Comprehensive Cross-Domain Taxonomic Classification of Microbiotas using Partitioned Amplification Multiplexed Amplicon Sequencing

Understanding microbial ecosystems requires accurate identification and quantification of species across all domains of life - bacteria, archaea, and eukaryotes. However, traditional sequencing approaches are biased toward prokaryotes, often overlooking eukaryotic microbes due to technical limitations and high costs. This gap in taxonomic analysis hinders our ability to fully comprehend microbial diversity, interactions, and ecosystem functions. To address this challenge, we develop Partitioned Amplification Multiplex Amplicon Sequencing (PAMA-seq), a method that can simultaneously quantify microorganisms across the prokaryotic and eukaryotic domains. In PAMA-seq, rRNA genes from different species are partitioned into nanoliter water-in-oil droplets and independently amplified via droplet PCR. Each droplet contains primer pairs targeting either 16S rRNA for prokaryotes or 18S rRNA for eukaryotes, with sample dilution ensuring that only a single target gene is present per droplet. Following PCR amplification to saturation, the amplicon molecule count for any given species is proportional to the number of droplets containing that species, and thus reflects the initial rRNA gene concentration. PAMA-seq is validated using synthetic microbial community with both bacteria and yeast, as well as ocean and human gut microbiome samples. Overall, PAMA-seq provides a scalable and cost-effective approach for cross-domain taxonomic classification, suitable for complex microbiomes analysis.