Investigation of viruses in the environment often requires the amplification of viral DNA before sequencing of viral metagenomes. In this study, two of the most widely used amplification methods, the linker amplified shotgun library (LASL) and multiple displacement amplification (MDA) methods, were applied to a sample from the seawater surface. Viral DNA was extracted from viruses concentrated by tangential flow filtration and amplified by these two methods. 454 pyrosequencing was used to read the metagenomic sequences from different libraries. The resulting taxonomic classifications of the viruses, their functional assignments, and assembly patterns differed substantially depending on the amplification method. Only double-stranded DNA viruses were retrieved from the LASL, whereas most sequences in the MDA library were from single-stranded DNA viruses, and double-stranded DNA viral sequences were minorities. Thus, the two amplification methods reveal different aspects of viral diversity.

Little is known about the archaeal diversity of fermented seafood; most of the earlier studies of fermented food have focused on lactic acid bacteria (LAB) in the fermentation process. In this study, the archaeal and bacterial diversity in seven kinds of fermented seafood were culture-independently examined using barcoded pyrosequencing and PCR–denaturing gradient gel electrophoresis (DGGE) methods. The multiplex barcoded pyrosequencing was performed in a single run, with multiple samples tagged uniquely by multiplex identifiers, using different primers for Archaea or Bacteria. Because PCR–DGGE analysis is a conventional molecular ecological approach, this analysis was also performed on the same samples and the results were compared with the results of the barcoded pyrosequencing analysis. A total of 13 372 sequences were retrieved from 15 898 pyrosequencing reads and were analyzed to evaluate the diversity of the archaeal and bacterial populations in seafood. The most predominant types of archaea and bacteria identified in the samples included extremely halophilic archaea related to the family Halobacteriaceae; various uncultured mesophilic Crenarchaeota, including Crenarchaeota Group I.1 (CG I.1a and CG I.1b), Marine Benthic Group B (MBG-B), and Miscellaneous Crenarchaeotic Group (MCG); and LAB affiliated with genus Lactobacillus and Weissella. Interestingly, numerous uncultured mesophilic Crenarchaeota groups were as ubiquitous in the fermented seafood as in terrestrial and aquatic niches; the existence of these Crenarchaeota groups has not been reported in any fermented food. These results indicate that the archaeal populations in the fermented seafood analyzed are diverse and include the halophilic and mesophilic groups, and that barcoded pyrosequencing is a promising and cost-effective method for analyzing microbial diversity compared with conventional approaches.

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 φ29 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.

Viruses are recognized as the most abundant biological components on Earth, and they regulate the structure of microbial communities in many environments. In soil and marine environments, microorganism-infecting phages are the most common type of virus. Although several types of bacteriophage have been isolated from fermented foods, little is known about the overall viral assemblages (viromes) of these environments. In this study, metagenomic analyses were performed on the uncultivated viral communities from three fermented foods, fermented shrimp, kimchi, and sauerkraut. Using a high-throughput pyrosequencing technique, a total of 81,831, 70,591 and 69,464 viral sequences were obtained from fermented shrimp, kimchi and sauerkraut, respectively. Moreover, 37 to 50% of these sequences showed no significant hit against sequences in public databases. There were some discrepancies between the prediction of bacteriophages hosts via homology comparison and bacterial distribution, as determined from 16S rRNA gene sequencing. These discrepancies likely reflect the fact that the viral genomes of fermented foods are poorly represented in public databases. Double-stranded DNA viral communities were amplified from fermented foods by using a linker-amplified shotgun library. These communities were dominated by bacteriophages belonging to the viral order Caudovirales (i.e., Myoviridae , Podoviridae , and Siphoviridae ). This study indicates that fermented foods contain less complex viral communities than many other environmental habitats, such as seawater, human feces, marine sediment, and soil.