Complete genome sequence of the hyperthermophilic archaeon Saccharolobus islandicus Y08.82.36 isolated from a Yellowstone hot spring

TL;DR

This paper presents the full genome sequence of a heat-loving archaeon from Yellowstone hot springs.

Contribution

The novel contribution is the complete genome assembly of Saccharolobus islandicus Y08.82.36.

Findings

The genome of Saccharolobus islandicus Y08.82.36 has been fully sequenced and assembled.

The archaeon was isolated from a hot spring in Yellowstone National Park.

Abstract

Members of the order Sulfolobales are known to thrive in acidic, high-temperature hot springs. Here, we report the sequencing and complete genome assembly of a hyperthermophilic archaeon Saccharolobus islandicus Y08.82.36, isolated from a hot spring in the Norris Geyser Basin, Yellowstone National Park, USA, in 2008.

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ANNOUNCEMENT

Saccharolobus islandicus (formerly Sulfolobus islandicus) is an aerobic, hyperthermophilic archaeon that was first isolated by Zillig et al. from a hot spring in Iceland (1). Subsequently, S. islandicus has been found in hot springs worldwide, establishing it as one of a few archaeal species suitable for studying population biology and evolutionary genomics (2–4). We previously reported an S. islandicus isolate Y08.82.36 that serves as a host for propagating a range of archaeal viruses (5–7) but lacked a complete genome necessary for studying host-virus interactions. Here, we use PacBio and Illumina sequencing to resolve the full genome of this strain.

Strain Y08.82.36 was originally isolated from a hot spring sample collected in 2008 from the Norris Geyser Basin, Yellowstone National Park, USA (44.7276°N, 110.7144°W). Hot spring sediment samples were collected in sterile Falcon tubes and stored at room temperature during transport. For isolation, 1 mL of sample was inoculated into 19 mL of DT medium (8) and incubated at 76°C with shaking for 10 days. Enrichment cultures were serially diluted and plated to obtain a single colony using the double-layer plating approach (9), which was then grown in 1 mL DT medium for 3 days without shaking. This culture was transferred into 19 mL of fresh medium and incubated for another 3 days before preparing frozen stocks for storage at −80 °C in 8% (vol/vol) DMSO. High-molecular-weight (HMW) DNA was obtained using the Nanobind CBB Kit (Pacific Biosciences), and total genomic DNA for Illumina sequencing was extracted from the same culture using the DNeasy Blood & Tissue Kit (Qiagen).

For PacBio sequencing, 1 µg of HMW DNA was sheared to an average fragment length of 10 kb using a Megaruptor 3 (Diagenode) and converted into a library using a SMRTbell Express Template Prep Kit 3.0 (Pacific Biosciences). This library was pooled and sequenced on a SMRT cell 25M on a PacBio Revio. Demultiplexing and circular consensus sequence generation were performed using SMRT Link v13.1, requiring three polymerase passes and a read quality of at least 0.99, yielding 24,276 HiFi reads. Filtlong v0.2.1 (10) was used to retain the best 95% of bases by quality, resulting in 22,778 HiFi reads (average length = 6,115 bp; N50 = 6,534 bp; average read quality = 30.2).

Illumina sequencing libraries were prepared from 150 ng DNA using an Illumina DNA Prep Kit (Illumina) and sequenced using a MiSeq with v2 reagents (paired-end; 2 × 250 bp) to produce 496,069 raw reads that were analyzed with FastQC v0.11.9 (11). Adapter removal and quality filtering were performed using Trimmomatic v0.39 (12), yielding 431,982 reads with an average read quality ≥33.0.

All tools were run using default parameters. HiFi reads were assembled with the Autocycler v0.4.0 pipeline (13). Autocycler subsampled the reads and assembled each independent subset using Canu v2.4 (14), Flye v2.9.5 (15), Miniasm v0.3 (16) and Minipolish v0.1.3 (17), NextDenovo v2.5.2 (18) and NextPolish v1.4.1 (19), and Raven v1.8.3 (20). The assembled genome was polished using the Illumina reads and Polypolish v0.6.0 (21). Read mapping to the polished assembly was validated using Breseq v0.39.0 (22). The assembly produced a single, circular contig with a total size of 2,633,435 bp, 53× (PacBio) and 74× (Illumina) coverage, and an average GC content of 35.33%.

Y08.82.36 was predicted to have 2,973 genes using the NCBI Prokaryotic Genome Annotation Pipeline v6.10 (23), including 47 tRNAs and 3 rRNAs. Analysis by CRISPRCasTyper v1.8.0 (24) identified three arrays associated with a complete Type I-D CRISPR-Cas system containing 20, 34, and 77 spacers and an orphan Type III-B system. Notably, Y08.82.36 lacks a Type I-A CRISPR-Cas system, which is typically present in S. islandicus strains.