| Viruses are the most abundant and diverse microflora,which have been discovered in various organisms and the environment,and play an important role in the environmental and human health.In recent years,the study on viruses has produced a qualitative leap with the continuous development of science and technology.The advent of viral metagenomics has broken the shackles of traditional methods for the isolation and identification of viruses,accelerating the pace of the study on viruses.With the rapid development of sequencing techniques and progressive reduction of the relevant costs,viral metagenomics technique has become one of the most important means for the discovery of novel viruses.In a laboratory aiming at discovering new animal viruses and conducting early warning for emerging infections based on the viral metagenomics study,the efficiency and accuracy of data analysis is essential to fulfill these aims,but which cannot being done if the process is completely depended on a sequencing company.Therefore,it's urgent to establish a local viral metagenomics analysis platform to provide technical support for the early warning of new outbreaks.To establish a localized animal viral metagenomics analysis platform,firstly,a standardized and well-compatible viral reference database was established using Perl program and MySQL database language.This database contains more than 110,000 complete genome sequences of currently known viruses from 89 families or genera.Secondly,according to the process of metagenomics data analysis,software for quality control,data cleaning,sequence assembling,sequence annotation,function prediction,and genetic evolution analysis were modified and installed on a high-performance computing node equipped with a Linux system.To improve sensitivity or specificity in different situations,two annotated strategies for gene sequences were established: including assembly-free analysis and assembly-based analysis.The former can improve the sensitivity of annotation and facilitate the discovery of low-copy viral genome sequences,while the latter can improve the specificity of annotation to ensure the accuracy of the annotation results.To verify the performance of the platform in field practice,metagenomics sequence data of nasal swab and serum samples of pigs collected in Anhui province were analyzed.A total of 31,490,033 and 41,208,780 pair-reads were obtained from the nasal and serum samples respectively after removing the joint sequences and low-quality sequences using data quality control and cleaning methods.The obtained reads were then assembled using MEGAHIT software,resulting in 415,117 contigs from nasal swabs samples and 81,026 contigs from serum samples.The obtained contigs were used to perform gene annotation.A total of 307 contigs were annotated into 1,942 virus sequences in the nasal swab samples,of them 48.9% belonging to the family Anelloviridae,20.2% belonging to the family Parvoviridae,16.2% belonging to the family Circoviridae,and 11.1% belonging to the family Coronaviridae.Other viruses,such as mammalian astrovirus,poxvirus,and leukemia virus,were also found in the sample.A total 10,012 contigs were annotated to 1,376 virus sequences in the serum samples,66.5% belonging to the family Parvoviridae,29.5% belonging to the family Anelloviridae,and 1.3% belonging to the family Circoviridae.The results indicate that significant differences in virus diversity existed between nasal swab and serum samples.Furthermore,assembly-free analysis strategy took shorter time(1.5 d)than it done by assembly-based analysis method(3.5 d).In conclusion,we have established a local platform for animal viral metagenomics analysis.This platform performed efficiently and accurately in viral metagenomics analysis,and meets completely the needs of surveillance and early warning of emerging animal virus diseases,with a potential value in the application of animal disease prevention and control in China. |
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