Development and Evaluation of Whole Cell- and Genomic DNA-based Microbiome Reference Standards Credible leads to Incredible™ Application Note Juan Lopera 1 , Monique Hunter 1 , Ray-Yuan Chuang 1 , Stephen King 1 , Megan Amselle 1 , Brian Chase 1 , Maria Mayda 1 , Kevin Zinn 1 , Samuel S. Minot 2 , Nicholas B. Greenfeld 2 , and Dev Mittar 1 . 1 ATCC, Manassas, VA 2 One Codex, San Francisco, CA Abstract ATCC has developed ATCC® Microbiome Standards for use in a broad array of applications ranging from method optimization to data inter- pretation. These standards are fully sequenced, characterized, and authenticated mock microbial communities that mimic mixed metagenomic samples. They were developed as whole cell or nucleic acid preparations with even or staggered genomic DNA abun- dance, and medium or high levels of mock community complexity rang- ing from 10 to 20 strains per sample. Here, we explore the development and use of the ATCC® Microbiome Standards. Introduction Advancement and accessibility of next-generation sequencing technologies have influenced microbiome analyses in tremen- dous ways, opening up applications in the areas of clinical, diagnostic, therapeutic, industrial, and environmental re- search. However, due to the complexity of 16S rRNA and metagenomic sequencing analysis, signifcant challenges can be posed by biases introduced during sample prepara- tion, DNA extraction, PCR amplifcation, library preparation, sequencing, or data interpretation. Many researchers have published studies on these biases, and leaders in the microbi- ome feld have highlighted the need for standardization 1-4 . One of the primary challenges in assay standardization is the limited availability of reference materials. To address these biases and provide a measure of stan- dardization within microbiome research and appli- cations, ATCC has developed a set of mock microbial communities, which includes lyophilized whole cells or genomic DNA, for use as microbiome reference standards. These standards mimic mixed metagenomics samples and comprise fully sequenced, characterized strains selected on the basis of select phenotypic and genotypic attributes. To further enhance the use of microbiome reference standards and eliminate the bias associated with data analysis, One Co- dex has developed a data analysis module in collaboration with ATCC that provides simple output in the form of true-positive, relative abundance, and false-negative scores for 16S rRNA com- munity profling and shotgun metagenomic sequencing.