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 whole exome sequencing data, next-generation sequencing data, or whole genome 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 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.
DNA extraction from saliva, whole blood, FFPE tissues, buccal swabs, buffy coats, platelet-depleted whole blood, plasma, FFPE blocks, microscope slides, hair, nails, etc. 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.
Assays have been developed to detect and quantify Epstein–Barr virus in relation to Burkitt lymphoma, as well as for a pseudogene associated with Chronic Granulomatous Disease, and the EPAS1 gene for 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.
The Lymphoma Subtyping Test (LST) was developed to run on the NanoString platform and used to subtype patients as a companion diagnostic in a large clinical trial (NCT02285062) to identify patients of the ABC type who will respond to the drug lenalidomide. It is a highly complex test involving extraction of RNA from biopsy tissue specimens and requires a technician to “cut” material from slides to capture tumor sample. Other RNA assays can be developed as well, utilizing the Nanostring nCounter Flex Dx instrument in our clean room facility.
Illumina MiSeqDx for next-generation sequencing
Leica BondRx stainer for RNAScope assays
Thermo Fisher QuantStudio for qPCR
Illumina NextSeq for next-generation sequencing
Beckman FXp robotic liquid handler and Hamilton Microlab Prep for automation
Microm Microtome for sectioning formalin-fixed paraffin-embedded (FFPE) tissues
We have installed a Sciex Citrine Mass spectrometer to expand our capabilities in the area of proteomics. This instrument is a high sensitivity triple quad mass spectrometer that has been FDA registered as a general-purpose reader so it can be used for a variety of assays. We are working closely with NCI collaborators to validate a number of LC MS/MS and immunoMRM assays. The integration of this information with genomics assays will allow us to enter the new field of proteogenomics, offering genomic-guided proteomics. The CLIA lab anticipates this capability to be added later in 2024.
*Please note: Custom assays can be developed and CLIA validated as needed. Contact us for more information.
Other Capabilities/Instruments:
List of genes optimized for traditional PCR and Sanger sequencing:
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To request services from this NCI Frederick core facility, you must submit your requisition through: NAS.
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.