B-240 Development of AcroMetrixTM Human Microbiome Quality Control Materials

Abstract

Abstract Background The human microbiome is receiving significant attention given its critical roles in the maintenance of human health. The compositional and functional alterations of the human microbiota have been associated with various pathological conditions including colorectal cancer (CRC). As the number of human metagenomic data increases, the interest to utilize these metagenomic biomarkers for diagnosis of colorectal cancer and other diseases has also elevated. Studies that evaluated the defined fecal microbial markers for CRC detection showed the test accuracy could vary from 73% to 98% depending on the detection panels of microbiome biomarkers. The measurement inconsistencies between laboratories due to the use of different detection platforms, the coverage of various assay panels, the biases in different steps of microbiome workflow all indicated the critical need for reliable quality control materials. Currently, the commercially available microbial standards usually contain limited species and also mixed with fixed ratios which may not reflect the microbial composition in the clinical samples. There is no commutable reference material that recapitulates the dysbiotic microbial composition under healthy or pathological conditions (e.g., CRC). Therefore, in this study, we developed DNA quality control materials (QCMs) that recapitulate the dysbiotic microbial composition for colorectal cancer to assess the technical and non-technical variation for part of the microbiome workflow. Methods We first identified the specific bacterial species that either are enriched in CRC patients or healthy human gut based on comprehensive literature search and the other available dataset. We then created microbiome QCMs to recapitulate their dysbiotic microbial composition in comparison to the compositions in healthy condition. The QCM consists of a mixture of genomic DNA from the selected bacterial species, the copy number of DNA was determined by Bio-RadTM droplet digital PCR (ddPCR) with the assays that target to species-specific regions to reflect the microbial composition. In addition, the microbiome controls are further evaluated and validated by Microbiome Health Research Assay on Ion Ampliseq and GeneStudio S5 platforms. Results The AcroMetrixTM human microbiome QCMs have been carefully formulated to mimic dysbiotic microbial composition for colorectal cancer or healthy human gut. This CRC QCM covers 29 different species with a wide range of guanine-cytosine content sequences, belonging to 10 phyla and 20 genera. 25 out of these 29 species (86.2%) were covered by Microbiome Health Research Assay. To mimic the trends of dysbiotic microbial composition, the DNA frequency among these 29 species was targeted from 0% to 15% according to the microbial composition. Droplet digital PCR results confirmed the target frequencies within the accepted tolerance range and the results were compared with the data from the Next Generation Sequencing platform. Conclusions The human microbiome QCMs that cover 29 bacterial species and mimic dysbiotic microbial composition for colorectal cancer or healthy human gut have been designed and developed. These QCMs could provide essential elements to verify, validate and monitor test performance, technical and non-technical variation of microbiome workflows, as well as product QC releases and method development for ddPCR & NGS assays.