Micelle PCR reduces chimera formation in 16S rRNA profiling of complex microbial DNA mixtures.

Stefan A Boers,John P Hays,Ruud Jansen

doi:10.1038/srep14181

Abstract

16S rRNA gene profiling has revolutionized the field of microbial ecology. Many researchers in various fields have embraced this technology to investigate bacterial compositions of samples derived from many different ecosystems. However, it is important to acknowledge the current limitations and drawbacks of 16S rRNA gene profiling. Although sample handling, DNA extraction methods and the choice of universal 16S rRNA gene PCR primers are well known factors that could seriously affect the final results of microbiota profiling studies, inevitable amplification artifacts, such as chimera formation and PCR competition, are seldom appreciated. Here we report on a novel micelle based amplification strategy, which overcomes these limitations via the clonal amplification of targeted DNA molecules. Our results show that micelle PCR drastically reduces chimera formation by a factor of 38 (1.5% vs. 56.9%) compared with traditional PCR, resulting in improved microbial diversity estimates. In addition, compartmentalization during micelle PCR prevents PCR competition due to unequal amplification rates of different 16S template molecules, generating robust and accurate 16S microbiota profiles required for comparative studies (e.g. longitudinal surveys).

Highlights

Sequences can be filtered out of next-generation sequencing (NGS) results using specialized software[7,8], the generation of chimeric products can still seriously reduce the amount of useful information obtained in a single sequencing run[9]
To evaluate the ability of Micelle PCR (micPCR) to increase the accuracy of 16S rRNA sequencing, universal 357F and 926R primers were used to amplify the 16 S rRNA V3–V5 region from a synthetic microbial community containing equimolar 16S rRNA operon counts derived from 20 different bacterial species (HM-782D supplied by BEI Resources)[13]
In this report we show that the use of micelle PCR is suitable for 16 S microbiota profiling experiments and strongly reduces the formation of the chimeric 16 S rRNA amplicons that are a major source of unidentifiable operational taxonomic units (OTUs) in microbiome studies

Summary

Introduction

Sequences can be filtered out of NGS results using specialized software[7,8], the generation of chimeric products can still seriously reduce the amount of useful information obtained in a single sequencing run[9] And this is seldom appreciated by users of NGS technologies, PCR is a competitive reaction meaning that the presence of multiple PCR targets in a single amplification reaction may lead to the preferential amplification of a particular subset of 16S rRNA gene copies[10]. The results could be biased by factors related to the amplification efficiency of particular 16S rRNA amplicons rather than the relative abundance of 16S rRNA genes in the test sample To overcome these sample-independent limitations, we developed and evaluated a micelle based amplification strategy targeting the 16S rRNA gene that greatly reduces chimera production during PCR amplification and prevents the formation of PCR competition products. NGS platforms such as Ion Torrent (Life Technologies) and 454 (Roche) have adopted emulsion-based amplification strategies in their standard NGS workflows to clonally re-amplify DNA sequencing libraries, as their molecular detection methods are not sensitive enough for single molecule sequencing and to prevent mixed sequences

Methods

Results

Conclusion