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Genome Sequences

Complete Genome Sequence of Bacillus mycoides TH26, Isolated from a Permafrost Thaw Gradient

Tracie Haan, Scout McDougall, Devin M. Drown
Catherine Putonti, Editor
Tracie Haan
aDepartment of Biology and Wildlife, University of Alaska Fairbanks, Fairbanks, Alaska, USA
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Scout McDougall
aDepartment of Biology and Wildlife, University of Alaska Fairbanks, Fairbanks, Alaska, USA
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Devin M. Drown
aDepartment of Biology and Wildlife, University of Alaska Fairbanks, Fairbanks, Alaska, USA
bInstitute of Arctic Biology, University of Alaska Fairbanks, Fairbanks, Alaska, USA
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  • ORCID record for Devin M. Drown
Catherine Putonti
Loyola University Chicago
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DOI: 10.1128/MRA.00507-19
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ABSTRACT

Here, we describe the complete genome assembly of a Bacillus mycoides isolate collected from a boreal forest soil associated with permafrost thaw. Using only long-read Nanopore sequences and VolTRAX library preparation, we assembled two circular contigs totaling 5,789,722 bp (N50, 5,306,036), a complete chromosome with one plasmid.

ANNOUNCEMENT

We collected soil from the Fairbanks Permafrost Experiment Station in Alaska (64.875646°N, 147.668981°W). We have been sampling at this site to understand how belowground microbial communities mediate the effect of permafrost thaw on aboveground plant communities in boreal and arctic ecosystems. The psychrotolerant Bacillus mycoides isolate described here comes from an undergraduate-driven project on the coselection of heavy metal tolerance and antibiotic resistance, and it is associated with a permafrost thaw feature.

We homogenized the active layer from two soil cores and added 1 g of homogenized sample to tryptic soy broth (TSB). After 48 h at 22°C, we plated a dilution on tryptic soy agar and used the streak plate method three times to purify our randomly selected isolate. From this purified isolate, we inoculated a liquid culture of TSB, incubated it at 22°C overnight, and used 1.8 ml to extract DNA using the DNeasy UltraClean microbial kit (Qiagen).

We used 1 μg of DNA as input for the VolTRAX V2 (Oxford Nanopore Technologies [ONT]) to prepare a sequencing library (VSK-VSK002 workflow). We sequenced the prepared library using a MinION device (ONT) and an r9.4.1 flow cell (FLO-MIN106) for 48 h (VSK002 script). We base called the raw data using Guppy v2.3.1 (ONT), specifying the flipflop model (–c dna_r9.4.1_450bps_flipflop.cfg) and default parameters (Table 1). Before assembly, we created a quality-controlled data set using Porechop v0.2.4 (https://github.com/rrwick/Porechop) with default parameters to trim adaptors and discard sequences with middle adapters (-discard_middle) and Filtlong v0.2.0 (https://github.com/rrwick/Filtlong) to filter by lengths of ≥1,000 bp (-min_length 1000) and quality (Q score) of ≥10 (-min_mean_q 90).

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TABLE 1

Summary of sequencing data statistics

Before genome assembly, we subsampled 1 Gb of the quality-controlled data using Filtlong (-length_weight 10 -target_bases 1000000000) and used the longer reads for assembly. We assembled the genome using Canu v1.8 (1) (coverage, 200×) with default parameters, specifying the estimated genome size (-genomeSize=5m) and Nanopore reads (-nanopore-raw). Before using medaka (https://github.com/nanoporetech/medaka) for polishing, ONT recommends 4 rounds of Racon v1.3.1 (2) polishing with the following parameters: score for matching bases (-match 8), score for mismatching bases (-mismatch -6), threshold for average base quality of windows (-quality-threshold -1), default gap penalty (-gap -8), and default window (-window-length 500). We ran a final polish with medaka v0.6.1, specifying the flipflop model (-m r941_flip) and default parameters. For both Racon and medaka polishing, we used the entire 11-Gb quality-controlled data set (coverage, 1,958×).

Our 5,789,722-bp (N50, 5,306,036) polished assembly consists of 2 circular contigs, a 5,306,036-bp chromosome (GC content, 35.57%), and a 483,686-bp plasmid (GC content, 33.56%). We validated our assembly at multiple checkpoints using CheckM v1.0.11 (3) in the following lineage-specific workflow (lineage_wf): before polishing (completeness, 83.14%; contamination, 0.15%), after initial polishing with Racon (completeness, 94.67%; contamination, 0.07%), and after final Medaka polishing (completeness 98.21%; contamination, 0.15%).

We used PATRIC v3.5.36 (4) to assign 107 tRNAs, 28 rRNAs, and 6,458 coding sequences (CDS). PATRIC assigned the isolate as a member of the Bacillus cereus group, with close identity to the psychrotolerant species Bacillus mycoides. To assign taxonomy, we used blastn (5) to search against the NCBI 16S rRNA database and found 100% identity to Bacillus mycoides (strain DSM 11821). Therefore, we assign this isolate as Bacillus mycoides TH26.

Data availability.This whole-genome shotgun project has been deposited in GenBank under the accession no. CP037991 and CP037992. The versions described in this paper are the first versions, CP037991.1 and CP037992.1. Raw data for this project can be found in the GenBank SRA under accession no. PRJNA525875.

ACKNOWLEDGMENTS

Many thanks go to Ursel Schütte, Maddie McCarthy, Taylor Seitz, Jennie Humphrey, and Anne-Lise Ducluzeau for laboratory help. Tom Douglas from the Cold Regions Research and Engineering Laboratory (CRREL) Alaska provided field site access.

We acknowledge generous support from the Institute of Arctic Biology, Alaska INBRE, and the BLaST program. Research reported here was supported by BLaST through the National Institute of General Medical Sciences of the National Institutes of Health under awards UL1GM118991, TL4GM118992, and RL5GM118990. Research reported in this publication was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant P20GM103395.

FOOTNOTES

    • Received 1 May 2019.
    • Accepted 16 May 2019.
    • Published 13 June 2019.
  • Copyright © 2019 Haan et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

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Complete Genome Sequence of Bacillus mycoides TH26, Isolated from a Permafrost Thaw Gradient
Tracie Haan, Scout McDougall, Devin M. Drown
Microbiology Resource Announcements Jun 2019, 8 (24) e00507-19; DOI: 10.1128/MRA.00507-19

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Complete Genome Sequence of Bacillus mycoides TH26, Isolated from a Permafrost Thaw Gradient
Tracie Haan, Scout McDougall, Devin M. Drown
Microbiology Resource Announcements Jun 2019, 8 (24) e00507-19; DOI: 10.1128/MRA.00507-19
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