EnsembleSeq: A workflow towards real-time, rapid and simultaneous multi-kingdom amplicon sequencing for holistic and cost-effective microbiome research at scale

BackgroundBacterial communities are often concomitantly present with numerous microorganisms in the human body and other natural environments. Amplicon based microbiome studies have generally paid a skewed attention, that too at a rather shallow genus level resolution, to the highly abundant bacteriome, with interest now forking towards the other microorganisms, particularly fungi. Given the generally sparse abundance of other microbes in the total microbiome, simultaneous sequencing of amplicons targeting multiple microbial kingdoms could be possible even with full multiplexing. Guiding studies are currently needed for performing and monitoring multi-kingdom-amplicon sequencing and data capture at scale. MethodFull length bacterial 16S rRNA gene and entire fungal ITS region amplification was performed for human saliva samples (n=96, including negative and positive controls). Combined amplicon DNA libraries were prepared for nanopore sequencing using a major fraction of 16S molecules and a minor fraction of ITS amplicons. Sequencing was performed in a single run of an R10.4.1 flowcell employing the latest V14 chemistry. An approach for real time monitoring of the species saturation using dynamic rarefaction was designed as a guiding determinant of optimal run time. ResultsReal-time saturation monitoring for both bacterial and fungal species enabled the completion of sequencing within 30 hours, utilizing less than 60% of the total nanopores. [~]5 million HQ taxonomically assigned reads were generated ([~]4.2 million bacterial and 0.7 million fungal), providing a wider (beyond bacteriome) snapshot of human oral microbiota at species level resolution. Among the more than 400 bacterial and 240 fungal species identified in the studied samples, the species of Streptococcus (e.g. S. mitis, S. oralis) and Candida (e.g. C. albicans, C. tropicalis) were observed to be the dominating microbes in the oral cavity, respectively. This conformed well with the previous reports of the human oral microbiota. ConclusionEnsembleseq provides a proof-of-concept towards identification of both fungal and bacterial species simultaneously in a single fully multiplexed nanopore sequencing run in a time and resource effective manner. Details of this workflow are provided to enable large scale application for a holistic species level microbiome study.