Metagenome sequencing spike-ins ('sequins'). Soil and rhizosphere metagenomes

The complexity of microbial communities, combined with technical biases in next-generation sequencing (NGS), pose a challenge to metagenomic sequencing and analysis. Here, we have developed a set of 86 internal DNA standards, termed ‘sequins’ (sequencing spike-ins), that together constitute a synthetic community of artificial microbial genomes. Sequins are added to environmental DNA samples prior to library preparation, and undergo concurrent sequencing and analysis with the accompanying sample. Because they share no homology with known natural sequences, sequins can be readily distinguished from the accompanying sample, thereby comprising an internal, sample-specific control. We validate the use of sequins by comparison to mock microbial communities, and then demonstrate how they can be applied to profile real metagenome samples of unknown content. This demonstrates the use of sequins to measure fold-change differences in the size and structure of accompanying microbe communities, and perform quantitative normalization between multiple samples. Further, by sequencing them on Oxford Nanopore Technology’s MinION platform, we illustrate how sequins can be used to benchmark and optimize new sequencing technologies. We provide metagenome sequins, and an accompanying software toolkit that supports their analysis, as a reference standard to aid in metagenomic studies.