Genome sequencing and hybrid assembly of Edwardsiella ictaluri isolates recovered from various fish species

ABSTRACT

The bacterium Edwardsiella ictaluri causes disease in finfish and has been shown to infect multiple species. Eleven E. ictaluri genomes were sequenced, assembled, and annotated from a collection of isolates recovered from several different finfish species. This will serve as a valuable resource for comparative genomics studies.

ANNOUNCEMENT

The bacterium Edwardsiella ictaluri causes enteric septicemia of catfish (ESC). This pathogen can infect multiple fish species and can be devastating to aquaculture farms (1). Here, we sequenced, assembled, and annotated 11 genomes from a collection of isolates housed at the United States Department of Agriculture, Agricultural Research Service, Aquatic Animal Health Research Unit (USDA-ARS-AAHRU). The hosts from which these samples were collected included channel catfish, hybrid blue × channel catfish, walking catfish, and tadpole madtom (Table 1). Most isolates have been previously described elsewhere (2–7), except for a few, as follows. The “Curtis” isolate (Table 1) was recovered from a channel catfish with ESC from a farm in Hale County, Alabama, in 2003, and “T3-F2 AAHRU” (Table 1) was isolated from a channel catfish with ESC at our facility in 2021, both using standard methods for Edwardsiella spp (1). Bacterial isolates were grown from −80°C stocks that were briefly allowed to thaw and then aseptically streaked on sheep blood agar plates and grown at 26°C for 24 h. An individual colony from each isolate was picked by sterile loop, inoculated into brain-heart infusion (BHI) broth, and grown overnight to an approximate OD₆₀₀ of 0.5. A 1 mL aliquot of each isolate was taken and DNA isolated using the Wizard HMW DNA Extraction Kit (Promega, Madison, WI). We utilized a hybrid long-read (Nanopore) and short-read (Illumina) sequencing and assembly strategy, which aids in the generation of both high-quality chromosome and plasmid genome sequences (8). For Oxford Nanopore Technologies sequencing, we used the Native Barcoding Kit 24 V14 for library preparation and sequenced on the GridION using a FLO-MIN114 flow cell. DNA was not sheared/fragmented or size-selected per the manufacturer’s instructions; final library pool fragment size peak was >60 kb analyzed via an Agilent TapeStation with the Genomic DNA ScreenTape (Santa Clara, CA). Base calling was performed using Dorado with the “super accurate” configuration and raw data output to FASTQ format. For Illumina sequencing, 1 µg DNA was sent to a service provider (GENEWIZ, Azenta Life Sciences, South Plainfield, NJ), where the libraries were created using the NEBNext Ultra DNA Library Prep Kit for Illumina and the isolates were sequenced to ~1 Gb depth/sample using a 2 × 150 bp configuration on a NovaSeq X. Genomes were assembled using the Plassembler v1.6.2 pipeline (8). Briefly, short and long reads were filtered for quality control using Trimmomatic v0.39 (9) and LongQC v1.2.1 (10), respectively. Then, Flye assembler v2.9.4 (11) was used for chromosome clustering, and Unicycler v0.5.1 (12) was used for plasmid clustering. Each plasmid contig was then matched against The Plasmid Database (PLSDB) v2024_05_31_v2 using mash v2.3 (13). Genomes were annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) v2024-07-18.build7555 (14). All 11 chromosome sequences were able to be circularized, where the overlap was identified and trimmed automatically in Flye. Default parameters were used unless otherwise noted. All chromosome and plasmid sequences were further analyzed via BLASTn 2.16.0 and are considered circular and complete. A detailed summary of genome assembly statistics can be found in Table 1.

TABLE 1.

Summary statistics for the genome sequencing, assembly, and annotation of 11 Edwardsiella ictaluri isolates

Strain	Total no. of reads (Nanopore)	Genome coverage (Nanopore)	Read N50 (Nanopore)	Total no. of reads (Illumina)	Genome coverage (Illumina)	Genome size (Mbp)	GC content (%)	No. plasmids	No. of predicted genes	Host	16S top hit by BLASTn
EILO	200,764	130×	5,540	4.4M	345×	3.83	57.4	2	3,655	Clarius batrachus	Ei-39shipment9
RE-33	199,631	101×	5,640	4.5M	353×	3.83	57.4	2	3,659	Ictalurus punctatus	14-TAL-019-BIA
Curtis	8,059	23×	9,084	4.7M	395×	3.56	57.5	2	3,402	Ictalurus punctatus	Ei-39shipment9
AL-93-58	5,677	10×	13,266	4.9M	388×	3.83	57.4	3	3,695	Ictalurus punctatus	14-TAL-019-BIA
30IA New Jersey	7,679	15×	15,015	4.8M	383×	3.72	57.5	1	3,547	Noturus gyrinus	14-TAL-019-BIA
S94-1051	8,200	15×	12,990	3.7M	290×	3.84	57.4	3	3,660	Ictalurus punctatus	14-TAL-019-BIA
HCF #4 Smooth	7,763	14×	13,129	4.2M	326×	3.83	57.4	3	3,773	Ictalurus punctatus × I. furcatus	Ei-39shipment9
HCF #4Rough	8,486	16×	13,473	4.6M	360×	3.83	57.4	3	3,773	Ictalurus punctatus × I. furcatus	S07-698
NRRL B50348	6,591	14×	14,493	9.9M	777×	3.83	57.4	2	3,668	Ictalurus punctatus	S07-698
T3-F2 AAHRU	7,595	15×	12,045	10.5M	823×	3.84	57.4	2	3,679	Ictalurus punctatus	Ei-39shipment9
HCF-1	6,909	23×	14,478	10.6M	829×	3.85	57.4	2	3,672	Ictalurus punctatus × I. furcatus	14-TAL-019-BIA

DATA AVAILABILITY

Genome assemblies have been deposited to GenBank under the accession numbers CP180729, CP180732, CP180735, CP180738, CP180742, CP180746, CP180750, CP180752, CP180756, CP180759, CP180765.

Raw data (Illumina) can be found in the Sequence Read Archive under the accession numbers SRR32269917, SRR32269918, SRR32269919, SRR32269920, SRR32269921, SRR32269922, SRR32269923, SRR32269926, SRR32269936, SRR32269937, SRR32269938.

Raw data (Nanopore) can be found in the Sequence Read Archive under the accession numbers SRR32269924, SRR32269925, SRR32269927, SRR32269928, SRR32269929, SRR32269930, SRR32269931, SRR32269932, SRR32269933, SRR32269934, SRR32269935.