The Genome Sequencing Facility features the Illumina line of NGS Sequencing Systems.
The NovaSeq 6000 is Illumina’s latest advancement in NGS sequencing instrumentation. The NovaSeq is a dual flow cell instrument engineered to operate with 3 patterned flow cell formats providing 0.5 Tb – 6 Tb of total sequence data output. This scalable operation allows the Genomics Core to meet the demands of diverse sequencing projects from RNA-seq to Whole Genome Sequencing (WGS). The NovaSeq leverages Illumina’s proven Sequence by Synthesis (SBS) technology with superior run times and simplified RFID-encoded, cartridge based, reagent management for streamlined operation.
The patterned flow, cell first used on the HiSeq X system, has been adapted with improved density for the NovaSeq 6000. Each flow cell contains billions of nanowells in fixed locations producing even cluster spacing and uniform feature size to deliver extremely high cluster density. The high density afforded by the NovaSeq's patterned flow cell is combined with Illumina's proprietary onboard exclusion amplification clustering method (EXamp) which maximizes the nanowells that are occupied by DNA clusters originating from a single DNA template. This increased efficiency of flow cell utilization results in a significant increase in sequence data output.
The Illumina HiSeq2500 Sequencing System is a dual flow cell sequencing system which can operate in either a High Output or Rapid Read mode. In the High Output mode the 2500 has 16 lane capacity capable of generating sequence output up to 600 Gb for a 2x100bp run. In the Rapid Read mode the 2500 has 4 lane capacity capable of generating sequence output up to 90 Gb for a 2x150bp run. In the Rapid Read mode run times are significantly reduced. By comparison, a dual flow cell 2x100bp Rapid Read run is completed in ~30 hours versus ~10 days for a dual flow cell 2x100bp High Output run.
Fully automated clonal clustering of the High Output flow cell is performed using the cBOT Cluster Station which allows the amplification of > 96 samples in ~ 5 hours. The cBot also performs Rapid Read flow cell cluster of independent lanes allowing two unique sample pools to be run independently on the two available lanes.
Sequence data generated by the NovaSeq 6000 and HiSeq 2500 is processed by Real Time Analysis (RTA) software and deposited on the iCompute Server. The iCompute server combines Illumina analysis software with high-performance Dell blade servers and 36Tb Isilon clustered modular storage.
Additional equipment in support of the sequencing library preparation, validation and quantification includes the Covaris S2 Utrasonicator, Sage Science Pippin Prep preparative gel electrophoresis system, dual Agilent 2100 Bioanalyzer systems and a Roche LightCycler96 Real Time PCR system.
The Covaris S2 Ultrasonicator allows for computer controlled fragmentation of nucleic acids using Adaptive Focused Acoustic (AFA) technology. Used to prepare fragmented genomic DNA with the appropriate size distribution for generating whole genome and ChIP sequencing libraries.
The Pippin Prep allows for automated electrophoretic size selection of fragmented DNA for sequencing library construction. Primarily used for library sizing of ChIP-seq and Small RNA library preparations.
The Agilent 2100 Bioanalyzer provides a rapid assessment of the fragment sizing using a chip based capillary electrophoresis system. Integrity assessment of total RNA is also performed on all samples prior to RNA-seq library preparation. Library validation and sizing is also a primary function for the 2100 Bioanalyzers.
The Roche LightCycler Real Time PCR system is used quantitate each library preparation prior to sample pooling and sequencing. Real Time assays are performed using Roche FastStart Essential DNA Green Master Mix and KAPA Library Quant DNA Standards for Illumina sequencing. Accurate library concentrations allow the Genomics Core to tune the multiplexed sequencing strategies and obtain the targeted reads required to meet the needs of your project.