Promega, USA). DNA quantity and integrity were assessed using a NanoDrop 1000 spectrophotometer and Qubit 3.0 fluorometer (Thermo Fisher Scientific, USA) and a 2100 Bioanalyzer system (Agilent, USA), respectively.

For long-read sequencing, the DNA library was prepared using the rapid sequencing kit SQK-RAD004 on an R9.4.1 SpotON FLO-MIN106 flow cell and was sequenced with a MinION Mk1B device. Base calling was performed with Guppy v3.5.1. This generated 376,226 reads with a total length of 2,465,189,756 bp, an N₅₀ value of 13,374 bp, and genome coverage of 598×. The genome was de novo assembled using Flye v2.5 (6) and polished with Nanopolish v0.12.2a (7). For short-read sequencing, the same DNA as for Nanopore sequencing was used for library preparation. The DNA library was prepared using the NEBNext Ultra II FS DNA library preparation kit for Illumina with NEBNext multiplex oligonucleotides (New England Biolabs, USA). AMPure XP beads (Beckman Coulter, USA) were used for DNA size selection and purification. The DNA library was sequenced on a MiSeq instrument (Illumina, USA) with the 2 × 250-bp mode, yielding 1,877,134 paired-end reads. Duplicate read pairs were removed using a customized Perl script (https://github.com/Sunhh/NGS_data_processing/blob/master/drop_dup_both_end.pl) (8). Deduplicated reads were processed to remove the adaptors, low-quality sequences (Q < 20), and short reads (<50 bp) using Trimmomatic v0.39 (9). All software systems were run with their default settings unless otherwise noted. BWA v0.7.17 (10) and Pilon v1.22 (11) were used to align the resulting 1,590,269 high-quality cleaned Illumina read pairs to the draft genome and to correct the draft genome for three rounds, with a genome coverage of 128×. Circlator v1.5.5 was used with the fixstart argument to set the position number to start at the dnaA gene (12).

The circular Leifsonia sp. strain PS1209 genome is 4,091,164 bp long and was annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) v4.11 (13). It contains 3,926 genes in total, with 3,819 protein-coding genes, 52 pseudogenes, 2 5S rRNA-coding sequences, 2 16S rRNA-coding sequences, 2 23S rRNA-coding sequences, 46 tRNAs, and 3 noncoding RNAs. The GC content of PS1209 of 69.08% is 9% higher than that of 2Sb^T but similar to that of other Leifsonia spp. (4). The genome sequence of the type strain 2Sb is not publicly available but is needed to unambiguously determine the species of PS1209.