Preparation of robust Synthetic Control samples and their use in a metatranscriptomic clinical test

Metatranscriptomics (MT), or RNA sequencing, has the potential to revolutionize the field of molecular diagnostics. Due to the complexity of MT diagnostic models, positive and negative control materials for specific disease indications can be difficult to obtain. Controls must often be sourced directly from patients. This introduces logistical burdens, assay variability, and limits high throughput clinical laboratory operations. To overcome this limitation, we developed a method for generating Synthetic Control (SC) samples, which duplicate the nucleic acid signature of complex clinical specimens and produce the desired test outcome. SCs can be easily and cost-effectively produced in large quantities (>100,000 SCs per amplification cycle), enabling high throughput diagnostic testing. Here, we report the generation of Synthetic Positive Control (SPC) samples. SPCs were validated and implemented in a clinical laboratory. The SPCs produced robust positive signals (average OC risk score of 0.997) and high levels of reproducibility (%CV of 0.2%) in a high throughput automated CLIA laboratory. SCs are a novel and useful method for the generation of high quality controls for MT-based diagnostic tests, and their adoption could herald the widespread use of MT tests in molecular diagnostics. ### Competing Interest Statement All authors are employees of Viome Life Sciences, Inc. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. ### Funding Statement This study was funded by Viome Life Sciences ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The Viome IRB of Viome Life Sciences gave ethical approval for this work I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are proprietary to Viome Life Sciences but may be available upon reasonable request to the authors