Table 1.

Virus enrichment process prior to sequencing in metagenomic studies on human and animal samples.

Paper title	Author	Year	Journal	Aim of study	Sample	Step 1	Step 2	Step 3	Step 4	Step 5	Step 6	Step 7	Amplification	Sequencing
A virus discovery method incorporating DNase treatment and its application to the identification of two bovine parvovirus species	Allander et al.	2001	PNAS	Development of a method for discovery of unknown viruses and elimination of contaminating host DNA. Allowed the discovery of novel bovine parvoviruses.	Serum			–	–	–	–	–	Sequence-Independent Amplification	Cloning and sanger-method
Metagenomic analyses of an uncultured viral community from human faeces	Breitbart et al.	2003	Journal of Bacteriology	Metagenomic analyses of an uncultured viral community from human faeces	Faecal suspension				–	–	–	–	Sequence-independent Amplification	Cloning and sanger-method
Identification of a new human coronavirus	van der Hoek et al.	2004	Nature Medicine	Identification of a new human coronavirus	Suspension of LLC-MK2 cells			–	–	–	–	–	VIDISCA	cDNA-AFLP, cloning and sanger-method
Viral genome sequencing by random priming methods	Djikeng et al.	2008	BMC Genomics	Development of a method for rapid sequencing of whole genomes from new viruses	Bacterial growth media, plasma, cell culture supernatant, faecal suspension					–	–	–	Sequence-Independent Amplification	Cloning and sanger-method
A highly divergent Picornavirus in a marine mammal	Kapoor et al.	2008	Journal of Virology	Unidentified virus cultured from a seal	Supernatant from infected Vero cell culture					–	–	–	Sequence-Independent Amplification	Cloning and sanger-method
Rapid identification of known and new RNA viruses from animal tissues	Victoria et al.	2008	PLOS Pathogens	Unidentified viruses cultured in suckling mouse brains	Brain tissue homogenate from mice					–	–	–	Sequence-Independent Amplification	Cloning and sanger-method
Discovery of a novel single-stranded DNA virus from a Sea Turtle Fibropapilloma by using viral metagenomics	Ng et al.	2009	Journal of Virology	To investigate and purifiy the viruses associated with sea turtle fibropapillomatosis (FP)	External fibropapilloma homogenate						–	–	Sequence-Independent Amplification	Cloning and sanger-method
Laboratory procedures to generate viral metagenomes	Thurber et al.	2009	Nature Protocols	Development of laboratory procedure for making viral metagenomes	Various (i.e. soil, animal tissues, clinical samples)					–	–	–	As required. Genomiphi or Transplex	High-throughput sequencing
Identification and characterisation of deer astroviruses	Smits et al.	2010	Journal of General Virology	Detection of novel astroviruses in deer using a metagenomic approach	Faecal suspension				–	–	–	–	Sequence-Independent Amplification	Cloning and sanger-method
Metagenomic sequencing for virus identification in a public-health setting	Svraka et al.	2010	Journal of General Virology	Unidentified viruses cultured in in vitro cell lines	Cell culture suspension				–	–	–	–	Whole Transcriptome Amplification Kit (Qiagen) or GenomiPhi V2 (GE Healthcare)	Cloning and sanger-method
Human Picobirnaviruses identified by molecular screening of diarrhoea samples	Van Leeuwen et al.	2010	Journal of Clinical Microbiology	Viral metagenomic survey of human diarrhoea of unknown origin	Faecal suspension				–	–	–	–	Sequence-Independent Amplification	Cloning and sanger-method
A viral discovery methodology for clinical biopsy samples utilising massively parallel next generation sequencing	Daly et al.	2011	PLOS ONE	Development of a method for discovery of new viruses from clinical biopsy samples	Liver tissue homogenate from human and canine				–	–	–	–	Sequence-Independent Amplification	454 sequencing (Roche) and GAII sequencing (Illumina)
Identification and molecular characterisation of a new nonsegmented double-stranded RNA virus isolated from Culex mosquitoes in Japan	Isawa et al.	2011	Virus Research	Identification of 2 infectious agents from the mosquitoes Culex pipiens pallens and Culex inatomii	Mosquito homogenate					–	–	–	Sequence-Independent Amplification (Single Primer Amplification Technique – SPAT)	Sanger-method
Diversity and abundance of single-stranded DNA viruses in human faeces	Kim et al.	2011	Applied and Environmental Microbiology	Investigation of single-stranded DNA viruses in human faeces	Faecal suspension					–	–	–	GenomiPhi V2 (GE Healthcare)	454FLX sequencing (Roche)
Novel DNA virus isolated from samples showing endothelial cell necrosis in the Japanese eel, Anguilla japonica	Mizutani et al.	2011	Virology	Identification of novel eel virus	Supernantant from infected JEE culture cells			–	–	–	–	–	GenomePlex Whole Genome Amplification or GenomiPhi (GE Healthcare)	Sanger-method or 454FLX sequencing (Roche)
Broad surveys of DNA viral diversity obtained through viral metagenomics of mosquitoes	Ng et al.	2011	PLOS ONE	Viral diversity study in mosquito	Whole mosquito homogenate							–	GenomiPhi (GE Healthcare)	454 GS20 and 454FLX sequencing (Roche)
Exploring the diversity of plant DNA viruses and their satellites using vector-enabled metagenomics on whiteflies	Ng et al.	2011	PLOS ONE	To investigate the diversity of DNA viruses in whiteflies collected from different crops in 2 agriculturally important sites in Florida using vector-enabled metagenomics (VEM).	Whitefly homogenate	–							Genomiphi (GE Healthcare) and GenomePlex (Sigma–Aldrich)	Cloning and sanger-method
Random PCR and ultracentrifugation increases sensitivity and throughput of VIDISCA for screening of pathogens in clinical specimens	Tan et al.	2011	Journal of Infection in Developing Countries	Screening for unknown pathogens in clinical specimens	Clinical samples of plasma, throat swab, nasal pharyngeal aspirate.				–	–	–	–	VIDISCA	cDNA-AFLP, cloning and sanger-method
Metagenomic analysis of fever, thrombocytopenia and leukopenia syndrome (FTLS) in Henan Province, China: Discovery of a new bunyavirus	Xu et al.	2011	PLOS Pathogens	Discovery of a new bunyavirus in cases of fever, thrombocytopenia and leukopenia syndrome (FTLS)	Supernatant from infected Vero cell culture				–	–	–	–	Sequence-Independent Amplification	Cloning and sanger-method
Simultaneous identification of DNA and RNA viruses present in pig faeces using process-controlled deep sequencing	Sachsenröder et al.	2012	PLOS ONE	To establish a protocol for the simultaneous analysis of DNA and RNA viruses present in pig faeces	Faecal suspension								Transplex (WTA2, Sigma–Aldrich)	454FLX sequencing (Roche)
Sequence-independent VIDISCA-454 technique to discover new viruses in canine livers	van der Heijden et al.	2012	Journal of Virological Methods	Test the feasibility of VIDISCA-454 to obtain viral sequence information from idiopathic canine hepatitis liver biopsy.	Canine BDE cell culture supernatant			–	–	–	–	–	VIDISCA	454FLX sequencing (Roche)
Identification of a novel bat papillomavirus by metagenomics	Tse et al.	2012	PLOS ONE	Discovery and characterisation of a novel bat papillomavirus from rectal swabs of asymptomatic wild, food and pet animals using metagenomics	Animal rectal swabs				–	–	–	–	Rapisome pWGA (Biohelix)	454 GS FLX sequencing (Roche)
Complete genome sequence of an astrovirus identified in a domestic rabbit (Oryctolagus cuniculus) with gastroenteritis	Stenglein et al.	2012	Virology Journal	To screen samples from a gastroenteritis outbreak in a commercial rabbit colony	Faecal suspension				–	–	–	–	PCR	Cloning and sanger-method

Publications referred to include: Allander et al. (2001), Breitbart et al. (2003), Daly et al. (2011), Djikeng et al. (2008), Isawa et al. (2011), Kapoor et al. (2008), Mizutani et al. (2011), Ng et al. (2011a), Ng et al. (2009), Ng et al. (2011b), Sachsenroder et al. (2012), Smits et al. (2010), Svraka et al. (2010), Tan le et al. (2011), Thurber et al. (2009), van der Hoek et al. (2004), van Leeuwen et al. (2010), Victoria et al. (2008), Xu et al. (2011).

 Low-speed centrifugation.

 Filtration (excludes tangential flow).

 Ultracentrifugation.

 Nuclease treatment.

 Unclassified method.