Laboratory procedures to generate viral metagenomes.
This collection of laboratory protocols describes the steps to collect viruses from various samples with the specific aim of generating viral metagenome sequence libraries (viromes). Viral metagenomics, the study of uncultured viral nucleic acid sequences from different biomes, relies on several concentration, purification, extraction, sequencing and heuristic bioinformatic methods. No single technique can provide an all-inclusive approach, and therefore the protocols presented here will be discussed in terms of hypothetical projects. However, care must be taken to individualize each step depending on the source and type of viral-particles. This protocol is a description of the processes we have successfully used to: (i) concentrate viral particles from various types of samples, (ii) eliminate contaminating cells and free nucleic acids and (iii) extract, amplify and purify viral nucleic acids. Overall, a sample can be processed to isolate viral nucleic acids suitable for high-throughput sequencing in approximately 1 week.
Thurber
Rebecca V
RV
Department of Biology, San Diego State University, San Diego, California 92182, USA. rvegathurber@gmail.com
Haynes
Matthew
M
Breitbart
Mya
M
Wegley
Linda
L
Rohwer
Forest
F
D004267:DNA Viruses / Q000235:genetics* / Q000302:isolation & purification; D016679:Genome, Viral; D015698:Genomic Library; D023281:Genomics / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques; D014461:Ultracentrifugation; D014771:Virion / Q000235:genetics / Q000302:isolation & purification; D014776:Virus Cultivation