High-quality draft genome sequence of Thermobifida halotolerans DSM 44931

ABSTRACT

Here, we report the genome sequence of Thermobifida halotolerans DSM 44931, a bacterium that was originally isolated from a salt mine in the Yunnan Province of China. This genome was sequenced using Pacific Biosciences sequencing technology and was assembled into 2 contigs in 2 scaffolds. It has a total length of 5,506,851 bp and a GC content of 71.16%. Functional annotation of this genome provides further metabolic insight into this species.

ANNOUNCEMENT

Thermobifida halotolerans is an aerobic, gram-positive, and mesophilic bacterial species that was isolated from a salt mine in the Yunnan Province of China (1). It is a filamentous actinomycete whose genome encodes for enzymes that can degrade synthetic polymers and several types of biomass (2–5). It has previously been limited to a draft genome with 29× coverage, 74 scaffolds, and 371 contigs (GenBank accession number LIZN00000000) (6). This work presents an improved high-quality draft genome of T. halotolerans DSM 44931.

T. halotolerans DSM 44931 was purchased from the Leibniz Institute DSMZ and cultivated in 100 mL of Czapek peptone media for 7 days at 37°C. 20 mL of this culture was centrifuged at 4649 × g at room temperature for 8 minutes. High molecular weight genomic DNA was extracted from the pelleted sample using a modified cetyltrimethylammonium bromide protocol (7).

The quality and quantity of the extracted DNA were evaluated using an Agilent 2200 TapeStation and an Invitrogen Qubit 2.0 Fluorometer, respectively. The draft genome of T. halotolerans DSM 44931 was generated at the Department of Energy (DOE) Joint Genome Institute (JGI) using the Pacific Biosciences (PacBio) sequencing technology (8). Default parameters were used for all software unless otherwise specified. A >10 kb PacBio SMRTbellTM library was constructed and sequenced on the PacBio Sequel platform, which generated 81,163 high-fidelity CCS reads totaling 763,311,996 bp (Fig. 1). BBDuk in BBTools v38.98 was used to filter out low-quality reads (9). The input read coverage was 139.4×. Reads >5 kb were assembled with Flye v2.8.3 (10). The final draft assembly contained 2 contigs in 2 scaffolds, totaling 5,506,851 bp in size with a GC content of 71.16% (Table 1). The contigs were determined to be linear by Circlator v1.5.5 (11). The quality of this assembly was evaluated using tRNAscan-SE v2.0.4 (12) to count tRNAs; Barrnap v0.9-Dev (13) to determine the presence of the 5S, 16S, and 23S rRNA genes; and CheckM2 v1.1.0 (14) to estimate completeness and contamination (Table 1).

Fig 1.

A histogram of the length frequencies of the CCS reads generated by the PacBio Sequel platform.

TABLE 1.

Genome assembly statistics, quality assessment, and annotation statistics for T. halotolerans DSM 44931

Metric	Assembly statistic
Total number of scaffolds	2
Total number of contigs	2
Total scaffold sequence length (bp)	5,506,851
Total contig sequence length (bp)	5,506,851
Scaffold N50 (bp)	5,450,929
Contig N50 (bp)	5,450,929
Pre-filtered CCS read N50 (bp)	11,849
Largest contig (bp)	5,450,929
Number of scaffolds >50 kb	2
Percent of genome in scaffolds >50 kb	100%
Percent of reads assembled	100%

^a GC percentage shown as count of G's and C's divided by the total number of bases. The total number of bases is not necessarily synonymous with a total number of G's, C's, A's, and T's.

^b Regulatory or miscellaneous genes are genes that are not classified as a protein-coding gene, a type of RNA, or a pseudogene, but as 'unknown' or 'other' by the source provider.

The genome draft assembly was annotated using the JGI Integrated Microbial Genomes (15) annotation pipeline v5.1.9 (16). INFERNAL v1.1.2 (17) was used to search against the Rfam 13.0 (18) database (excluding tRNA and CRISPR models) to identify structural RNAs and regulatory motifs; GeneMark.hmm-2 v1.05 (19) and Prodigal v2.6.3 (20) to identify protein-coding genes; tRNAscan-SE v2.0.4 (12) to identify tRNAs; and CRT v1.8.2 (21) to predict CRISPR arrays. Functional annotation was assigned to the genes via lastal 1256 (22) and HMMER v3.1b2 (23) using the following databases: IMG-NR 20211118 (24), SMART 01_06_2016 (25), COG 2003 (26), TIGRFAM v15.0 (27), SuperFamily v1.75 (28), Pfam v34.0 (29), and Cath-Funfam v4.2.0 (30). Topological annotation of protein-coding genes was assigned using SignalP 4.1 (31) and decodeanhmm 1.1g (32). These annotations categorize this genome into protein-coding genes, regulatory and miscellaneous features, and RNA genes; furthermore, they reveal predicted metabolic roles and clustering of the protein-coding genes (Table 1).

Overall, this high-quality draft genome improves our understanding of T. halotolerans biology and our ability to use this species and other actinomycetes for biotechnological applications.

DATA AVAILABILITY

The genome sequence was deposited to GenBank under the accession number JBGBYW000000000. The raw reads have been deposited in the NCBI SRA under the accession number SRP583730. Additional data can be explored or downloaded from the JGI Integrated Microbial Genomes with Microbiomes (IMG/M) portal using the NCBI BioProject accession number PRJNA1115251.