Sequencing platform and library preparationchoices impact viral metagenomes

© F.Latreille

Tara Oceans

Sergei A. SOLONENKO, J. César IGNACIO-ESPINOZA, Adriana ALBERTI, Corinne CRUAUD, Steven HALLAM, Kostas KONSTANTINIDIS, Gene TYSON, Patrick WINCKER and Matthew B. SULLIVAN

BMC Genomics 2013, 14:320

 

Abstract

Background: Microbes drive the biogeochemistry that fuels the planet. Microbial viruses modulate their hostsdirectly through mortality and horizontal gene transfer, and indirectly by re-programming host metabolisms duringinfection. However, our ability to study these virus-host interactions is limited by methods that are low-throughputand heavily reliant upon the subset of organisms that are in culture. One way forward are culture-independentmetagenomic approaches, but these novel methods are rarely rigorously tested, especially for studies ofenvironmental viruses, air microbiomes, extreme environment microbiology and other areas with constrainedsample amounts. Here we perform replicated experiments to evaluate Roche 454, Illumina HiSeq, and Ion TorrentPGM sequencing and library preparation protocols on virus metagenomes generated from as little as 10pg of DNA.

Results: Using %G + C content to compare metagenomes, we find that (i) metagenomes are highly replicable,(ii) some treatment effects are minimal, e.g., sequencing technology choice has 6-fold less impact than varyinginput DNA amount, and (iii) when restricted to a limited DNA concentration (<1μg), changing the amount ofamplification produces little variation. These trends were also observed when examining the metagenomes forgene function and assembly performance, although the latter more closely aligned to sequencing effort and readlength than preparation steps tested. Among Illumina library preparation options, transposon-based librariesdiverged from all others and adaptor ligation was a critical step for optimizing sequencing yields.

Conclusions: These data guide researchers in generating systematic, comparative datasets to understand complexecosystems, and suggest that neither varied amplification nor sequencing platforms will deter such efforts.