Various microorganisms play key roles in the nitrogen (N) cycle. Quantitative PCR (qPCR) and PCR amplicon sequencing of N cycle functional genes allow us to analyze the abundance and diversity of microbes responsible for N-transforming reactions in various environmental samples. However, analysis of multiple target genes can be cumbersome and expensive. PCR-independent analysis, such as metagenomics and metatranscriptomics, is useful but expensive, especially when we analyze multiple samples and try to detect N cycle functional genes present at a relatively low abundance. Here, we present the application of microfluidic qPCR chip technology to simultaneously quantify and prepare amplicon sequence libraries for multiple N cycle functional genes as well as taxon-specific 16S rRNA gene markers for many samples. This approach, named the nitrogen cycle evaluation (NiCE) chip, was evaluated by using DNA from pure and artificially mixed bacterial cultures and by comparing the results with those obtained by conventional qPCR and amplicon sequencing methods. Quantitative results obtained by the NiCE chip were comparable to those obtained by conventional qPCR. In addition, the NiCE chip was successfully applied to examine the abundance and diversity of N cycle functional genes in wastewater samples. Although nonspecific amplification was detected on the NiCE chip, this can be overcome by optimizing the primer sequences in the future. As the NiCE chip can provide a high-throughput format to quantify and prepare sequence libraries for multiple N cycle functional genes, this tool should advance our ability to explore N cycling in various samples.
Bibliographical noteFunding Information:
We thank Reiko Hirano, Akiyoshi Ayumi, and Nora Powers for technical assistance and Hirotsugu Fujitani and Yuga Hirakata for providing AOA and NOB cultures and anaerobic granular sludge. We also thank Kelly Duhn for scientific editing of the manuscript. This work was supported in part by Mitsui Co. & Ltd., the Environment Fund (to S.I.), the MnDRIVE Initiative of the University of Minnesota (to S.I.), grants-in-aid for scientific research (no. 26281017 and 17H01854) from the Japan Society for the Promotion of Science (JSPS) (to T.S.), and the National Institute of Polar Research (NIPR) through general collaboration project no. 27-29 (to M.O.). Part of the computations was performed by using a supercomputer maintained by the National Institute of Genetics (NIG), Research Organization of Information and Systems (ROIS), Japan.
- Amplicon sequencing
- Microfluidic chip
- Nitrogen cycle
- Quantitative PCR
- Wastewater treatment