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Genome Sequences

High-Quality Draft Single-Cell Genome Sequences of Two Gammaproteobacteria Strains Sampled from Soil in a Strawberry Farm

Takuya Yoda, Koji Arikawa, Tatsuya Saeki, Ayumi Matsuhashi, Masahito Hosokawa
J. Cameron Thrash, Editor
Takuya Yoda
abitBiome, Inc., Tokyo, Japan
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Koji Arikawa
abitBiome, Inc., Tokyo, Japan
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Tatsuya Saeki
abitBiome, Inc., Tokyo, Japan
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Ayumi Matsuhashi
abitBiome, Inc., Tokyo, Japan
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Masahito Hosokawa
abitBiome, Inc., Tokyo, Japan
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J. Cameron Thrash
University of Southern California
Roles: Editor
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DOI: 10.1128/MRA.00743-20
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ABSTRACT

Here, we present high-quality draft single-cell genome sequences of Gammaproteobacteria strains BBSC-SA01 and BBSC-SA02, obtained from uncultivated cells of soil in a strawberry farm using the single-cell sequencing platform bit-MAP. These draft genomes putatively represent novel species within Gammaproteobacteria and allow further investigation into the soil microbiome.

ANNOUNCEMENT

Gammaproteobacteria is a class of Gram-negative bacteria comprising 17 orders and plays an important role in crop and animal health in various soil microbiomes (1, 2). Although their universal distribution and prevalence in the soil environment are revealed by 16S rRNA gene amplicon sequencing and shotgun metagenomic sequencing, most of them remain uncultivated at this time (3). Therefore, an expansion of the reference genomes of Gammaproteobacteria would lead to an improved understanding of their genetic diversity and relationship with the environment and other microbial species. In our present study, we sequenced two uncultivated strains of Gammaproteobacteria using the single-cell genome sequencing platform bit-MAP, originally named SAG-gel (4).

Soil sampling was conducted in two strawberry cultivation greenhouses before harvest in December 2019 (Okawa, Fukuoka, Japan). A total of 20 g of soil sample was collected at a depth of 5 to 10 cm around the seedlings. To extract the bacterial fraction, 1.5 g of the soil samples was suspended in 3 ml of Dulbecco’s phosphate-buffered saline (DPBS). The mixture was vortexed and allowed to settle for 15 min and then filtered to separate the soil particles. Single cells were isolated into gel beads, and their genomes were amplified using the REPLI-g kit (Qiagen) following the SAG-gel method (4). Single gel beads harboring a single-cell amplified genome (SAG) were sorted into individual wells in a 96-well microplate (Axygen) using BD FACSMelody (BD Biosciences) and then reamplified with the REPLI-g kit. A pooled paired-end sequence library (300 × 2 bp) of 24 SAGs was prepared using the Nextera XT DNA sample preparation kit (Illumina) and sequenced using a MiSeq sequencer (Illumina). Default settings were used for data analysis unless otherwise noted. Sequence reads were filtered using BBduk v38.79 (https://sourceforge.net/projects/bbmap/) with the parameters qtrim=r, trimq=10, minlength=40, maxns=1, and minavgquality=15; they were assembled with SPAdes v3.14.0 (5) using the settings –t 4, --sc, --careful, and --disable-rr, and contigs of >1,000 bp were retained for the following analysis. Completeness and contamination were calculated using CheckM v1.1.2 (6) using the option --reduced_tree. The number of contigs, N50 value, and total length of SAGs were evaluated with QUAST v5.0.2 (7). The number of tRNAs was examined with Prokka v1.14.6 (8) using the option --mincontiglen 200. The 16S rRNA gene sequence was extracted with Prokka and assigned to RefSeq with a BLASTn v2.9.0+ search (9). The SAGs were taxonomically classified with GTDB-Tk v1.1.1 (10), and the average nucleotide identities (ANI) were calculated with sendsketch.sh v38.79 (https://www.biostars.org/p/234837/) against the RefSeq genome database (NCBI).

The genome statistics are given in Table 1. Based on the operational standards for SAGs (11), they are classified as high-quality draft genomes. They were classified as Legionellales bacterium UBA4722 (strain BBSC-SA01) and Gammaproteobacteria bacterium RIFCSPHIGHO2_12_FULL_45_12 (strain BBSC-SA02) with GTDB-tk. Comparison with 16S rRNA gene sequences using RefSeq found the two most closely related 16S rRNA sequences to be Legionella dresdenensis strain W03-356 (GenBank accession number NR_115062.1, 88.38%) and Aquicella siphonis strain SGT-108 (NR_025764.1, 94.553%), respectively. With respect to the maximum ANI among all available RefSeq genomes, the highest similarities of BBSC-SA01 and BBSC-SA02 were observed with Methylophaga sulfidovorans (73.4%) and Aquicella lusitana (78.2%), respectively. The ANI between the two genomes was only 63.4%, suggesting that BBSC-SA01 and BBSC-SA02 were distantly related to each other.

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TABLE 1

Genome statistics of BBSC-SA01 and BBSC-SA02

Data availability.The genome sequences reported in this article were deposited in DDBJ/ENA/GenBank under BioProject number PRJDB10155 and the accession numbers given in Table 1. The versions described in this paper are the first versions.

ACKNOWLEDGMENTS

This work was supported by the Ministry of Agriculture, Forestry, and Fisheries.

The supercomputing resource was provided by the Human Genome Center (University of Tokyo).

FOOTNOTES

    • Received 13 July 2020.
    • Accepted 10 August 2020.
    • Published 27 August 2020.
  • Copyright © 2020 Yoda et al.

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

REFERENCES

  1. 1.↵
    1. Fierer N
    . 2017. Embracing the unknown: disentangling the complexities of the soil microbiome. Nat Rev Microbiol 15:579–590. doi:10.1038/nrmicro.2017.87.
    OpenUrlCrossRefPubMed
  2. 2.↵
    1. Benhizia Y,
    2. Benhizia H,
    3. Benguedouar A,
    4. Muresu R,
    5. Giacomini A,
    6. Squartini A
    . 2004. Gamma proteobacteria can nodulate legumes of the genus Hedysarum. Syst Appl Microbiol 27:462–468. doi:10.1078/0723202041438527.
    OpenUrlCrossRefPubMedWeb of Science
  3. 3.↵
    1. Pham V,
    2. Kim J
    . 2012. Cultivation of unculturable soil bacteria. Trends Biotechnol 30:475–484. doi:10.1016/j.tibtech.2012.05.007.
    OpenUrlCrossRefPubMed
  4. 4.↵
    1. Chijiiwa R,
    2. Hosokawa M,
    3. Kogawa M,
    4. Nishikawa Y,
    5. Ide K,
    6. Sakanashi C,
    7. Takahashi K,
    8. Takeyama H
    . 2020. Single-cell genomics of uncultured bacteria reveals dietary fiber responders in the mouse gut microbiota. Microbiome 8:5. doi:10.1186/s40168-019-0779-2.
    OpenUrlCrossRef
  5. 5.↵
    1. Bankevich A,
    2. Nurk S,
    3. Antipov D,
    4. Gurevich A,
    5. Dvorkin M,
    6. Kulikov A,
    7. Lesin V,
    8. Nikolenko S,
    9. Pham S,
    10. Prjibelski A,
    11. Pyshkin A,
    12. Sirotkin A,
    13. Vyahhi N,
    14. Tesler G,
    15. Alekseyev M,
    16. Pevzner P
    . 2012. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 19:455–477. doi:10.1089/cmb.2012.0021.
    OpenUrlCrossRefPubMed
  6. 6.↵
    1. Parks D,
    2. Imelfort M,
    3. Skennerton C,
    4. Hugenholtz P,
    5. Tyson G
    . 2015. CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. Genome Res 25:1043–1055. doi:10.1101/gr.186072.114.
    OpenUrlAbstract/FREE Full Text
  7. 7.↵
    1. Gurevich A,
    2. Saveliev V,
    3. Vyahhi N,
    4. Tesler G
    . 2013. QUAST: quality assessment tool for genome assemblies. Bioinformatics 29:1072–1075. doi:10.1093/bioinformatics/btt086.
    OpenUrlCrossRefPubMedWeb of Science
  8. 8.↵
    1. Seemann T
    . 2014. Prokka: rapid prokaryotic genome annotation. Bioinformatics 30:2068–2069. doi:10.1093/bioinformatics/btu153.
    OpenUrlCrossRefPubMedWeb of Science
  9. 9.↵
    1. Altschul SF,
    2. Gish W,
    3. Miller W,
    4. Myers EW,
    5. Lipman DJ
    . 1990. Basic local alignment search tool. J Mol Biol 215:403–410. doi:10.1016/S0022-2836(05)80360-2.
    OpenUrlCrossRefPubMedWeb of Science
  10. 10.↵
    1. Parks D,
    2. Chuvochina M,
    3. Waite D,
    4. Rinke C,
    5. Skarshewski A,
    6. Chaumeil P,
    7. Hugenholtz P
    . 2018. A standardized bacterial taxonomy based on genome phylogeny substantially revises the tree of life. Nat Biotechnol 36:996–1004. doi:10.1038/nbt.4229.
    OpenUrlCrossRefPubMed
  11. 11.↵
    1. Bowers RM,
    2. Kyrpides NC,
    3. Stepanauskas R,
    4. Harmon-Smith M,
    5. Doud D,
    6. Reddy TBK,
    7. Schulz F,
    8. Jarett J,
    9. Rivers AR,
    10. Eloe-Fadrosh EA,
    11. Tringe SG,
    12. Ivanova NN,
    13. Copeland A,
    14. Clum A,
    15. Becraft ED,
    16. Malmstrom RR,
    17. Birren B,
    18. Podar M,
    19. Bork P,
    20. Weinstock GM,
    21. Garrity GM,
    22. Dodsworth JA,
    23. Yooseph S,
    24. Sutton G,
    25. Glöckner FO,
    26. Gilbert JA,
    27. Nelson WC,
    28. Hallam SJ,
    29. Jungbluth SP,
    30. Ettema TJG,
    31. Tighe S,
    32. Konstantinidis KT,
    33. Liu W-T,
    34. Baker BJ,
    35. Rattei T,
    36. Eisen JA,
    37. Hedlund B,
    38. McMahon KD,
    39. Fierer N,
    40. Knight R,
    41. Finn R,
    42. Cochrane G,
    43. Karsch-Mizrachi I,
    44. Tyson GW,
    45. Rinke C,
    46. Lapidus A,
    47. Meyer F,
    48. Yilmaz P,
    49. Parks DH,
    50. Eren AM,
    51. Schriml L,
    52. Banfield JF,
    53. Hugenholtz P,
    54. Woyke T, Genome Standards Consortium
    . 2017. Minimum information about a single amplified genome (MISAG) and a metagenome-assembled genome (MIMAG) of bacteria and archaea. Nat Biotechnol 35:725–731. doi:10.1038/nbt.3893.
    OpenUrlCrossRef
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High-Quality Draft Single-Cell Genome Sequences of Two Gammaproteobacteria Strains Sampled from Soil in a Strawberry Farm
Takuya Yoda, Koji Arikawa, Tatsuya Saeki, Ayumi Matsuhashi, Masahito Hosokawa
Microbiology Resource Announcements Aug 2020, 9 (35) e00743-20; DOI: 10.1128/MRA.00743-20

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High-Quality Draft Single-Cell Genome Sequences of Two Gammaproteobacteria Strains Sampled from Soil in a Strawberry Farm
Takuya Yoda, Koji Arikawa, Tatsuya Saeki, Ayumi Matsuhashi, Masahito Hosokawa
Microbiology Resource Announcements Aug 2020, 9 (35) e00743-20; DOI: 10.1128/MRA.00743-20
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