Studies have demonstrated that SARS-CoV-2 RNA can be detected in the feces of infected individuals. This finding spurred investigation into using wastewater-based epidemiology (WBE) to monitor SARS-CoV-2 RNA and track the appearance and spread of COVID-19 in communities. SARS-CoV-2 is present at low levels in wastewater, making sample concentration a prerequisite for sensitive detection and utility in WBE. Whereas common methods for isolating viral genetic material are biased toward intact virus isolation, it is likely that a relatively low percentage of the total SARS-CoV-2 RNA genome in wastewater is contained within intact virions. Therefore, we hypothesized that a direct unbiased total nucleic acid(TNA) extraction method could overcome the cumbersome protocols, variability and low recovery rates associated with the former methods. This led to development of a simple, rapid, and modular alternative to existing purification methods. In an initial concentration step, chaotropic agents are added to raw sewage allowing binding of nucleic acid from free nucleoprotein complexes, partially intact, and intact virions to a silica matrix. The eluted nucleic acid is then purified using manual or semi-automated methods. RT-qPCR enzyme mixes were formulated that demonstrate substantial inhibitor resistance. In addition, multiplexed probe-based RT-qPCR assays detecting the N1, N2 (nucleocapsid) and E (envelope) gene fragments of SARS-CoV-2 were developed. The RT-qPCR assays also contain primers and probes to detect Pepper Mild Mottle Virus (PMMoV), a fecal indicator RNA virus present in wastewater, and an exogenous control RNA to measure effects of RT-qPCR inhibitors. Using this workflow, we monitored wastewater samples from three wastewater treatment plants (WWTP) in Dane County, Wisconsin. We also successfully sequenced a subset of samples to ensure compatibility with a SARS-CoV-2 amplicon panel and demonstrated the potential for SARS-CoV-2 variant detection. Data obtained here underscore the potential for wastewater surveillance of SARS-CoV-2 and other infectious agents in communities.

Studies have demonstrated that SARS-CoV-2 RNA can be detected in the feces of infected individuals. This finding spurred investigation into using wastewater-based epidemiology (WBE) to monitor SARS-CoV-2 RNA and track the appearance and spread of COVID-19 in communities. SARS-CoV-2 is present at low levels in wastewater, making sample concentration a prerequisite for sensitive detection and utility in WBE. Whereas common methods for isolating viral genetic material are biased toward intact virus isolation, it is likely that a relatively low percentage of the total SARS-CoV-2 RNA genome in wastewater is contained within intact virions. Therefore, we hypothesized that a direct unbiased total nucleic acid extraction method could overcome the cumbersome protocols, variability and low recovery rates associated with the former methods. This led to development of a simple, rapid, and modular alternative to existing purification methods. In an initial concentration step, chaotropic agents are added to raw sewage allowing binding of nucleic acid from free nucleoprotein complexes, partially intact, and intact virions to a silica matrix. The eluted nucleic acid is then purified using manual or semi-automated methods. RT-qPCR enzyme mixes were formulated that demonstrate substantial inhibitor resistance. In addition, multiplexed probe-based RT-qPCR assays detecting the N1, N2 (nucleocapsid) and E (envelope) gene fragments of SARS-CoV-2 were developed. The RT-qPCR assays also contain primers and probes to detect Pepper Mild Mottle Virus (PMMoV), a fecal indicator RNA virus present in wastewater, and an exogenous control RNA to measure effects of RT-qPCR inhibitors. Using this workflow, we monitored wastewater samples from three wastewater treatment plants (WWTP) in Dane County, Wisconsin. We also successfully sequenced a subset of samples to ensure compatibility with a SARS-CoV-2 amplicon panel and demonstrated the potential for SARS-CoV-2 variant detection. Data obtained here underscore the potential for wastewater surveillance of SARS-CoV-2 and other infectious agents in communities.

Characterization of the somatic sequence variations that accrue in cells is critical for understanding the pronounced cellular and clinical heterogeneity observed in cancer. The ability to efficiently detect variations in tumors can help to identify biomarkers which may be relevant to clinical trials, support more accurate prognosis, and help guide more effective choices of therapy. Next-generation sequencing (NGS) has become a valuable tool for discovering somatic mutations in cancers. Here we present an alternative approach to current methodologies for addressing these needs. HEAT-Seq (High Efficiency Amplification of Targets for Sequencing) is a targeted NGS method based on optimized, multiplexed molecular inversion probes (MIPs). This is a convenient, sensitive and cost-effective target enrichment technology for SNP discovery and SNP validation in cancer-related genes. HEAT-Seq probes target both DNA strands and were designed to facilitate bioinformatic error correction. Molecule identifiers (UIDs) incorporated into the probes tag PCR duplicates and support ultra-sensitive detection of low frequency variants, reduction of false positives, and accurate assessment of molecular complexity free of amplification bias. Sensitivities for allele detection have been measured to below 1%. The HEAT-Seq workflow from input DNA to sequence-ready sample can be completed in ≤8 hours without any requirement for a separate library preparation step, which saves both time and cost. Additionally, the capture, amplification and sample clean up steps are performed in a single reaction tube. This eliminates the need to transfer samples to subsequent reaction tubes, preserves sample identity, prevents cross contamination, limits sample loss, and makes the HEAT-Seq protocol easy to automate. A comparison of current PCR-based targeted sequencing with the new HEAT-Seq technology indicates significant advantages for HEAT-Seq in the accurate quantification of low frequency cancer variants. In summary, HEAT-Seq technology offers a rapid, convenient, automatable option to identify genomic DNA variants and enable advances in cancer research. Citation Format: Keynttisha Jefferson, Heather Halvensleben, Dawn Green, Ryan Bannen, Michael Brockman, Todd Richmond, Daniel Burgess. A novel molecular inversion probe (MIP) system for the streamlined identification of germline and somatic sequence variants in cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5223.

Next generation sequencing (NGS) of enriched targets is increasingly being used to discover and track mutations in genes implicated in cancer. Amplicon-based target enrichment approaches are frequently used with cancer samples because there is no sample library preparation needed and primer-based approaches are typically efficient with sequencing reads. However, traditional amplicon approaches don't scale up well and may have PCR and coverage uniformity bias. As the number of samples is increased, a fast, reliable, and cost-effective target enrichment method is a critical tool for mutation discovery and tracking. Existing target enrichment approaches based on molecular inversion probes (MIPs) have presented challenges with probe design and dependability. We have developed HEAT-Seq (High Efficiency Amplification of Targets for Sequencing) using advanced versions of MIPs which enable library-free enrichment, improved scalability, and the ability to remove PCR duplicates. We have also developed companion command line software which trims primer sequences and removes PCR duplicates. This software uses community standard file formats (FASTQ, SAM/BAM) and is intended to be compatible with common “best practice” NGS analysis pipelines. Here we describe two HEAT-Seq cancer panels which have been optimized to improve coverage uniformity and probe reliability. The HEAT-Seq Oncology Panel targets both strands for all protein coding bases in 60 cancer-related genes where possible. The target for this design is 245 kb. With 2 million 2×76bp reads for each of 8 replicates, we observe an on-target read rate of >95%. After duplicate removal, the% of target bases with at least 20x coverage is >90%. We observe uniformity (percent of probes > = 20% of the target mean) of ∼90%. The HEAT-Seq Ultra Hot-Spot Panel is a mutation-focused design that provides ultra-deep sequencing coverage capability for sequencing of mutation hot-spots in 53 cancer-related genes to detect low frequency variants in heterogeneous samples. The target for this design is 30.5 kb. With 500,000 2×76bp reads for each of 24 replicates, we observe an on-target read rate of ∼80%. After duplicate removal, the% of target bases with at least 20x coverage is >95%. We observe uniformity (percent of probes > = 20% of the target mean) of ∼95%. We also observed detection of validated low frequency variants down to 1%. In summary, both panels have been shown to capture cancer-related genes and target regions at depths sufficient to identify and track genomic variants. Citation Format: Ryan Bannen, Michael Brockman, Mark D’Ascenzo, Keynttisha Jefferson, Dawn Green, Heather Halvensleben, Kurt Heilman, Todd Richmond, Daniel Burgess. Cancer target enrichment panels using advanced molecular inversion probes (MIPs) with ability to reduce amplification bias and detect low frequency variants. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5217.

PurposeTypically, before Y90 radioembolization procedure undergoes, a CT is completed and the Barbeau test followed by radial artery ultrasound is used to determine if the artery is sufficiently large for vascular access [1-4]. 2.5 mm is the average radial artery diameter, and a vessel measurement of 2.0 mm is the recommended minimum diameter for safe vessel access, but a diameter of 1.5–2.0 mm can be accessed [4-9]. Our study explores using common femoral artery measurements from the pre-procedure CT abdomen/pelvis to assess in a binary manner if the vessel is sufficiently large to use for radial artery access. Materials and MethodsAll computed tomography scans of yttrium-90(Y90) radioembolization of the liver tumor procedures from January 1, 2015 - December 31, 2019 were retrospectively reviewed. Medical records were used from 47 procedures to gather patients' age, gender, Avastin use, femoral artery size (mm), administer Y90 (%), history of diabetes, and smoking status were recorded. ResultsThe minimum femoral artery size in patients who underwent transradial artery Y90 liver tumor radioembolization was 6 mm, with a mean femoral artery size of 10 mm. A comparative analysis of Y90 liver tumor uptake revealed no significant difference in radioembolization tumor uptake based on the initial site of procedure, transfemoral or transradial artery, (p > 0.81229). ConclusionThe study suggests that femoral arteries can predict radial artery diameter and that a femoral artery diameter of 10 mm should yield high confidence that the patient will be a candidate for transradial approach.