Optimisation_of_high_throughput_16S_rRNA_gene_amplicon_sequencing__conditional_release. Optimisation_of_high_throughput_16S_rRNA_gene_amplicon_sequencing__conditional_release

16S rRNA gene sequencing has been fundamental in characterising human and environmental microbiomes that contribute to health and disease. It remains a cost-effective and computationally less-demanding alternative to shotgun metagenomic sequencing for taxonomic profiling. As the method is reference library independent, it can be employed for samples across hosts and environments ranging from high to low microbial biomass in environmental and clinical settings, including those containing previously unidentified species. Furthermore, quantity both increases statistical power and enables the ascertainment of complex relationships between different species, including antagonistic relationships and (mutualistic) trophic networks. Further optimizing the cost-effectiveness of 16S rRNA sequencing so that thousands of samples can be readily studied would benefit research projects that are otherwise limited by funds and time. Efficient sequencing at scale requires greater efficiency of 16S rRNA gene PCR library preparation than is currently possible. This could be explored by identifying rate-limiting steps that can be streamlined, allowing the automation of laboratory processes. These are key to reducing operator time and costs, while increasing throughput, but without impacting quality of results. We identified and interrogated several steps that have limited evidence for their absolute necessity in our current 16S rRNA gene library preparation protocol. These were: (1) the need for multiple PCR reactions per sample with subsequent pooling of PCR reactions to reduce PCR drift (i.e. the potential over-amplification of specific PCR products due to stochasticity in the PCR reaction), and (2) the use of a manually prepared master mix, which increases liquid handling. Here, we evaluate the impact of pooling of PCR reactions (single, duplicate, and triplicates) and the use of manual versus premixed mastermix on the observed microbial diversity using 16S rRNA gene PCR from human nasal samples and a mock microbial community. We use a replicate experiment to examine the consistency of results. Data shared for replication of results for protocol development and not research purposes. No metadata is held for these samples This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/