Towards quantitative metagenomics of wild viruses and other ultra‐low concentration DNA samples: a rigorous assessment and optimization of the linker amplification method

Abstract

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 artefacts, 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 G + C content 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 first of which we tested and present data for here.

Shotgun metagenomics indicates novel family A DNA polymerases predominate within marine virioplankton

“…The combination of hydroxyapatite chromatography for selective isolation of ssDNA molecules along with linker amplification may have enabled detection of ssDNA viral groups that have evaded detection in MDA-based library preparation techniques. Recent work has demonstrated that viral metagenomes prepared using linker amplification more accurately preserve the underlying distributions of viruses within a community (Duhaime et al, 2012).…”

The Number of Target Molecules of the Amplification Step Limits Accuracy and Sensitivity in Ultradeep-Sequencing Viral Population Studies

“…At the viral community level, the linker amplification shotgun library (LASL) method (1) amplifies only double-stranded DNA (dsDNA) viruses (therefore missing single-stranded DNA [ssDNA] and RNA viruses) and suffers from differential amplification rates related to the size of amplicons (16) and GC content (17). The multipledisplacement amplification method (MDA), based on RCA (18), tends to overrepresent small circular genomes (e.g., ssDNA viruses) (19,20) and, depending on the kit, tends to overrepresent sequences rich in GC content or frequent sequences in the population (21).…”

New Approaches Indicate Constant Viral Diversity despite Shifts in Assemblage Structure in an Australian Hypersaline Lake

“…However, we stress a number of points in support of this approach. (i) Metagenomic libraries were prepared without multiple-displacement amplification, reducing biases in the sequencing data prior to assembly (9,52). (ii) Because no reference haloviral genomes were detected in LT metagenomes, a de novo method for characterizing these assemblages was required.…”