Q&A based on the presentation Detection and Characterization of Viral Pathogens and Their Impact on the Immune System by Bill Hunt, MS, Director, Microfluidics Product Management at Standard BioTools
As researchers around the world mount an aggressive and sustained response to the COVID-19 pandemic, it is important to build on past successes and harness insights from current efforts to better detect and characterize pathogens, identify treatments, and prepare for future outbreaks. Standard BioTools™ microfluidics-based genomic analysis tools can support and accelerate these goals by leveraging the benefits of nanoliter-scale automated microfluidics technology.
D3™ can design primers for targeted next-generation sequencing (NGS), gene expression and SNP genotyping panels.
Assay design specialists are available for assistance through the D3 website or by emailing email@example.com. The team can provide assistance in designing a panel to user-specified genomes.
If the viral genome is polyadenylated, as SARS-CoV-2 is, it should be detectable based on our oligo-dT capture workflow, depending on the concentration of the viral genome in the sample. The kit supports inputs of 10–100 ng of total RNA and has demonstrated robust performance over a range of RNA integrity number (RIN) values.
Detection of Human and Viral Full-Length RNA Using a Nanoscale Microfluidic Platform: Download the poster.
Analytical Validation of an Automated Full-Length mRNA Sequencing Library Preparation Method on a Microfluidic Circuit: Download the application note.
A group at the Icahn School of Medicine at Mount Sinai has been investigating this. In addition to looking at the virus itself and its direct RNA-based signature, they are looking at the host reaction to the virus being present. The aim is to identify a set of biomarkers, whether RNA-based or epigenetic-based, looking at the host response to the virus in the system. If we can understand whether a host response is more immunological or epigenetic in nature, we can determine the type of changes occurring, such as a methylation change. This is an area of interest in research because the ability to detect the virus quickly will allow for better triage of the individual and also aid in our understanding of what response is needed if the virus is detected in someone who is developing COVID-19 or is asymptomatic.
This is an active area of development for us and for our customers, including members or groups working on viral load applications using Standard BioTools technology. Currently, technologies are setting goal standards to detect 5 copies or less per reaction, and this is the target we are aiming to achieve as well.
This is an area in which we are waiting for input from a consortium led by Mount Sinai. They are working to come up with the content, and we will work with them to develop the assays based on the targets they provide.
Each microfluidics workflow can vary depending on the integrated fluidic circuit (IFC) and protocol chosen. Specific scripts are available to support a turnaround time of approximately 2 hours, while others can take longer. Our protocol for Real-Time PCR for Viral RNA Detection (FLDM-00102) analyzes 192 samples in a 3-hour 20-minute run time using fast PCR.
One of the big advantages of microfluidics technology is the capacity of throughput. The IFC structure can process up to 192 samples at once. Combined with a 2-hour run time, it is can analyze thousands of samples in a day. In contrast to the Abbott point-of-care test, the microfluidics workflows are high-throughput and high-capacity. A direct comparison of the two platforms demonstrates that in a 4-hour span, the Biomark™ HD system can test 192 samples where a point-of-care test, such as the Abbott system, can process 48 samples.
The 192.24 IFC provides 4,608 datapoints per run. The 96.96 Dynamic Array™ IFC can get up to 9,216 datapoints per run.
Several institutions have published their primer/probe sequences, including the CDC. Labs are using those sequences as part of their designs for laboratory-developed tests that are either run in-house or have been submitted for emergency use authorization. NCBI genomes are available to the public and can be accessed through their website. For example, viral genomes can be found at ncbi.nlm.nih.gov/genome/viruses/
In Standard BioTools systems, microfluidics refers to the structure, the reaction chambers and the architecture of the devices. Pre-processing of a variety of samples, including sample extraction, upstream of the IFC itself is somewhat agnostic. Several systems support the generation of purified nucleic acid, which serves as the input for use on the IFCs. Commercially available kits encompass manual or automated sample extraction and preparation upstream of the microfluidic device.
Currently, our customers are using a variety of commercial products available on the market today. In the future, we may develop sample extraction methods using our technologies.
The microfluidics system can be used across a variety of chemistries. Standard BioTools offers a chemistry for SNP detection, as well as Delta Gene™ chemistry for gene expression detection. We also support chemistries from other suppliers, including the application of TaqMan® chemistry workflows to our microfluidics, as well as chemistries from Integrated DNA Technologies (IDT™). Once a new chemistry becomes available, and possibly in collaboration with external partners, we can adapt those chemistries to be used with our microfluidics.
Cost is one of the system’s biggest advantages, because microfluidics works at such small concentrations and volumes of material. Reagent volume can be a large cost for many applications, so using much smaller amounts substantially reduces the cost of an individual reaction. Microfluidics systems also offer high-throughput and high-multiplex capacity, providing more results from a single test run. Moreover, the system can generate multiple datapoints for each of those samples. On the 192.24 IFC, for example, each of the 192 samples is interrogated by the 24 assays in the central matrix of the IFC, for a total of 4,608 datapoints for one IFC run. The library prep systems apply the same combinatorial concept, but each assay inlet represents a pool of assays, which can number in the hundreds. This allows the relatively quick generation of complex libraries for interrogation by NGS.
The approach you use depends on the questions you are asking. The advantages of the Standard BioTools microfluidics platform is performing high-throughput studies at a relatively low cost. For questions around therapeutic stratification or disease progression and immune profiling, mass cytometry is of particular interest. The genomics platforms can be used for looking at changes in viral load or epigenetic changes associated with alterations in the immune system. It really depends on the questions you are trying to address as you look at treatment, detection and progression of the disease.
Using the 192.24 Dynamic Array IFC for Gene Expression, the limit of detection was measured at 1 copy per µL of RNA (5 copies total).
Gene expression studies can also be done on Dynamic Array IFCs using TaqMan gene expression assays or Standard BioTools Delta Gene assays.
In the 192.24 Dynamic Array IFC for Gene Expression and the IDT 2019-nCoV CDC qPCR Probe Assay protocol, the starting amount is 5 µL of purified RNA. The exact amount of RNA may vary from sample to sample because some RNA comes from the host and some from the pathogens that may be present in the sample.
NGS could be used to detect mutations occurring in the viral genome. A custom-targeted NGS assay panel for the SARS-CoV-2 genome can be designed through the D3 Assay Design Group and prepared for sequencing on Illumina® systems using Advanta™ NGS Library Prep chemistry.