simultaneous identification of dna and rna viruses present in pig faeces using process-controlled deep sequencing同时识别dna和rna病毒在猪的粪便使用过程控制深度测序.pdfVIP

Simultaneous Identification of DNA and RNA Viruses Present in Pig Faeces Using Process-Controlled Deep Sequencing ¨ 1 2 1 1 2 1 Jana Sachsenroder , Sven Twardziok , Jens A. Hammerl , Pawel Janczyk , Paul Wrede , Stefan Hertwig , Reimar Johne1* 1 Federal Institute for Risk Assessment, Berlin, Germany, 2 Institute for Molecular Biology and Bioinformatic, Charite, Berlin, Germany Abstract Background: Animal faeces comprise a community of many different microorganisms including bacteria and viruses. Only scarce information is available about the diversity of viruses present in the faeces of pigs. Here we describe a protocol, which was optimized for the purification of the total fraction of viral particles from pig faeces. The genomes of the purified DNA and RNA viruses were simultaneously amplified by PCR and subjected to deep sequencing followed by bioinformatic analyses. The efficiency of the method was monitored using a process control consisting of three bacteriophages (T4, M13 and MS2) with different morphology and genome types. Defined amounts of the bacteriophages were added to the sample and their abundance was assessed by quantitative PCR during the preparation procedure. Results: The procedure was applied to a pooled faecal sample of five pigs. From this sample, 69,613 sequence reads were generated. All of the added bacteriophages were identified by sequence analysis of the reads. In total, 7.7% of the reads showed significant sequence identities with published viral sequences. They mainly originated from bacteriophages (73.9%) and mammalian viruses (23.9%); 0.8% of the sequences showed identities to plant viruses. The most abundant detected porcine viruses were kobuvirus, rota