Amplification methods bias metagenomic libraries of uncultured single-stranded and double-stranded DNA viruses

Abstract

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.

Fig. 1.

Classification of metagenomic sequences from the three libraries based on biological groups (A) and viral genome types and viral families (B) from BLASTX analysis. Among total reads analyzed in the LASL, MDAH, and MDAX libraries, only 14.2%, 6.0%, and 9.3%, respectively, showed hits against the NCBI nonredundant protein database (A) and only 9.8%, 6.2%, and 9.8%, respectively, against the CAMERA viral protein database (B).