Linker amplification for quantitative metagenomics of wild viruses and other ultra-low concentration DNA samples
Metagenomics generates and tests hypotheses about dynamics and mechanistic drivers in wild populations, yet commonly suffers from insufficient (<1 ng) starting genomic material for sequencing. Current solutions for amplifying sufficient DNA for metagenomics analyses include linear amplification for deep sequencing (LADS), which requires more DNA than is normally available, linker amplified shotgun libraries (LASLs), which is prohibitively low throughput, and whole genome amplification, which is significantly biased and thus non-quantitative. Here, we adapt the LASL approach to next generation sequencing by offering an alternate polymerase for challenging samples, developing a more efficient sizing step, integrating a "reconditioning PCR" step to increase yield and minimize late-cycle PCR artifacts, and empirically documenting the quantitative capability of the optimized method with both laboratory isolate and wild community viral DNA. Our optimized linker amplification method requires as little as 1 pg of DNA and is the most precise and accurate available, with amplification biases less than 1.5-fold, even for complex samples as diverse as a wild virus community. While optimized here for 454 sequencing, this linker amplification method can be used to prepare metagenomics libraries for sequencing with next-generation platforms, including Illumina and Ion Torrent. The sequences submitted to CAMERA are all those described in the publication. |  |  | |