Design and evaluation of primers targeting genes encoding NO-forming nitrite reductases: implications for ecological inference of denitrifying communities.

Lea Wittorf,Christopher M Jones,Sara Hallin,Germán Bonilla-Rosso

doi:10.1038/srep39208

Lea Wittorf, Christopher M Jones + Show 2 more

Open Access

https://doi.org/10.1038/srep39208

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Abstract

The detection of NO-forming nitrite reductase genes (nir) has become the standard when studying denitrifying communities in the environment, despite well-known amplification biases in available primers. We review the performance of 35 published and 121 newly designed primers targeting the nirS and nirK genes, against sequences from complete genomes and 47 metagenomes from three major habitats where denitrification is important. There were no optimal universal primer pairs for either gene, although published primers targeting nirS displayed up to 75% coverage. The alternative is clade-specific primers, which show a trade-off between coverage and specificity. The test against metagenomic datasets showed a distinct performance of primers across habitats. The implications of clade-specific nir primers choice and their performance for ecological inference when used for quantitative estimates and in sequenced-based community ecology studies are discussed and our phylogenomic primer evaluation can be used as a reference along with their environmental specificity as a guide for primer selection. Based on our results, we also propose a general framework for primer evaluation that emphasizes the testing of coverage and phylogenetic range using full-length sequences from complete genomes, as well as accounting for environmental range using metagenomes. This framework serves as a guideline to simplify primer performance comparisons while explicitly addressing the limitations and biases of the primers evaluated.

Highlights

The detection of nitric oxide (NO)-forming nitrite reductase genes has become the standard when studying denitrifying communities in the environment, despite well-known amplification biases in available primers
We found no primer displaying both complete coverage and specificity at the same time, and the continuous increase in diversity with the addition of novel sequences plus the fact that no novel target regions exist for either gene, indicate that it may not be possible to obtain a single ideal universal primer pair for each of the nir genes
Primer design is complicated due to synonymous substitutions in the third codon position in protein-coding genes and there will likely be a non-conserved site every three nucleotides, even if there are strong selection forces acting towards the conservation of that particular amino acid residue

Summary

Introduction

The detection of NO-forming nitrite reductase genes (nir) has become the standard when studying denitrifying communities in the environment, despite well-known amplification biases in available primers. In an attempt to circumvent this problem, Wei and collaborators[18] recently developed primer variants that target individual nir sequence clades, similar to what was done for the nosZ gene encoding the nitrous oxide reductase[19] This greatly simplifies primer design, the usage of clade-specific primers brings additional considerations that need to be addressed due to the demand of such primer sets. Our approach includes an in silico analysis of 35 available nirS and nirK primers as well as 121 new primers designed in this study and was facilitated by the recent publication of robust and comprehensive phylogenies for both genes[7] We used both fully sequenced genomes and 47 metagenomes to evaluate phylogenetic coverage and specificity as well as performance against environmental sequences lacking PCR-amplification bias

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