Project description:An interventional, prospective clinical performance study protocol, for the testing of DNA extracted from tumor tissue biopsy samples, using the therascreen KRAS RGQ PCR Kit, from patients with Non-Small Cell Lung Cancer and Colorectal Cancer, screened in Amgen’s clinical trial (Protocol No. 20170543).
Project description:Purpose: To develop a methodology to obtain high-quality expression data from matched primary tumours and metastases by utilizing LCM to isolate pure cellular populations Methods: Snap frozen biopsies of matched primary tumour, metastasis and the associated normal tissues were microdissected to obtain stromal and epithelial components. RNA was extracted from the respective microdissected samples and subsequently subjected to RNA sequencing. Results: We demonstrated an optimised LCM protocol which reproducibly delivered intact RNA used for RNA sequencing and quantitative polymerase chain reaction (qPCR). This framework helped to uncover multiple differentially expressed cancer-associated genes and qPCR verification of 9 chosen targets confirmed the high fidelity of the RNA sequencing process. Conclusion: This study suggests that the combination of matched tissue specimens with tissue microdissection and NGS provides a viable platform to unmask hidden biomarkers and provides insight into tumour biology at a higher resolution.
Project description:Monitoring the spread of viral pathogens in the population during epidemics is crucial for mounting an effective public health response. Understanding the viral lineages that constitute the infections in a population can uncover the origins and transmission patterns of outbreaks and detect the emergence of novel variants that may impact the course of an epidemic. Population-level surveillance of viruses through genomic sequencing of wastewater captures unbiased lineage data, including cryptic asymptomatic and undiagnosed infections, and has been shown to detect infection outbreaks and novel variant emergence before detection in clinical samples. Here, we present an optimised protocol for quantification and sequencing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in influent wastewater, used for high-throughput genomic surveillance in England during the COVID-19 pandemic. This protocol utilises reverse compliment PCR for library preparation, enabling tiled amplification across the whole viral genome and sequencing adapter addition in a single step to enhance efficiency. Sequencing of synthetic SARS-CoV-2 RNA provided evidence validating the efficacy of this protocol, while data from high-throughput sequencing of wastewater samples demonstrated the sensitivity of this method. We also provided guidance on the quality control steps required during library preparation and data analysis. Overall, this represents an effective method for high-throughput sequencing of SARS-CoV-2 in wastewater which can be applied to other viruses and pathogens of humans and animals.