GENOME SEQUENCES

Complete Genome Sequencing of the Attenuated Strain

Mycoplasma synoviae 5-9
Xiaorong Zhang,a,b Yang Chen,a Zehua Wei,a Shuang Ma,b Mengjiao Guo,a Dianfeng Chu,b Chengcheng Zhang,a Yongzhong Cao,c
Yantao Wua,c

a Jiangsu Co-Innovation Center for the Prevention and Control of Animal Infectious Disease and Zoonoses, College of Veterinary Medicine, Yangzhou University,
Yangzhou, Jiangsu, China
b State Key Laboratory of Genetically Engineered Veterinary Vaccines, Qingdao Yebio Biological Engineering Co., Ltd., Qingdao, Shandong, China
c The International Joint Research Laboratory of Agricultural and Agri-product Safety, Yangzhou University, Yangzhou, Jiangsu, China

Xiaorong Zhang, Yang Chen, and Zehua Wei contributed equally to this article. Order of authors was ranked by contributions.

ABSTRACT Here, we present the complete genome sequence of Mycoplasma synoviae

strain 5-9. Strain 5-9 was attenuated by chemical mutagenesis from a field strain isolated
from egg breeders in Ningxia, China. It was completely sequenced and its genome anno-
tated; it is presented with the relevant data as a potential vaccine candidate.

M ycoplasma synoviae is an important pathogen infecting chickens, causing acute

or chronic respiratory diseases, infectious synovitis, air sacculitis (1–3), and egg-
shell apex abnormalities (4). Ningxia/2017-1 is the virulent parent strain of 5-9, isolated
in Ningxia, China. M. synoviae isolation and identification using Mycoplasma broth (MB)
and Mycoplasma agar (MA) were conducted as previously described (5). A Ningxia/
2017-1 culture was exposed to 100 m g/ml N-methyl-N9-nitro-N-nitrosoguanidine (NTG) for
15 min and continuously passaged 5 times in 10 m g/ml NTG at 33°C before being plated
onto MA. Single colonies were selected to grow in MB at 33°C (6).
High-quality genomic DNA (optical density at 260/280 nm [OD260/280] = 1.8 ; 2.0)
from 5-9 was extracted for Illumina and ONT sequencing using a bacterial genomic
DNA kit (CWBIO Biotech, China) until the color changed from red to orange-yellow
when cultured at 33°C and passaged 2 times in MB (pH 6.9). For next-generation
sequencing (NGS), paired-end libraries with insert sizes of ;400 bp were prepared
using the Illumina TruSeq Nano DNA high-throughput library prep kit. The purified
DNA was sheared into smaller fragments of the desired size (Covaris). Finally, the quali-
fied Illumina paired-end library was used for Illumina NovaSeq 6000 sequencing
(2  150-bp format; Shanghai BIOZERON Co., Ltd., China). A total of 2,333 Mb raw data
was produced, and we obtained 2,222 Mb clean data after filtering out the low-quality reads
using Trimmomatic (7) (http://www.usadellab.org/cms/?page=trimmomatic) (Table 1). The
raw paired-end reads were trimmed and quality controlled using Trimmomatic v0.36 (7)
(http://www.usadellab.org/cms/?page=trimmomatic) (parameters, SLIDINGWINDOW:4:15 Editor David Rasko, University of Maryland
MINLEN:75). School of Medicine
Copyright © 2021 Zhang et al. This is an open-
A Nanopore library was prepared using the ONT 1D ligation sequencing kit (SQK-LSK108)
access article distributed under the terms of
with the native barcoding expansion kit (EXP-NBD103). At least 1 m g DNA was treated with the Creative Commons Attribution 4.0
the end-repair/dA-tailing module, but the DNA was eluted in 24 m l following AMPure XP International license.
bead cleanup. Following the barcode ligation reaction, the DNA was cleaned again using Address correspondence to Yantao Wu,
[email protected].
AMPure XP beads and elution in 10 m l. The run was performed on a MinION MK1b device
The authors declare no conflict of interest.
using the NC_48 h_Sequencing_Run_FLOMIN106_SQK-LSK108 protocol.
Received 2 October 2021
The Illumina data were used to evaluate the complexity of the genome and correct the Accepted 17 November 2021
Nanopore long reads. First, we used ABySS v2.02 (8, 9) (http://www.bcgsc.ca/platform/bioinfo/ Published 9 December 2021
software/abyss) to perform the genome assembly with multiple kmer parameters and obtain

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Zhang et al.

TABLE 1 Genome assembly, Nanopore sequencing, and Illumina sequencing data

Illumina sequencing

Nanopore sequencing Raw data Clean data

Total N50 length G+C Coverage No. of No. of Avg length Total no. Total Total no. Total
Sample length (bp) (bp) content (%) (×) reads bases (bp) of reads size (Mb) of reads size (Mb)
5-9 786,872 786,872 28.47 1,015 125,369 798,728,344 6,371 15,553,532 2,333 14,881,648 2,222.2

optimal assembly results. Second, Canu v2.0 (10) (https://github.com/marbl/canu) was used to
assemble the Nanopore corrected long reads. Finally, GapCloser v1.12 (11) (https://
sourceforge.net/projects/soapdenovo2/files/GapCloser/) was applied to fill the remaining
local inner gaps and correct single-base polymorphisms for the final assembly results. The
complete circularized genome was drawn using Circos v0.64 (12) (http://circos.ca/). Default
parameters were used for all software unless otherwise specified. The consensus assembly
generated one contig of 786,872 bp (1,015-fold coverage). The G1C content was 28.47%
(Table 1).
Gene models were identified using GeneMark. Then, all gene models were searched
using blastp against the NCBI nonredundant (NR), Swiss-Prot (http://uniprot.org), KEGG
(http://www.genome.jp/kegg/), and COG (http://www.ncbi.nlm.nih.gov/COG) data-
bases. Additionally, tRNAs were identified using tRNAscan-SE v1.23 (13) (http://lowelab
.ucsc.edu/tRNAscan-SE), and rRNAs were determined using RNAmmer v1.2 (14) (https://
services.healthtech.dtu.dk/service.php?RNAmmer-1.2). There were 652 protein-coding
genes and 44 RNA genes (34 tRNAs, 3 5S rRNAs, 2 16S rRNAs, 2 23S rRNAs, and 3 non-
coding RNA [ncRNA] genes).
Data availability. The complete genome sequence and annotation of M. synoviae
strain 5-9 have been deposited at GenBank under accession number CP083748. The raw
data were deposited in the Sequence Read Archive (SRA) database under the accession
numbers SRR16005423 (Oxford Nanopore) and SRR16005422 (Illumina). The BioProject
accession number is PRJNA763075. The BioSample accession number is SAMN21422653.

ACKNOWLEDGMENTS
This work was supported by the Key Special Project “Science and Technology
Promote Economy of 2020” of the National Key Research and Development Program
(SQ2020YFF0426460), China Agriculture Research System of MOF and MARA (CARS-
40), the State Key Laboratory of Genetically Engineered Veterinary Vaccines (AGVSKL-
ZD-202001), and a project funded by the Priority Academic Program Development of
Jiangsu Higher Education Institutions (PAPD).

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