Next Generation Sequencing

The Lineberger Comprehensive Cancer Center's (LCCC) Pre-Clinical Genomic Pathology Laboratory (gPATH) is a medium throughput facility providing comprehensive genomic services for researchers employing Next Generation Sequencing (NGS) technology in the performance of various types of genomic studies.

Next Generation Sequencing Services:

  • Free Project Consultation (gpath@unc.edu).
  • Laboratory Information Management System (LIMS) Sample Tracking.
  • Automated Nucleic Acid Extraction (Promega Maxwell 16 MDx): DNA, RNA from frozen tissue, FFPE tissue, blood/buffy coat, cheek swabs and custom samples
  • Nucleic Acid Quality/Quantity Assessment: UV, Fluorometric, Agilent TapeStation
  • Acoustic Fragmentation of Genomic DNA: Covaris E220
  • Automated Next Generation Sequencing Library Preparation: DNA-Seq (whole exome; with capture), RNA-Seq (whole transcriptome; with capture) using Agilent Technologies Bravo Workstations.
  • Next Generation Sequencing: Illumina MiSeq, NextSeq 500 and HiSeq 2000/2500 (High Throughput Sequencing Facility)

Services are available to Lineberger Comprehensive Cancer Center members and non-members as well as UNC and non-UNC clients.

- For a free project consultation, please complete the Project Consultation Request Form and email the form to gpath@unc.edu.

- For project initiation, please complete the Project Submission Form and email the form to gpath@unc.edu.

Services Overview:

gPATH extracts nucleic acids from a wide range of sample types including frozen tissue, blood, buccal swabs, cells and formalin-fixed, paraffin-embedded (FFPE) tissue. H&E examination and assessment by a qualified pathologist, as well as macrodissection services are provided, as needed.  gPATH employs two dedicated Promega Maxwell MDx16TM instruments (Promega, Inc., Madison, WI.) for automated extraction of RNA or DNA from up to 16 samples per hour per instrument. A Maxwell RSCTM instrument is employed for ccfDNA extraction from 0.2-1.0 ml of plasma. Subsequent quality assessments using ultraviolet (UV) absorbance and quantity assessments (for dsDNA) using Picogreen (PG) assays are performed on all samples via either a SpectraMaxTM Model M2 dual mode microplate reader  (Molecular Devices, Inc., Sunnyvale, CA.) using recommended 96 well sample plates (Thermo-Fisher, Waltham, MA,  #07-200-623 (UV); VWR, Radnor, PA., #82050-754 (PG)) or a Qubit 2.0TM (Invitrogen) or QuantusTM (Promega) fluorometer. For RNA samples that are to be processed for RNASeq, each sample is also evaluated for fragment length, quality and molar concentration via an Agilent Tapestation 2200TM using manufacturer supplied RNA Screentapes (Agilent, Cat#5067-5576).

For extracted and OC'd samples that are to be moved forward through gPATH's automated library preparation workflow, 0.2-2 µg of DNA is acoustically fragmented to approximately 180-225 base pairs using a Covaris E220 focused ultrasonicator instrument (Covaris, Inc., Woburn, Mass.). Using specialized 96 well acoustic microplates (or single micro tubes), this instrument performs automated fragmentation of up to 96 separate DNA samples sequentially at an approximate rate of one sample every 4-6 minutes. Post-fragmentation, the sample is enriched for appropriate sized fragments using an automated separation step employing AMPure beads (Beckman Coulter, #A63882, Indianapolis, IN.). Fragment size enrichment and subsequent DNA or RNA library preparation steps requiring precise liquid handling steps are performed using the Agilent Bravo A and/or Bravo B (with automated reagent and consumables feeds) robots (Agilent Technologies, Santa Clara, CA).

gPATH generates NGS libraries using any of the following formats:

DNA-Seq Libraries:

  • Agilent SureSelectXT custom capture (exome capture) UNCSeq Version 8.0 design (Agilent Technologies, Santa Clara, CA, #5190-4828, #G9641B, #5500-0075, #5190-3730, #5190-4394).
  • Agilent SureSelectXT Human All Exon capture.

RNA-Seq Libraries:

  • Illumina TruSeq RNA V2 (Illumina, Inc, San Diego, CA., #RS-122-2001).
  • Agilent SureselectXT RNA (Agilent Technologies, Santa Clara, CA., #G9692A) capture (UNCSeq Version 8.0).
  • Illumina TruSeq Stranded Total RNA w/Ribo-Zero Gold (Illumina, #RS122-2301).
  • Agilent SureselectXT RNA FFPE (includes Epicenter Ribo-Zero Gold) capture (UNCseq Version 8.0).
  • Illumina TruSeq RNA Access (Illumina, RS-301-2002).

Unique single indexing of libraries is achieved using a set of 96 separate, 8-mer indices (Agilent, Santa Clara, CA, SureSelectXT, #5190-5454) which allows multiplexed sequencing of libraries.

During the course of the library preparation procedure, the quality of each library is assayed at 3 different stages - once after fragmentation and the initial AMPure bead clean-up, once after adapter ligation and pre-capture PCR, and once after all steps are completed. The Agilent TapeStation 2200 (Agilent Technologies, Santa Clara, CA, #G2965AA) using either D1K Screentapes and reagents (#5067-5582, #5067-5583) or High Sensitivity D1K Screentapes and reagents (#5067-5584, #5067-5585) is employed to concurrently assay 16 samples at a time. These assays provide important metrics including fragment size, concentration and molarity and, in the case of RNA assays, RINs.

Completed libraries are normalized and pooled to the desired density and concentration using Bravo robots guided by vWorksTM automation control software (Agilent Technologies, Santa Clara, CA.). To evaluate library quality, a sample pool of all libraries created in a single automation batch run is generated and sequenced on an Illumina MiSeq sequencer using a MiSeq Reagent Nano Kit, v2 (300 cycles) (Illumina, Cat. #MS-103-1001). This relatively short run confirms appropriate cluster density and verifies accuracy of library indexing prior to the initiation of a more expensive and time-consuming deep sequencing procedure; eg., NextSeq500TM, HiSeq2500TM, or HiSeq4000TM. Two genomic controls are run with every library preparation batch.

Routinely libraries generated by gPATH are pooled and transferred to the UNC High Throughput Sequencing Facility (HTSF) for deep sequencing using an Illumina HiSeq2500TM or a HiSeq4000TM. Alternatively, gPATH can perform NGS using either the Illumina MiSeq or NextSeq500TM sequencers, as appropriate, and offers full benchtop sequencing capability at a variety of read lengths and output levels. Bioinformatics analysis support is provided by the Lineberger Comprehensive Cancer Center Bioinformatics Core.

 All sample information and metrics are recorded and maintained in the gPATH Lab Information Management System (LIMS) designed and maintained by the LCCC Bioinformatics Core.