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A quantitative sequencing method using synthetic internal standards including functional and phylogenetic marker genes.

ABSTRACT: 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.

SUBMITTER: Koike K

PROVIDER: S-EPMC10667660 | biostudies-literature | 2023 Dec

REPOSITORIES: biostudies-literature

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A quantitative sequencing method using synthetic internal standards including functional and phylogenetic marker genes.

Koike Kazuyoshi K Honda Ryo R Aoki Masataka M Yamamoto-Ikemoto Ryoko R Syutsubo Kazuaki K Matsuura Norihisa N

Environmental microbiology reports 20230718 6

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 t ...[more]

PMID: 37465846

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Project description:An inherent issue in high-throughput rRNA gene tag sequencing microbiome surveys is that they provide compositional data in relative abundances. This often leads to spurious correlations, making the interpretation of relationships to biogeochemical rates challenging. To overcome this issue, we quantitatively estimated the abundance of microorganisms by spiking in known amounts of internal DNA standards. Using a 3-year sample set of diverse microbial communities from the Western Antarctica Peninsula, we demonstrated that the internal standard method yielded community profiles and taxon cooccurrence patterns substantially different from those derived using relative abundances. We found that the method provided results consistent with the traditional CHEMTAX analysis of pigments and total bacterial counts by flow cytometry. Using the internal standard method, we also showed that chloroplast 16S rRNA gene data in microbial surveys can be used to estimate abundances of certain eukaryotic phototrophs such as cryptophytes and diatoms. In Phaeocystis, scatter in the 16S/18S rRNA gene ratio may be explained by physiological adaptation to environmental conditions. We conclude that the internal standard method, when applied to rRNA gene microbial community profiling, is quantitative and that its application will substantially improve our understanding of microbial ecosystems.IMPORTANCE High-throughput-sequencing-based marine microbiome profiling is rapidly expanding and changing how we study the oceans. Although powerful, the technique is not fully quantitative; it provides taxon counts only in relative abundances. In order to address this issue, we present a method to quantitatively estimate microbial abundances per unit volume of seawater filtered by spiking known amounts of internal DNA standards into each sample. We validated this method by comparing the calculated abundances to other independent estimates, including chemical markers (pigments) and total bacterial cell counts by flow cytometry. The internal standard approach allows us to quantitatively estimate and compare marine microbial community profiles, with important implications for linking environmental microbiomes to quantitative processes such as metabolic and biogeochemical rates.

Dataset Information

A quantitative sequencing method using synthetic internal standards including functional and phylogenetic marker genes.

Publications

A quantitative sequencing method using synthetic internal standards including functional and phylogenetic marker genes.

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