ABSTRACT BY SPONSOR: Progress towards standardizing metagenomics: application of metagenomic reference materials to develop a reproducible microbial lysis methodology with minimum bias

Michael M. ^1,2 David C. Danko ^3,4 Elaine Wolfe ¹ Shuiquan Tang ¹ Karen G. Jarvis ⁵ Christopher J. Grim ⁵ Venu Lagishetty ⁶ Jonathan P. Jacobs ⁶ Jason W. Arnold ⁷ Ryan Kemp ¹ Christopher Mason ^4,8

¹Department of Bioinformatics, Zymo Research Corp., USA
²Molecular, Cell, and Developmental Biology/quantitative and Computational Biosciences Institute, University of California, USA
³Tri-Institutional Computational Biology and Medicine Program, Weill Cornell Medicine of Cornell University, USA
⁴Institute for Computational Biomedicine, Department of Physiology and Biophysics, Weill Cornell Medicine of Cornell University, USA
⁵Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, USA
⁶Microbiome Core, Division of Digestive Diseases, Department of Medicine, University of California, USA
⁷Microbiome Core, School of Medicine, University of North Carolina, USA
⁸The Feil Family Brain and Mind Research Institute, Weill Cornell Medicine of Cornell University, USA

Rapid growth of metagenomics research over the past decade has contributed to a lack of reproducibility between different methods and laboratories, which limits our ability to compare between studies and decreases confidence in previous conclusions.

To address the diversity of methods available, we compared the performance of many commercially- and academically-sourced lysis protocols. Methods were evaluated using mock microbial community standards with defined composition to serve as a ground truth for measurements. To facilitate comparisons, we developed the Measurement Integrity Quotient (MIQ), providing a single numerical score that describes the accuracy of an observed composition relative to a known standard. Utilizing this method, we compared the effects of different variables on lysis efficiency, including thermal, enzymatic, and mechanical (bead) lysis as well as over 40 different bead material/size combinations and cell disruptor type/intensity/run time combinations. Additionally, typical sample types (feces, soil, skin, saliva, urine) were tested with different lysis methods with replicates at different laboratories for over 1500 samples tested.

Hard, dense ceramic beads of mixed size on an appropriate cell disruptor with a validated protocol created the least biased lysis of all examined methods. The use of MIQ score provided rapid, easy to understand analysis of accuracy and has been publicly released. The use of these data and methods can help create more reproducible metagenomics pipelines with results that better represent the true composition of the sample. These methods achieved minimal amounts of deviation from expected composition and a high degree of run-to-run and interlab reproducibility.