Complete Genome Sequences of Two Mycoplasma gallisepticum F-Strain Variants.

Mycoplasma gallisepticum pathology in poultry is preventable by vaccination with live M. gallisepticum vaccines. Research has suggested possible differences in host response between F-strain-based vaccines. The genomes of the AviPro vaccine and F99 parent strains were sequenced for comparison with the already sequenced F-strain vaccine.

ANNOUNCEMENT

Mycoplasma gallisepticum infection of poultry can cause significant pathology and severe economic losses for poultry producers, but its effects can be mitigated by vaccination with live M. gallisepticum vaccines (1, 2). Comparative research between two commercial F-strain vaccines and the parent strain suggested possible differences in host response to and protection afforded by the vaccines (3). One commercial vaccine strain was previously sequenced (4). The parent strain (F99) and commercial vaccine strain (AviPro) were sequenced for comparison with the previously sequenced commercial vaccine strain from Schering-Plough Animal Health (FVAX) (4).

The F99 strain was obtained from Stan Kleven (5). The AviPro-MG-F strain was cultured from a commercial vaccine (Lohmann Animal Health, Winslow, ME, USA). Both strains were grown on Frey’s agar (6), and a single colony of each strain was grown in Frey’s broth. Bacteria were grown to mid-log phase as indicated by the orange color of the phenol red indicator and pelleted by centrifugation (20,000 × g), and the pellets were stored at –80°C (7). DNA for genome sequencing was isolated from bacterial pellets using a DNeasy blood and tissue kit (Qiagen, Inc., Germantown, MD, USA) according to the manufacturer’s instructions. DNA quantity and purity (260/280 nm and 260/230 nm ratios) were assessed using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

Illumina sequencing was done by the USDA ARS Genomics and Bioinformatics Research Unit (Stoneville, MS, USA). DNA samples were sheered to 500-bp fragments, libraries were prepared with an Illumina NeoPrep instrument, and 2 × 150-bp paired-end sequencing was performed using a NextSeq 500 sequencer (Illumina Biotechnology Company, San Diego, CA, USA). Sequences were trimmed with FastX trimmer, and bases 9 to 144 were retained (FastX toolkit version 0.0.14 [http://hannonlab.cshl.edu/fastx_toolkit/]). Sickle version 1.33 was used to filter paired sequences with quality and length thresholds of 25 and 20, respectively (8). All software packages were used with default parameters unless otherwise specified. The average sequence quality was 36, and the average length was 136 bases for all sequences. A total of 15,517,562 paired-end reads for F99 and 12,723,542 for AviPro were used for assembly.

Genomic DNA for MinION sequencing (Oxford Nanopore Technologies, Oxford, UK) was performed as described previously using frozen bacterial cell pellets of the same passage number as that used for Illumina sequencing. Genomic DNA was barcoded and prepared for sequencing using the kits EXP-NBD103 and SQK-LSK108, and 1D sequencing was performed for 48 hours using a FLO-MIN106 flow cell. DNA base calling and barcode sorting were performed using Albacore version 2.0.1 (Oxford Nanopore Technologies). Porechop version 0.2.4 was used to remove barcode and adapter sequences (9). Fastqutils from the NGSUtils package version 0.5.9 (https://github.com/ngsutils/ngsutils) was used to remove sequences shorter than 3,000 bases to facilitate assembly (10). Average sequence length postfiltering for MinION sequences was >8,000 bases for both genome samples, with 9,494/122,987 and 8,397/109,975 sequences retained for F99 and AviPro, respectively. This resulted in greater than 70× coverage for each MinION data set. Genome assembly using both the Illumina and MinION data was performed using the hybrid assembly mode of Unicycler version 0.4.1 (https://github.com/rrwick/Unicycler) (11).

Each genome was assembled into a single circular contig. The AviPro genome is 975,069 bases long, while the F99 genome is 975,073 bases long. Both genomes have a GC content of 31.4%. Notable differences between the genomes include an additional vlha gene (MGF 4707) in FVAX that is not present in either F99 or AviPro, the addition of two ACACCA repeats in the F99 MGF 4275 homologue, and the lack of the premature stop codon in the MGF 0748 homologues of F99 and AviPro.

Data availability.

These genome sequences were deposited in GenBank under the accession numbers CP028147 (AviPro) and CP028146 (F99). The raw sequence data have been deposited at the Sequence Read Archive under the accession numbers SRR8647640 (AviPro Illumina data), SRR8647639 (AviPro MinION data), SRR8647642 (F99 Illumina data), and SRR8647641 (F99 MinION data).