CCR Genomics Core lists over 900 registered members in its iLab Management and scheduling software with 582 Principle Investigators/Research groups and 318 individual users. In FY 17, 282 Principal Investigators utilized Core services of which 211 are from NCI. Of the 47 labs/branches listed under CCR, 33 utilize our services. Those labs include: Basic Research Laboratory, Cancer and Developmental Biology Laboratory, Cancer Inflammation Program, Dermatology Branch, Developmental Therapeutics Branch, Endocrine Oncology Branch, Experimental Immunology Branch, Experimental Transplantation and Immunology Branch, Genetics Branch, HIV and AIDs Malignancy Branch, HIV Dynamics and Replication, Laboratory of Biochemistry and Molecular Biology, Laboratory of Cancer Biology and Genetics, Laboratory of Cell and Developmental Signaling, Laboratory of Cell Biology, Laboratory of Cellular and Molecular Biology, Laboratory of Cellular Oncology, Laboratory of Genome Integrity, Laboratory of Human Carcinogenesis, Laboratory of Immune Cell Biology, Laboratory of Metabolism, Laboratory of Molecular Biology, Laboratory of Protein Dynamics and Signaling, Laboratory of Receptor Biology and Gene Expression, Lymphoid Malignancies Branch, Molecular Imaging Program, Molecular Targets Laboratory, Radiation Biology Branch, Surgery Branch, Thoracic and Gastrointestinal Oncology Branch, Urologic Oncology Branch, Vaccine Branch, and Women's Malignancies Branch. The Core also provides services to investigators in the Clinical Center as well as 11 other NIH institutes including NIAID, NHLBI, NIDDK, NEI, NIAMS, NIMH, NINDS, NIAAA, NHLBI, NIA and NICHD. Available Core services include: Sanger Sequencing carried out using two ABI 16-capillary 3130XLs, one 96 capillary 3730XL. These instruments are capable of reading up to 800 base pairs with 95% accuracy. Data are analyzed and made available within one business day. The current charges are $2.00 for electrophoresis only and $7.00 for the sequencing reactions, clean-up and electrophoresis. The Core is in the process of upgrading the two 16-capillary 3130XLs to the 3500xL instruments. The 3500xL offers numerous advantages over the 3130xL including 24 vs 16 capillaries increasing our turn-around time, improved thermal control with new door seal improving data precision, updated software which monitors consumables and 20% smaller footprint. This acquisition with replacements of our obsolete machines will ensure that CCR investigators have rapid access to state-of-the art, most accurate and longest read length Sanger sequencers and will improve core efficiency. Illumina Next Generation Sequencing is performed with two different platforms, the MiSeq Desktop Sequencer featured in the Core since 2012 and NextSeq 500 which was recently added. Researchers can submit pre-made libraries or purified DNA/RNA for library construction and subsequent sequencing. The Core will also perform quality control (QC) on all libraries prior to sequencing. Individuals meet with Core members to discuss the best experimental designs and costs are offset by subsidies provided by the Office of Science and Technology Resources (OSTR). NanoString Digital Gene Expression Analysis System is capable of direct quantification of individual mRNAs in a biological sample without the use of enzymes or amplification. NanoString designs each CodeSet and then investigators bring their samples to the core to be processed. The cost of the Codesets is subsidized by the OSTR. Core charges range from $5/sample to $24/sample for NCI staff depending on what services and supplies are necessary. Non-NCI staff pays $24 to $48/sample. Bio-Rad QX200 Droplet Digital PCR System provides absolute quantification of target DNA, including measurement of viral loads and microbial quantification; detection of copy number variations and rare mutations; absolute quantification of mRNA and miRNA and quantification of NGS libraries. Typically, after discussion with Corestaff and if necessary BioRad for assay development, customers bring prepared samples in a sealed plate ready to be applied to Automatic Droplet Generator. Results are usually available within 24 h. Data analysis software is available for use on customers' own computer. Current costs $5/sample. Fluidigm C1 Single Cell Autoprep System for genomic analysis of single cells is an automated single-cell isolation and preparation system that provides an easy and reproducible workflow to process 96 single cells for genomic analysis including RNA-Seq, Whole Genome Amplification and sequencing and Gene Expression Profiling on Biomark HD qPCR. Customers contact the Core manager to schedule training and access to the instrument. The core also provides access to the Nexcelom Vision CBA image cytometry system to determine cell size, number and viability prior to C1single cell isolation as well as Keyence All-in One microscope for annotation. Subsidies for this technology are provided by the OSTR. Agilent TapeStation 2200 allows for reproducible and user-independent quality control of DNA and RNA samples and libraries. QC services are provided to customers obtaining NGS services from our Core as well as the CCR Sequencing Facility at the Frederick National Laboratory. The Core charges range from $3/sample to $4/sample for NCI staff depending on number of samples and what supplies are necessary. The Core will soon be adding the 4200 TapeStation instrument to its QC repertoire. The 4200 will allow for unattended walk away operation with fully automated sample processing for up to 96 samples permitting processing of samples after hours. In addition, reliable results can be obtained in as little as 1-2 minutes per sample, or less than 90 minutes for 96 samples and will require only 1-2 uL of sample per run. Moreover, Agilent 4200 will maintain the same footprint as the Agilent 2200 and will save valuable bench space. In addition, the CCR Genomics Core has Qubit Fluorometric Quantitation and the Sage Science PippinHT available for use by any investigator that wants to use them. The Keyence BZ-X700 microscope when not in use by the Fluidigm system is also available for use. The fluorescence microscope has a large motorized stage that can house multi-well plates or flasks and is upgraded to support image stitching/optical sectioning, cell counting and measurement. Bench space is furnished by the Core and the staff maintains a sign-up calendar on a first-come, first-served basis. So far in FY17, the Core has managed over 3138 iLab requests and processed 28,379 samples for Sanger sequencing, 1448 for Nanostring, 7394 for ddPCR and 6404 for tapestation. The Core has been involved in 53 different NGS projects featuring the MiSeq and NextSeq 500 platforms. Partnerships with NIDCD and Dr. Jing Huang in NCI continue with single cell isolation using the Fluidigm C1 with the 96 well IFC. The Big Dye Program where the Core purchases Big Dye in bulk discounted quantities and then aliquots it and sells it to Core users at cost continues to thrive and save the NCI money. Thus far in in FY17 we have had 30 requests totaling $15,045 in sales. The program is available for all investigators within CCR, even if they do not use the core for sequencing. Our users were asked to select the area of research for each submission to the core. There were over 92 different areas selected. The top 20 areas of research based on the number of total submissions for all platforms are: molecular biology, immunology, leukemia, genomics, biotechnology research, breast cancer - basic, microbial genetics, chemotherapy, infectious diseases, kidney disease - research, DNA repair, genetic diseases, sarcomas - soft tissue, liver cancer, ALS, hematology, aging, oncogenes, colon/rectum and gene mapping - human.
