Defining precision medicine and its aim
Precision medicine—occasionally referred to as “personalized medicine”—is an approach or medical model that promotes customizing and tailoring healthcare based on an individual’s underlying biological traits. The driving force behind precision medicine is the progress being made in the “big data” movement including genomics, transcriptomics, metabolomics, microbiomics, and the accompanying bioinformatics analysis pipelines. By surveying and incorporating underlying biological information from a patient, the aim of precision medicine is to be able to better classify patients into subpopulations to provide more “precise” or accurate care.
This year’s annual Precision Medicine World Conference (PMWC) took place in-person in Silicon Valley or Santa Clara, California from June 28-30, 2022. OraSure Technologies, Inc. was represented at this conference by several speakers, including at least one from each of our three subsidiary companies: Diversigen (Dr. Tonya Ward, Sr. Director of Bioinformatics & Laboratory Operations), DNA Genotek (Dr. Heloise Breton, Senior Product Manager – Microbiome and Dr. Rafal Iwasiow, Vice President of Innovation and Technology), and Novosanis (Jhana Hendrixkc, Scientific & Clinical Affairs Specialist). In case you missed any of these four presentations, below we provide summaries highlighting the key points.
Metatranscriptomics: enabling biomarker discovery — Dr. Tonya Ward, Sr. Director of Bioinformatics & Laboratory Operations at Diversigen
High-throughput sequencing efforts have focused on DNA sequencing of entire communities using either targeted approaches like amplicon sequencing of marker genes (e.g., 16S ribosomal RNA genes) or shotgun metagenomic sequencing of all available DNA from the sample. Metagenomics can comprehensively identify microbial community members while at the same time inferring microbe function through gene alignment to curated reference databases. However, functional inference through metagenomics does not distinguish between active and inactive microbiome members, making it difficult to pinpoint active microbial functions contributing to observed microbiome behavior.
Unlike metagenomics, metatranscriptomics measures the dynamic characteristic of gene expression through messenger RNA (mRNA) sequencing. Metatranscriptomics provides valuable insights into microbial taxonomy and community function by profiling expressed genes that can be used to distinguish between active from inactive microbes. In addition, metatranscriptomics can also provide insights into non-coding RNA and RNA viruses. While microbiome diversity has been intensively studied, the application of metatranscriptomics to human health, including biomarker discovery, is still relatively new.
Dr. Tonya Ward, Sr. Director of Bioinformatics & Lab Operations at Diversigen, gave an oral presentation at PMWC 2022 on how metatranscriptomics enables biomarker discovery. In her presentation, Dr. Ward discussed the development of Diversigen’s metatranscriptomic service to overcome several existing challenges in metatranscriptomic workflows, including extraction and stability of RNA, the presence of ribosomal RNA, and the development of an appropriate bioinformatics annotation pipeline.
Both the storage medium and extraction method can influence microbial profiles generated from an RNA sample. As RNA samples readily degrade, the conversion of RNA to stable, complementary DNA (cDNA) needs to be fast and efficient to avoid potentially losing the sample or introducing bias. Several factors must be considered in terms of extraction and stability including:
- Compatibility: not all storage buffers are made equal in terms of compatibility; some may preserve well but are not compatible with optimized extraction methods
- Yield: obtaining sufficient high-quality, intact mRNA that is representative of the community
- Contaminants: Samples can contain high levels of substances that inhibit extraction and stability
- RNA half-life: RNAses in the environment degrade RNA readily, thus a quick and RNAse-free workflow is needed.
A second existing challenge is the fact that ribosomal RNA (rRNA) can easily represent >90% of the RNA in a sample, thus blocking out the signal of other types of RNA in the sample, including mRNA. Solutions to overcome this issue include ribosomal depletion and/or deep sequencing. Ribosomal depletion involves using either subtractive hybridization using rRNA-specific probes to capture rRNA or exonuclease digestion, both of which ultimately remove rRNA from the sample. Meanwhile, deep sequencing involves sequencing samples to a high level of reads to become sensitive enough to detect microbial mRNA beyond the rRNA signal.
Finally, in terms of developing an appropriate bioinformatics pipeline to evaluate and conduct annotation of metatranscriptomics data, Diversigen utilizes comprehensive analysis, including gene expression profiles, taxonomic profiles, and diversity metrics through a curated database containing over 190,000 genomes and 100 million unique genes. Diversigen developed this solution through several combined factors including:
- Adapter & quality control filtering: Trimming of adapters and removal of low-quality reads
- Host depletion & rRNA depletion: Removal of hosts reads via alignment to host-specific database and rRNA sequence removal via alignment to an rRNA-specific database
- Database pre-filtering: Creation of OER-sample database prior to filtering
- Database refinement: Refinement of sample-specific databases to refine and minimize false positives
- Full alignment: Generation of taxonomic and functional profiles using more stringent parameters
- Filtering & processing: Functional and taxonomic outputs are filtered based on coverage
- Report generation: Annotations aggregated and compiled for browsing and downstream analysis
Dr. Ward emphasized that Diversigen’s metatranscriptomic service has been validated for performance and accuracy through in silico mock community profiling. Through testing of seven metagenomic samples as the basis, taxonomic compositions were determined using a random subset of 20% of genomes from samples. By testing the annotation performance, it was shown that Diversigen’s metatranscriptomic service has a 98.57% true positive rate, 98.6% accuracy, and 98.6% sensitivity.
Diversigen’s metatranscriptomic service provides robust results through a standardized high-throughput workflow and comprehensive analysis that generates repeatable and reproducible reports including gene expression profiles, taxonomic profiles, and diversity metrics.
OMNIgene™•SKIN: a validated skin microbiome collection system — Dr. Heloise Breton, Senior Product Manager – Microbiome at DNA Genotek
Research into the human microbiome has been advancing stepwise with gains in sequencing technology. The microbes present in and on our bodies play a critical role in our general health and have been implicated in the onset, regulation, and progression of several diseases to date. Accurately surveying the human microbiome—traditionally consisting of the gut and/or skin—will be an important step in forging our understanding of how best to tailor healthcare to individuals.
For example, to assess the skin microbiome of individuals, harvesting or collecting a sample of the microbes present on the skin is a critical step in the process. For skin microbial data to be biologically accurate, sample collection must be site-specific, effective in terms of storing, stabilizing, and transporting microbial DNA for downstream analysis, and ultimately reproducible. During her oral presentation at PMWC 2022, Dr. Heloise Breton focused on a specific product that addresses this exact need. Developed by DNA Genotek, the OMNIgene™•SKIN device is an all-in-one system for the collection, stabilization, transportation, and storage of microbial DNA from skin samples. Throughout her presentation, Dr. Breton highlighted the capabilities of this collection device, including evidence on how OMNIgene•SKIN device provides:
- An accurate representation of the microbiome profile; this is thanks to the device preserving the relative abundance of microbial species during storage and shipping.
- High quality DNA for downstream analysis.
- Stabilized DNA at ambient temperature for easy storage and transportation.
- Easy user instructions for naïve donors, validated for successful self-collection for microbiome analysis.
- Skin site-specific microbial signatures that avoid bias. To capture site-specific microbiome profiles, test samples were collected from the three major types of skin sites as described in the literature: sebaceous (face, scalp), dry (forearm), and wet (toe web).
Dr. Breton also detailed what we found to be the best microbial DNA extraction method to use with samples collected using OMNIgene•SKIN devices to optimize sample yields. In this case, the specific, high throughput protocol she highlighted—developed by the sister company of DNA Genotek, Diversigen—recovers over 100 times more microbial DNA than traditional extraction methods.
One of the steps tested using Diversigen’s protocol involved assessing different input amounts from skin samples to see if DNA quantity altered the final skin microbial profile. Sample collection, when done by multiple different people, can introduce a degree of variability. Thus, it was important to test whether the OMNIgene•SKIN device could still produce consistent data when supplied with varying amounts of starting material. Representative face samples were collected and diluted to show that the protocol can generate similar profiles and consistent results across a range of skin sample DNA quantities. Here, the diluted samples represent either poor collection or potential donor differences in microbial content. Our results showed that even after diluting the skin sample input, the profiles generated are comparable to those undiluted, demonstrating consistency.
OMNIgene™•GUT Dx: a new benchmark for gut microbiome collection — Dr. Rafal Iwasiow, Vice President of Innovation and Technology at DNA Genotek
In his presentation, Dr. Rafal Iwasiow, Vice President of Innovation and Technology at DNA Genotek, presented the latest gut microbiome collection device by DNA Genotek—the OMNIgene•GUT Dx kit, the first and only U.S. Food and Drug Administration (FDA)-authorized device for gut microbiome profiling. The OMNIgene•GUT Dx kit is specifically designed to collect human fecal samples to assess the gut microbiome profile and has been granted FDA De Novo classification for the preservation and stabilization of the relative abundance of microbial nucleic acids in clinical samples.
As a diagnostic device, Dr. Iwasiow talked about how OMNIgene•GUT Dx device has been tested and has demonstrated the following:
- Ability to preserve microbial relative abundance for accurate microbiome profiling
- Sample stability: the microbiome profile of fecal samples collected using the OMNIgene•GUT Dx collection device do not change during ambient temperature storage for up to 30 days.
- Reproducibility: assessed by validation testing, including a whole genome sequencing assay workflow.
- Ease-of-use: robustly tested through usability studies with naïve donors.
Usability testing for the OMNIgene•GUT Dx device demonstrated that the product can be used by any member of the population and lead to the successful collection of a fecal sample. Two studies were conducted as part of the usability testing:
- A study using simulated samples aimed at confirming that the user instructions were fit for purpose and could be understood by most of the population. This meant testing participants of various ages, genders, and levels of education. This study was conducted by a contract organization and allowed us to optimize the instructions based on user feedback.
- A second study that required users to collect a fecal sample with the OMNIgene•GUT Dx kit at their homes and answer the same comprehension questions as the simulated samples study.
Pointing to the results of the usability studies, Dr. Iwasiow stated that users asked to collect a sample using the OMNIgene•GUT Dx device would be able to provide sufficient sample for successful downstream analysis, return their collected sample by mail (if required), and ensure that samples will be well-homogenized within the device (i.e., mixing with the stabilization chemistry is key for sample stability and reproducibility).
In his final slides, Dr. Iwasiow emphasized that discovery potential is only as strong as your sample signal; the more accurate your signal, the better your data, and the better your conclusions. Sample signals can vary based on noise and technical biases; Dr. Iwasiow stated that the OMNIgene GUT•Dx device addresses both challenges by reducing noise and technical bias in datasets.
Urine as a liquid biopsy for oncology applications — Jhana Hendrickx, Scientific & Clinical Affairs Specialist at Novasanis
Highlighting the current challenges with traditional cancer screening and detection methods, Jhana Hendrickx, Scientific & Clinical Affairs Specialist at Novosanis, spoke about the use of minimally invasive procedures such as liquid biopsies as an alternative sample for oncology applications.
At the start of her presentation, she explained why urine is an exciting and promising sample in cancer research. A lot of work is being done to evaluate urine for detection and monitoring of urological and systemic cancers. For example, the number of scientific and peer-reviewed publications focusing on the association between “urine and oncology”, or “urine and liquid biopsies” has increased significantly over the last three decades.
Urine is attractive for many reasons—the sample is easily accessible, non-invasive, available in larger quantities and applicable for home collection. The presentation further explained the increasing number of biomarker candidates that can be found in urine, including DNA, RNA, proteins, exosomes, and metabolites for several cancer types, including for bladder, cervical, kidney, prostate, breast, colon, and lung cancer. Additionally, several diagnostic assays using urine are commercially available for prostate cancer.
However, for effective clinical applications, standardization of preanalytical conditions for the handling of urine specimens is required. This is where Novosanis’ urine collection device, Colli-Pee™ fits in. Urine collected with Colli-Pee offers improved diagnostic sampling accuracy and patient comfort compared to a regular urine cup. Additionally, Novosanis and DNA Genotek, have developed a novel stabilization chemistry, UAS™ for the preservation of urine. Urine collected with Colli-Pee containing UAS™, which is available for research use only (RUO), has shown to prevent degradation of EV-RNA and cfDNA.
Novosanis has also received several European grants to address the performance of Colli-Pee collected urine for key cancer types. URODETECT, in collaboration with the University of Antwerp, focuses on urine as a potential sample for breast and prostate cancer. CASUS aims to offer a molecular screening solution for cervical cancer screening based on first-void urine self-sampling.