Quantitative PCR provides a simple and accessible method for quantitative microbiota profiling

PLoS One. 2020 Jan 15;15(1):e0227285. doi: 10.1371/journal.pone.0227285. eCollection 2020.

Abstract

The use of relative abundance data from next generation sequencing (NGS) can lead to misinterpretations of microbial community structures, as the increase of one taxon leads to the concurrent decrease of the other(s) in compositional data. Although different DNA- and cell-based methods as well as statistical approaches have been developed to overcome the compositionality problem, and the biological relevance of absolute bacterial abundances has been demonstrated, the human microbiome research has not yet adopted these methods, likely due to feasibility issues. Here, we describe how quantitative PCR (qPCR) done in parallel to NGS library preparation provides an accurate estimation of absolute taxon abundances from NGS data and hence provides an attainable solution to compositionality in high-throughput microbiome analyses. The advantages and potential challenges of the method are also discussed.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacteria / classification
  • Bacteria / genetics
  • Bacteria / isolation & purification*
  • DNA, Bacterial / genetics
  • DNA, Bacterial / isolation & purification
  • Feasibility Studies
  • Gene Library
  • High-Throughput Nucleotide Sequencing
  • High-Throughput Screening Assays / methods*
  • Humans
  • Microbiota / genetics*
  • RNA, Ribosomal, 16S / genetics
  • Randomized Controlled Trials as Topic
  • Real-Time Polymerase Chain Reaction*

Substances

  • DNA, Bacterial
  • RNA, Ribosomal, 16S

Grants and funding

The work was funded by grants from the University of Helsinki (AS, KK, PL), the EU/EFPIA Innovative Medicines Initiative Joint Undertaking (EMIF grant no. 115372, HY-J), the EPoS (Elucidating Pathways of Steatohepatitis) consortium funded by the Horizon 2020 Framework Program of the European Union under Grant Agreement 634413 (HY-J), Academy of Finland (KK, HY-J), Otto A. Malm (CJ), Sigrid Juselius (HY-J), EVO (HY-J), Finnish Medical (PL), Alfred Kordelin (PL), Paulo (AS and PL), Jalmari and Rauha Ahokas and Novo Nordisk (HY-J), Mary and Georg C. Ehnrooth (AS) as well as Biocodex Microbiota (AS) Foundations. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.