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The method of spiking synthetic internal standard genes (ISGs) to samples for amplicon sequencing, generating sequences and converting absolute gene numbers from read counts has been used only for phylogenetic markers and has not been applied to functional markers. In this study, we developed ISGs, including gene sequences of the 16S rRNA, pmoA, encoding a subunit of particulate methane monooxygenase and amoA, encoding a subunit of ammonia monooxygenase. We added ISGs to the samples, amplified the target genes and performed amplicon sequencing. For the mock community, the copy numbers converted from read counts using ISGs were equivalent to those obtained by the quantitative real‐time polymerase chain reaction (4.0 × 104 versus 4.1 × 104 and 3.0 × 103 versus 4.0 × 103 copies μL‐DNA−1 for 16S rRNA and pmoA genes, respectively), but we also identified underestimation, possibly due to primer coverage (7.8 × 102 versus 3.7 × 103 μL‐DNA−1 for amoA gene). We then applied this method to environmental samples and analysed phylogeny, functional diversity and absolute quantities. One Methylocystis population was most abundant in the sludge samples [16S rRNA gene (3.8 × 109 copies g−1) and the pmoA gene (2.3 × 109 copies g−1)] and were potentially interrelated. This study demonstrates that ISG spiking is useful for evaluating sequencing data processing and quantifying functional markers.

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