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

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).

Microbiological diagnosis of device‐related biofilm infections

“…Other factors associated with NGS that may influence results, such as chimera formation of amplicons and library preparation methodology may also be problematic . Boers et al.…”

“…Other factors associated with NGS that may influence results, such as chimera formation of amplicons (72) and library preparation methodology may also be problematic (73). Boers et al nicely summarized the potential pitfalls in NGS-based microbiome study and urged everyone working with NGS data to be more critical (74).…”

Scaling up: A guide to high‐throughput genomic approaches for biodiversity analysis

“…Digital droplet PCR (ddPCR) is when digital PCR is carried out in micelle droplets, and this can be used for endpoint or real‐time DNA quantification depending on the platform. In digital PCR, a single template is amplified on its own and can avoid problems from mixed‐template PCR such as the generation of chimeric sequences, primer‐template bias and template competition (Boers, Hays, & Jansen, ; Williams et al., ). ddPCR has been used to estimate eDNA concentration, fish abundance and biomass (Doi et al., ).…”

“…As shown in Table 1, this is another fluorescence-based detection method that can be used to quantify individuals or be used as a target enrichment method prior to HTS. Digital PCR is an alternative to traditional qPCR where a sample is separated into thousands of parallel PCRs each with a single-or no-template molecule (Vogelstein & Kinzler, 1999 (Boers, Hays, & Jansen, 2015;Williams et al, 2006). ddPCR has been used to estimate eDNA concentration, fish abundance and biomass (Doi et al, 2015).…”

Long-read DNA metabarcoding of ribosomal rRNA in the analysis of fungi from aquatic environments

“…Multiple PCR reactions (up to 50) were required for each environmental sample to ensure sufcient product for library preparation (1 µmg purifed PCR product). We also included a two-step emulsion PCR (emPCR) of the mock community in order to test whether emPCR could reduce chimera formation rate by the physical isolation of DNA template molecules (Boers 2015). The Micellula DNA Emulsion kit (Roboklon GmbH, Berlin) was used for a two-step PCR: a frst amplifcation of 25 cycles, with 2µml of the cleaned template used in a second 25 cycle PCR.…”