Thirteen draft genome assemblies of Bacillus spp. isolated from HLB-impacted citrus trees

Abstract

We report the draft genome assembly, annotation and phylogenetic placement of 13 Bacillus spp. isolated from citrus groves under high (Florida) or low (California) Huanglongbing (HLB) disease pressure.

Introduction

Bacillus is a well-studied bacterial genus of Gram-positive and spore-forming bacteria, commonly soil-borne and plant-associated [1-3]. Many Bacillus species produce a range of secondary metabolites and volatile organic compounds that are beneficial to plants through various mechanisms, such as pathogen suppression or plant growth promotion [4-7]. Genome availability is the initial step in unlocking the beneficial metabolic potential of plant-associated Bacillus spp. by enabling the identification of bioactive compound-producing gene clusters. Here we announce the draft genome assemblies of thirteen Bacillus spp. isolated either from the root, leaf or stem tissue of citrus trees in areas of high (Florida) or low (California) Huanglongbing (HLB) disease pressure.

Publication

The publication by Vieira et al. can be found here: in preparation for submission to Microbiology Resource Announcements.

External Data Availability

The Sequence Read Archive and genomic data has been deposited in GenBank BioProject under no. PRJNA1046128 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1046128). The version described in this paper is the first version.

1. Background and Experimental Methods

Sample Collection

Sampling of plant material and bacterial isolation are described in [8] as part of a bioprospecting effort designed to generate a citrus-associated microbial repository. Briefly, leaves, stems, and roots from citrus trees were collected from citrus orchards in Florida and in California. Each sample was macerated with 1X PBS and 100 ul of the slurry was plated on tryptic soy agar (TSA) and potato dextrose agar plates with 0.1 g/liter tetracycline hydrochloride (PDA). Plates were incubated at 28C for 4 days. The microbial consortia on each plate were scrapped after adding 1 ml of 1X PBS to generate bulk culture tubes. Isolates were recovered from bulk culture tubes by plating on TSA and PDA and incubating plates at 28C for up to 5 days. Single colony of bacterial isolates were streaked onto TSA plates until pure cultures were obtained. Long-term stocks of each isolate were made in 15% glycerol (final concentration) and stored at -80C.

Isolation

For each isolate, DNA was extracted from a single colony grown overnight at 28C in tryptic soy broth (TSB), using the Wizard® Genomic DNA Purification Kit (Promega Corporation, Madison, Wisconsin, USA), following manufacturer’s instructions for Gram-positive bacteria.

Genome Sequencing

Library preparation and sequencing were performed by SeqCenter (Pittsburgh, Pennsylvania, USA). Sample libraries were prepared using the Illumina DNA Prep kit and IDT 10 bp unique dual indexing (UDI) indices, and sequenced on an Illumina NovaSeq 6000, producing 2x151 bp reads. Demultiplexing, quality control and adapter trimming was performed with bcl-convert (v4.0.3) [9]. The total of paired-end raw reads recovered ranged from 2,560,758 to 13,949,264.

2. Import

Raw reads were imported through the staging area into the narrative as non-interleaved reads.

from biokbase.narrative.jobs.appmanager import AppManager
AppManager().run_app_batch(
    [{
        "app_id": "kb_uploadmethods/import_fastq_noninterleaved_as_reads_from_staging",
        "tag": "release",
        "version": "5b9346463df88a422ff5d4f4cba421679f63c73f",
        "params": [{
            "fastq_fwd_staging_file_name": "CB0069_S156_R1_001.fastq.gz",
            "fastq_rev_staging_file_name": "CB0069_S156_R2_001.fastq.gz",
            "name": "CB0069v2_reads"
        }, {
            "fastq_fwd_staging_file_name": "CB0742_S157_R1_001.fastq.gz",
            "fastq_rev_staging_file_name": "CB0742_S157_R2_001.fastq.gz",
            "name": "CB0742_reads"
        }],
        "shared_params": {
            "sequencing_tech": "Illumina",
            "single_genome": 1,
            "read_orientation_outward": 0,
            "insert_size_std_dev": None,
            "insert_size_mean": None
        }
    }],
    cell_id="e77909e3-3e87-4be5-b112-54d13dfcae65",
    run_id="531891a7-d530-43d0-aa32-4c30da6b3fd7"
)

from biokbase.narrative.jobs.appmanager import AppManager
AppManager().run_app_batch(
    [{
        "app_id": "kb_uploadmethods/import_fastq_noninterleaved_as_reads_from_staging",
        "tag": "release",
        "version": "5b9346463df88a422ff5d4f4cba421679f63c73f",
        "params": [{
            "fastq_fwd_staging_file_name": "CB0069_1.fq.gz",
            "fastq_rev_staging_file_name": "CB0069_2.fq.gz",
            "name": "CB0069_reads"
        }],
        "shared_params": {
            "sequencing_tech": "Illumina",
            "single_genome": 1,
            "read_orientation_outward": 0,
            "insert_size_std_dev": None,
            "insert_size_mean": None
        }
    }],
    cell_id="ade04936-e295-489b-87b2-a5a9fd34bf2d",
    run_id="918efa19-54e2-47c1-a347-d6fb677a4e45"
)

from biokbase.narrative.jobs.appmanager import AppManager
AppManager().run_app_batch(
    [{
        "app_id": "kb_uploadmethods/import_fastq_noninterleaved_as_reads_from_staging",
        "tag": "release",
        "version": "5b9346463df88a422ff5d4f4cba421679f63c73f",
        "params": [{
            "fastq_fwd_staging_file_name": "893_S98_R1_001.fastq.gz",
            "fastq_rev_staging_file_name": "893_S98_R2_001.fastq.gz",
            "name": "CB893_reads"
        }, {
            "fastq_fwd_staging_file_name": "676_S96_R1_001.fastq.gz",
            "fastq_rev_staging_file_name": "676_S96_R2_001.fastq.gz",
            "name": "CB676_reads"
        }],
        "shared_params": {
            "sequencing_tech": "Illumina",
            "single_genome": 1,
            "read_orientation_outward": 0,
            "insert_size_std_dev": None,
            "insert_size_mean": None
        }
    }],
    cell_id="81509afe-ef90-4bd5-b7c8-0da15afcac08",
    run_id="72bf7f52-a547-4210-bf69-8385bff2b906"
)

from biokbase.narrative.jobs.appmanager import AppManager
AppManager().run_app_batch(
    [{
        "app_id": "kb_uploadmethods/import_fastq_noninterleaved_as_reads_from_staging",
        "tag": "release",
        "version": "5b9346463df88a422ff5d4f4cba421679f63c73f",
        "params": [{
            "fastq_fwd_staging_file_name": "Copy of cb0027_1.fq.gz",
            "fastq_rev_staging_file_name": "Copy of cb0027_2.fq.gz",
            "name": "CB0027_reads"
        }, {
            "fastq_fwd_staging_file_name": "Copy of cb0021_1.fq.gz",
            "fastq_rev_staging_file_name": "Copy of cb0021_2.fq.gz",
            "name": "CB0021_reads"
        }, {
            "fastq_fwd_staging_file_name": "Copy of cb0019_1.fq.gz",
            "fastq_rev_staging_file_name": "Copy of cb0019_2.fq.gz",
            "name": "CB0019_reads"
        }],
        "shared_params": {
            "sequencing_tech": "Illumina",
            "single_genome": 1,
            "read_orientation_outward": 0,
            "insert_size_std_dev": None,
            "insert_size_mean": None
        }
    }],
    cell_id="3a92803f-718b-4ab2-9950-7f66ee3cadbd",
    run_id="594621e9-628f-432a-b0b0-268dfaaa24ef"
)

from biokbase.narrative.jobs.appmanager import AppManager
AppManager().run_app_batch(
    [{
        "app_id": "kb_uploadmethods/import_fastq_noninterleaved_as_reads_from_staging",
        "tag": "release",
        "version": "5b9346463df88a422ff5d4f4cba421679f63c73f",
        "params": [{
            "fastq_fwd_staging_file_name": "CB909_S144_R1_001.fastq.gz",
            "fastq_rev_staging_file_name": "CB909_S144_R2_001.fastq.gz",
            "name": "CB909"
        }, {
            "fastq_fwd_staging_file_name": "CB687_S152_R1_001.fastq.gz",
            "fastq_rev_staging_file_name": "CB687_S152_R2_001.fastq.gz",
            "name": "CB687"
        }, {
            "fastq_fwd_staging_file_name": "CB912_S156_R1_001.fastq.gz",
            "fastq_rev_staging_file_name": "CB912_S156_R2_001.fastq.gz",
            "name": "CB912"
        }],
        "shared_params": {
            "sequencing_tech": "Illumina",
            "single_genome": 1,
            "read_orientation_outward": 0,
            "insert_size_std_dev": None,
            "insert_size_mean": None
        }
    }],
    cell_id="538b7113-c0a0-4bae-811d-fbbde312b5fc",
    run_id="ca75ce03-d77d-4918-9de0-a83f872c4612"
)

from biokbase.narrative.jobs.appmanager import AppManager
AppManager().run_app_batch(
    [{
        "app_id": "kb_uploadmethods/import_fastq_noninterleaved_as_reads_from_staging",
        "tag": "release",
        "version": "5b9346463df88a422ff5d4f4cba421679f63c73f",
        "params": [{
            "fastq_fwd_staging_file_name": "CB729_S153_R1_001.fastq.gz",
            "fastq_rev_staging_file_name": "CB729_S153_R2_001.fastq.gz",
            "name": "CB729_reads"
        }, {
            "fastq_fwd_staging_file_name": "CB902_S142_R1_001.fastq.gz",
            "fastq_rev_staging_file_name": "CB902_S142_R2_001.fastq.gz",
            "name": "CB902_reads"
        }, {
            "fastq_fwd_staging_file_name": "CB904_S143_R1_001.fastq.gz",
            "fastq_rev_staging_file_name": "CB904_S143_R2_001.fastq.gz",
            "name": "CB904_reads"
        }],
        "shared_params": {
            "sequencing_tech": "Illumina",
            "single_genome": 1,
            "read_orientation_outward": 0,
            "insert_size_std_dev": None,
            "insert_size_mean": None
        }
    }],
    cell_id="d9a39030-1c57-400b-9577-b1dd20961451",
    run_id="5cc84383-2a8d-40ae-8b2e-efd135c67424"
)

3. Genome Assembly

QC

Sequence analysis from quality control to annotation was performed on the KBase web service [10] and the publicly available narrative containing all analyses and data can be found at https://narrative.kbase.us/narrative/157793. Default parameters were used except where otherwise noted. Quality of raw reads were assessed using FastQC (v0.12.1) [11] and quality control was performed using JGI RQCFilter pipeline BBTools (v38.22) [12] and PRINSEQ (v0.20.4) [13].

Assembly

Genomes were assembled using SPAdes (v3.15.3) [14] with k-mer sizes set for 21, 33, 55, 77, 91, 111. The quality of assemblies was assessed with QUAST (v4.4) [15] and CheckM (v1.0.18) [16], demonstrating that the number of contigs in the resulting assemblies ranged from 14 to 87 and the N50 value ranged from 164,068 to 1,085,110. The completeness of the genomes was between 99.38 to 99.59 and the GC content percentage ranged from 34.95 to 46.26.

4. Genome Annotation

The genomes were annotated using Prokka (v1.14.5) [17], revealing the largest genome size of 5,551,588 bp from Bacillus cereus, and the smallest genome size of 3,677,118 bp from Bacillus safensis. The number of predicted genes among the genomes ranged from 3,742 to 5,644.

5. Taxonomic Identification

Taxonomic identification was performed on GTDB-Tk (v2.3.2) [18]. A Bacillus phylogenetic tree with closely related species was constructed using FastTree2 (v2.1.11) [19] through SpeciesTree (v2.2.0), on KBase, and annotated on iTOL (v6.8.1) [20]

6. References

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Created Object Name	Type	Description
CB0069_RQC_prinseq_100923	PairedEndLibrary	Filtered Paired End Reads
CB0069_RQC_prinseq_100923_fwd_singletons	SingleEndLibrary	Filtered Forward Unpaired End Reads
CB0069_RQC_prinseq_100923_rev_singletons	SingleEndLibrary	Filtered Reverse Unpaired End Reads

Created Object Name	Type	Description
CB0019_RQC_prinseq_100923	PairedEndLibrary	Filtered Paired End Reads
CB0019_RQC_prinseq_100923_fwd_singletons	SingleEndLibrary	Filtered Forward Unpaired End Reads
CB0019_RQC_prinseq_100923_rev_singletons	SingleEndLibrary	Filtered Reverse Unpaired End Reads

Created Object Name	Type	Description
CB0021_RQC_prinseq_100923	PairedEndLibrary	Filtered Paired End Reads
CB0021_RQC_prinseq_100923_fwd_singletons	SingleEndLibrary	Filtered Forward Unpaired End Reads
CB0021_RQC_prinseq_100923_rev_singletons	SingleEndLibrary	Filtered Reverse Unpaired End Reads

Created Object Name	Type	Description
CB0027_RQC_prinseq_100923	PairedEndLibrary	Filtered Paired End Reads
CB0027_RQC_prinseq_100923_fwd_singletons	SingleEndLibrary	Filtered Forward Unpaired End Reads
CB0027_RQC_prinseq_100923_rev_singletons	SingleEndLibrary	Filtered Reverse Unpaired End Reads

Created Object Name	Type	Description
CB0687_RQC_prinseq_100923	PairedEndLibrary	Filtered Paired End Reads
CB0687_RQC_prinseq_100923_fwd_singletons	SingleEndLibrary	Filtered Forward Unpaired End Reads
CB0687_RQC_prinseq_100923_rev_singletons	SingleEndLibrary	Filtered Reverse Unpaired End Reads