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ABSTRACT Microbiome studies have the common goal of determining which microbial taxa are present, respond to specific conditions, or promote phenotypic changes in the host. Most of these studies rely on relative abundance measurements to drive conclusions. Inherent limitations of relative values are the inability to determine whether an individual taxon is more or less abundant and the magnitude of this change between the two samples. These limitations can be overcome by using absolute abundance quantifications, which can allow for a more complete understanding of community dynamics by measuring variations in total microbial loads. Obtaining absolute abundance measurements is still technically challenging. Here, we developed synthetic DNA (synDNA) spike-ins that enable precise and cost-effective absolute quantification of microbiome data by adding defined amounts of synDNAs to the samples. We designed 10 synDNAs with the following features: 2,000-bp length, variable GC content (26, 36, 46, 56, or 66% GC), and negligible identity to sequences found in the NCBI database. Dilution pools were generated by mixing the 10 synDNAs at different concentrations. Shotgun metagenomic sequencing showed that the pools of synDNAs with different percentages of GC efficiently reproduced the serial dilution, showing high correlation (r = 0.96; R2 ≥ 0.94) and significance (P |
