For PCR and Sanger sequencing both testing and bioinformatics interpretation are conducted. This method is currently being used in patients with chronic granulomatous disease (CGD), WHIM syndrome, and other autoimmune disorders. Because Sanger is a “general method,” it can also be and has been used for other mutation detection, including assays for inherited diseases and cancer-related genes, as well as verification of next-generation sequencing discoveries. Assays can be optimized for any new gene(s) of interest. We perform traditional polymerase chain reaction and Sanger sequencing for the discovery of de novo mutations, as well as for the confirmation of many forms of next-generation sequencing data. Assays for more than 90 genes have been developed for diseases such as dyskeratosis congenita, melanoma, colorectal cancer, xeroderma pigmentosum, and kidney disease.
The Pharmacoscan assay is used by several investigators at the NIH Clinical Center Pharmacy Department to test for genetic variations affecting the absorption, distribution, metabolism, and excretion of drugs. 4,627 ADME markers within approximately 1,200 genes are included in a single assay. Pharmacoscan is considered one of the panels that provide personalized medicine. Currently, there are only a limited number of genes of interest that are being used in the clinic, although the results of all are reported. The Applied Biosystems GeneTitan MC system is used for processing the Pharmacoscan assay. Other assays can also be developed on this multi-channel automated array instrument. We also provide a companion diagnostic assay with fragment analysis of the UGT1A1 gene.
As a companion to the Pharmacoscan assay, fragment analysis is used to more accurately genotype the UGT1A1 gene TA repeat region. This technique can also be used for other genes if needed; microsatellite instability testing and analysis is one example. This platform is precise, with easily interpretable plot and sizing data, and the run time is quick, allowing for rapid turnaround times if needed. The Applied Biosystems 3730XL DNA sequencers are used for processing fragment analysis samples.
Next-generation sequencing capabilities utilizing Illumina MiSeq, NextSeq, and NovaSeq platforms are at the forefront of new assay requests at the CMDL. Work with custom-designed panels as well as larger workflows such as whole genome sequencing are currently in development, with CLIA validation of a custom amplicon panel for HLA loss of heterozygosity underway and a future validation of a panel targeting Lynch Syndrome mononucleotide repeats planned. The CMDL also offers custom assays targeting both human and mouse p53, and has experience in targeted sequencing, RNA sequencing, whole exome sequencing, and whole genome sequencing to help aid any research efforts.
The CMDL is currently striding towards a new frontier with clinical proteomics via mass spectrometry. Utilizing an FDA-registered AB Sciex Citrine high sensitivity triple quad MS general purpose reader, the CMDL is nearing completion of validation for their first MS assay targeting Thyroglobulin in support of DCTD clinical trial efforts through an immune-enrichment multiple reaction monitoring approach. Talks are also currently underway to identify additional clinically actionable targets that would best serve NCI efforts and clinical trials. The integration of this information with genomics assays will allow us to offer proteogenomic interpretation, leading to deeper understanding of how genotypes support phenotypic presentation clinically.
DNA extraction from saliva, whole blood, FFPE tissues, buccal swabs, buffy coats, platelet-depleted whole blood, plasma, FFPE blocks, microscope slides, hair, nails, etc. as well as RNA extraction from FFPE slides and blocks has been performed under CLIA regulations. The high-quality nucleic acid can then be aliquoted, barcode labeled, and stored at the NCI-Frederick Repository for future studies and downstream applications, such as whole exome sequencing and whole genome sequencing or utilized for downstream efforts within the CMDL. Several extraction methods have been automated as well to increase throughput while reducing cost impact to customers.
Assays have been developed to detect and quantify Epstein–Barr virus in relation to Burkitt lymphoma, as well as for NCF1 and its pseudogene associated with Chronic Granulomatous Disease, and the EPAS1 gene for somatic mosaicism related to paragangliomas in patients. The Bio-Rad QX200 system is utilized. Assays for confirming known variants via other technologies such as qPCR can be implemented as well.
Plasmids and PCR products can be purified, cycle sequenced, and run on ABI 3730XL instruments. High-quality results in the form of .ab1 files are uploaded to a secure LIMS typically within 24-48 hours of sample receipt.
*Please note: Custom assays can be developed and CLIA validated as needed. Contact us for more information.
NIH Investigators may place a formal request for our lab's services at https://ncifrederick.cancer.gov/services/accessioning/Home/SignIn?ReturnUrl=%2fservices%2faccessioning%2f..