Assessing the accuracy of quantitative molecular microbial profiling
International Journal of Molecular Sciences
© 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).
The application of high-throughput sequencing in profiling microbial communities is providing an unprecedented ability to investigate microbiomes. Such studies typically apply one of two methods: amplicon sequencing using PCR to target a conserved orthologous sequence (typically the 16S ribosomal RNA gene) or whole (meta)genome sequencing (WGS). Both methods have been used to catalog the microbial taxa present in a sample and quantify their respective abundances. However, a comparison of the inherent precision or bias of the different sequencing approaches has not been performed. We previously developed a metagenomic control material (MCM) to investigate error when performing different sequencing strategies. Amplicon sequencing using four different primer strategies and two 16S rRNA regions was examined (Roche 454 Junior) and compared to WGS (Illumina HiSeq). All sequencing methods generally performed comparably and in good agreement with organism specific digital PCR (dPCR); WGS notably demonstrated very high precision. Where discrepancies between relative abundances occurred they tended to differ by less than twofold. Our findings suggest that when alternative sequencing approaches are used for microbial molecular profiling they can perform with good reproducibility, but care should be taken when comparing small differences between distinct methods. This work provides a foundation for future work comparing relative differences between samples and the impact of extraction methods. We also highlight the value of control materials when conducting microbial profiling studies to benchmark methods and set appropriate thresholds.
The authors acknowledge funding from the European Metrology Research Programme joint research project “INFECT MET” (http://infectmet.lgcgroup.com) (an EMRP project, jointly funded by the EMRP participating countries within EURAMET and the European Union) and the UK National Measurement System for funding of this work and for the support of Thomas Laver by the BBSRC Industrial Case Studentship award BB/H016120/1.
This is the final version of the article. Available from MDPI via the DOI in this record.
Vol. 15 (11), pp. 21476 - 21491
Place of publication