Computational protocol: Draft Genome Sequence of a Novel Thermofilum sp. Strain from a New Zealand Hot Spring Enrichment Culture

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Protocol publication

[…] Members of the crenarchaeal family Thermofilaceae () are widespread in terrestrial and marine hot springs (, ), with only two valid described species, Thermofilum pendens from Iceland () and T. uzonense from Kamchatka, Russia (). Both have sequenced genomes (, ). Additional genomes are available for Thermofilum spp. from Kamchatka, “T. adornatus” 1910b () and “T. carboxyditrophus” 1505 (GenBank assembly accession number GCA_000813245). With the exception of “T. librum” (), all Thermofilum spp. require Thermoproteales cell extracts or spent culture broth for growth. The extreme reduction in biosynthetic pathways for purines, amino acids, and cofactors in all Thermofilum genomes analyzed thus far suggests that these organotrophic thermophiles rely on other organisms and the environment for these small molecules. Here, we report the nearly complete draft genome of a new Thermofilum sp. strain, NZ13, obtained from a hot spring at Hell’s Gate (Tikitere) (38°03′47″S, 176°21′39″E; pH 6.0 and 74°C) in New Zealand.Enrichments were maintained stably for 4 days at 80°C in a modified DSMZ medium (number 88) containing yeast extract (0.5 g/liter), tryptone (0.5 g/liter), and a headspace of hydrogen (100% vol/vol). DNA was extracted from the enrichment using the Qiagen DNAeasy blood and tissue DNA kit. Nextera DNA libraries were sequenced using an Illumina MiSeq instrument. Adaptors and low-quality reads were removed using Trimmomatic () and assembled using IDBA-UD (). Contigs of less than 1 kb were removed. The contigs were binned using MaxBin (), resulting in the nearly complete genome of Thermofilum sp. strain NZ13. Three additional bins for a Fervidicoccus sp., a Desulfurococcaceae sp., and a Pyrobaculum sp. were also generated. Optimization of the Thermofilum sp. NZ13 assembly was achieved by mapping the reads back using Bowtie 2 () and SAMtools (, ), reassembled using IDBA-UD, and further validated with Emergent Self-Organizing Maps (ESOM) (). The draft genome (33 contigs) is 1.88 Mb and has 56.7% G+C content, which is higher than that of some Thermofilum genomes but similar to that of T. pendens. The genome completeness is about 99.3%, with very low (0.7%) contamination (CheckM []). The genome was annotated using Prodigal () and the arCOG database (, ). A total of 1,930 coding sequences, 47 tRNAs, and 5 clustered regularly interspaced short palindromic repeat (CRISPR) arrays were identified. Based on a RAxML concatenated phylogenetic tree of 16 ribosomal proteins and a 16S rRNA gene comparison using EzBioCloud () (98.4% 16S rRNA gene similarity to T. uzonense), Thermofilum sp. NZ13 probably represents a new species in the genus Thermofilum.Like its relatives, the Thermofilum sp. NZ13 genome encodes numerous peptidases, glucosidases, and an alpha amylase, suggesting that the organism can grow on a range of peptides, polysaccharides, and starches. Additionally, Thermofilum sp. NZ13 shows a large reduction in biosynthetic genes, indicating that, similar to T. pendens and T. uzonense, it likely relies on the environment for most amino acids, purines, and cofactors. The genome encodes a large number of ABC transporter genes, with a high proportion being membrane transport proteins. This draft genome from a Thermofilum sp. from New Zealand illustrates that globally, species in this genus share very similar ecological niches. […]

Pipeline specifications

Software tools Trimmomatic, IDBA-UD, MaxBin, Bowtie, SAMtools, CheckM, Prodigal, RAxML
Databases EzBioCloud
Applications Phylogenetics, De novo sequencing analysis
Chemicals Amino Acids, Purines