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Sequence Core Facility

Sequencing

Since 1999 the Netherlands Cancer Institute facilitates the Sequence Core where DNA sequencing and fragment analysis for research and diagnostic purposes is done.

The 3730 DNA Analyzer with 48 capillaries and the 3500xL Genetic Analyzer with 24 capillaries (both Life technologies) are used for analyzing DNA samples.  Both systems are based on polymer capillary electrophorese, with a maximum of  1,000 base read length. 

The reagent used  for sequencing is BigDyeTerminator V3.1/V1.1. The BDT sequencing reaction has to be performed by users themselves. The Sequence Core Facility will purify and analyze the samples on one of the sequencers. Beside DNA sequencing it is also possible to analyze PCR-products with Fragment analysis. The length of PCR-products is determined using a fluorescent labeled primer.

The Sequence Core Facility has a throughput of 110.000 samples a year. Within 24 hours, 90 percent of the samples is analyzed. The sequence data is available on a central server and will be stored after 6 months. 

Mass spectrometer

The Mass-Array system by Agena Bioscience is a detection platform utilizing mass spectrometry to accurately measure PCR-derived amplicons. It combines the benefits of a multiplexed primer extension reaction with MALDI-TOF mass spectrometry to characterize genotypes. The system is ideal for panels from 10 to 400 single nucleotide polymorphisms.

Workflow

Primers for PCR and single base extension for the SNP of interest are designed with the assay software. DNA is provided by the user and will be processed by the Sequence Core Facility:

  • Amplification of the region of interest with PCR
  • Purification and single base extension
  • Dispensing PCR-products on SpectroCHIP
  • MALDI-TOF Mass spectrometer Analysis.

This entire workflow can be completed in two days. 

Options

- Custom made assay design 

  • Panel for the clinical DNA-diagnostics laboratory (70 hotspot mutations in AKT, BRAF, DDR2, EGFR, KRAS, MEK1, NRAS, PIK3CA)
  • Your own panel for SNP/mutation detection

- Pre-designed panels 

  • Oncocarta: This panel includes more than 230 somatic mutations across 19 oncogenes.
  • Melacarta: This panel is designed specifically for metastatic melanoma profiling and offers 70 somatic mutations in 20 oncogenes.
  • Lungcarta: This panel provides a highly sensitive panel for evaluating more than 250 somatic mutations in 26 genes for targeted profiling in non-small cell lung cancer (NSCLC).
  • GyneCarta: This panel is designed for epithelial ovarian, cervical and endometrial cancer profiling. 
  • ColoCarta: This panel is designed for metastatic colorectal cancer.

 

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