justinshaffer / absolute_abundances_metagenomic_syndna

Codebase for synDNA from Zaramela et al. 2022

SynDNA – A synthetic DNA spike-in method for absolute quantification of shotgun metagenomic sequencing

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. An inherent limitation of relative values is 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 DNAs (synDNAs) spike-ins that enable precise and cost-effective absolute quantification of microbiome data by adding defined amounts of synDNAs into the samples. We designed 10 synthetic DNAs (synDNAs) with the following features: 2,000 bp length, variable GC content (26, 36, 46, 56, or 66% GC), and negligible identity with sequences found in the NCBI database. Dilution pools were generated by mixing the 10 synDNAs in different concentrations. Shotgun metagenomic sequencing showed that the pools of synDNAs with different %GC efficiently reproduced the serial dilution, showing high correlation (R2 ≥ 0.94) and significance (p-value < 0.01). Furthermore, we demonstrated that the synDNA can be used as DNA spike-ins to generate linear models and predict with high accuracy the absolute number of bacterial cells in complex microbial communities.