Amplification of uncultured single-stranded DNA viruses from rice paddy soil.
Viruses are known to be the most numerous biological entities in soil; however, little is known about their diversity in this environment. In order to explore the genetic diversity of soil viruses, we isolated viruses by centrifugation and sequential filtration before performing a metagenomic investigation. We adopted multiple-displacement amplification (MDA), an isothermal whole-genome amplification method with phi29 polymerase and random hexamers, to amplify viral DNA and construct clone libraries for metagenome sequencing. By the MDA method, the diversity of both single-stranded DNA (ssDNA) viruses and double-stranded DNA viruses could be investigated at the same time. On the contrary, by eliminating the denaturing step in the MDA reaction, only ssDNA viral diversity could be explored selectively. Irrespective of the denaturing step, more than 60% of the soil metagenome sequences did not show significant hits (E-value criterion, 0.001) with previously reported viral sequences. Those hits that were considered to be significant were also distantly related to known ssDNA viruses (average amino acid similarity, approximately 34%). Phylogenetic analysis showed that replication-related proteins (which were the most frequently detected proteins) related to those of ssDNA viruses obtained from the metagenomic sequences were diverse and novel. Putative circular genome components of ssDNA viruses that are unrelated to known viruses were assembled from the metagenomic sequences. In conclusion, ssDNA viral diversity in soil is more complex than previously thought. Soil is therefore a rich pool of previously unknown ssDNA viruses.
Kim
Kyoung-Ho
KH
Biological Resource Center, KRIBB, Daejeon 305-806, Korea.
Chang
Ho-Won
HW
Nam
Young-Do
YD
Roh
Seong Woon
SW
Kim
Min-Soo
MS
Sung
Youlboong
Y
Jeon
Che Ok
CO
Oh
Hee-Mock
HM
Bae
Jin-Woo
JW
D044822:Biodiversity; D003001:Cloning, Molecular; D004267:DNA Viruses / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D004277:DNA, Single-Stranded / Q000235:genetics*; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics*; D016679:Genome, Viral; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012275:Oryza / Q000821:virology*; D010802:Phylogeny; D017422:Sequence Analysis, DNA; D017385:Sequence Homology; D012988:Soil Microbiology; D014764:Viral Proteins / Q000235:genetics
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D014764:Viral Proteins