Functional genes to assess nitrogen cycling and aromatic hydrocarbon degradation: primers and processing matter

Targeting sequencing to genes involved in key ecofunctional pathways provides an opportunity to sample nature’s gene guild to greater depth and help link community structure to process-level outcomes. Vastly different approaches have been implemented for sequence processing and, ultimately, for taxonomic placement of these gene reads. The overall quality of next generation sequence analysis of functional genes is dependent on multiple steps and assumptions of unknown diversity. To illustrate current issues surrounding amplicon read processing we provide examples for three ecofunctional gene groups. A combination of in-silico, environmental and cultured strain sequences was used to test new primers targeting the dioxin and dibenzofuran degrading genes dxnA1, dbfA1, and carAa. The majority of obtained environmental sequences were classified into novel sequence clusters, illustrating the discovery value of the approach. Assessment and improvement of primer coverage is critical to enhancing comprehensiveness of ecofunctional gene recovery. Well-known nirK primers exhibited deficiencies in reference database coverage, illustrating the need to refine primer-binding sites and/or to design multiple primers, while nirS primers exhibited bias against the Chloroflexi, Deinococcus-Thermus, Aquificales and Bacteroidetes. Amino acid-based OTU clustering of the two N-cycle genes from soil samples yielded only 114 unique nirK and 45 unique nirS genera, likely a reflection of constricted primer coverage. Finally, supervised and non-supervised OTU analysis methods were compared using the nifH gene of nitrogen fixation, with generally similar outcomes, but the clustering (non-supervised) method yielded higher diversity estimates and stronger site-based differences. Current databases for most ecofunctional genes are less complete than for nifH, and hence clustering would be preferred. High throughput amplicon sequencing can provide inexpensive and rapid access to nature’s related sequences by circumventing the culturing barrier, but each unique gene requires individual considerations in terms of primer design, and sequence processing and classification.