Effect of primer mismatch, annealing temperature and PCR cycle number on 16S rRNA gene-targetting bacterial community analysis

Abstract

In the attempt to explore complex bacterial communities of environmental samples, primers hybridizing to phylogenetically highly conserved regions of 16S rRNA genes are widely used, but differential amplification is a recognized problem. The biases associated with preferential amplification of multitemplate PCR were investigated using 'universal' bacteria-specific primers, focusing on the effect of primer mismatch, annealing temperature and PCR cycle number. The distortion of the template-to-product ratio was measured using predefined template mixtures and environmental samples by terminal restriction fragment length polymorphism analysis. When a 1 : 1 genomic DNA template mixture of two strains was used, primer mismatches inherent in the 63F primer presented a serious bias, showing preferential amplification of the template containing the perfectly matching sequence. The extent of the preferential amplification showed an almost exponential relation with increasing annealing temperature from 47 to 61 degrees C. No negative effect of the various annealing temperatures was observed with the 27F primer, with no mismatches with the target sequences. The number of PCR cycles had little influence on the template-to-product ratios. As a result of additional tests on environmental samples, the use of a low annealing temperature is recommended in order to significantly reduce preferential amplification while maintaining the specificity of PCR.

Effect of marker choice and thermal cycling protocol on zooplankton DNA metabarcoding studies

“…The detection of additional taxa supports the use of low annealing temperatures to maximize taxonomic coverage for any given marker (Clarke et al., 2014; Sipos et al., 2007). Amplification and sequencing of COI amplicons generated using the low annealing temperature protocol was highly repeatable in spite of using different sequencing chemistries (v2 and v3) and number of sequencing cycles (2 × 250 and 2 × 300), with OTUs representing >0.1% of reads in one replicate almost always detected in the corresponding replicate.…”

“…To compare performance of the three markers, we evaluated taxonomic coverage and resolution, correspondence between morphology‐ and DNA‐based identification, and the ability to assess relative abundance of calanoid copepods from the proportion of HTS reads. For the COI marker, high annealing temperatures in the first rounds of the published touchdown PCR protocol (Leray et al., 2013) could bias PCR amplification toward taxa with less mismatches in the primer‐binding sites (Sipos et al., 2007); hence, we compared the number of taxa detected using the touchdown protocol to a protocol with a single low annealing temperature. We also explored the technical repeatability of taxon detection by re‐sequencing COI amplicons generated with identical PCR protocols.…”

Application of the denaturing gradient gel electrophoresis (DGGE) technique as an efficient diagnostic tool for ciliate communities in soil

“…Using an additional degenerate primer may solve this problem but could also result in differently migrating PCR products from the same template. Another possibility is to further lower the annealing temperature in order to reduce preferential amplification as suggested by recent studies (Sipos et al, 2007;Ishii and Fukui, 2001). We therefore recommend, that further small primer optimisation might be necessary before using this DGGE protocol as a monitoring tool for samples with an expected high abundance of Hymenostomatida, such as freshwater environments.…”

Use of bioinformatic tools in primer validation

“…Mismatches in the 3′ end affect the target amplification greater than mismatches toward the 5′ end or in the middle. 3,4 Second, the analysis performed by primer 3 software showed high Tm for the F′ primer, as well as high hairpin stability. Although it is possible to amplify templates with primers with high Tm using a two-step PCR, the methodology specifically states that they used the GC-Rich PCR system's instructions (Roche Applied Sciences, Rotkreuz, Switzerland), which follow a traditional PCR protocol.…”

“…This, combined with the calculated high hairpin stability, and the variant in the 3′ end could have had a negative effect on the PCR efficiency. 3,4 Because the conventional PCR was the first step in its three-step protocol, 1 and because it was the one that defined which samples would undergo a triplet PCR, this reduced efficiency would be undesirable.…”