The Applied Genomics Technology Center is equipped with an Illumina HiSeq 2500 Sequencer for next generation sequencing. This sequencer can be run in two modes: High Output mode which uses High Output flow cells containing eight lanes, and Rapid mode which uses Rapid flow cells containing two lanes. A 50 cycle (bp) paired-end High Output run typically generates an average of 150 Gb of data in about 5 days.
Our sequencing applications have typically included:
- Whole-genome sequencing (DNA-Seq)
- Targeted resequencing (Exome-Seq or custom-Seq)
- Transcriptome sequencing (RNA-Seq or small RNA-Seq)
- Epigenetics and gene regulation (ChIP-Seq or Whole-genome bisulfite sequencing)
The Core is committed to working with investigators to fulfill their scientific goals. Please refer to Illumina’s website for a complete list of applications and kits.
Library Prep and Sample Submission Requirements
The Core offers the investigator two options for library preps:
- Core-built libraries. The Core uses the Illumina TruSeq Sample Prep kit series for building libraries; however, you are able to request a specific supplier and kit if you wish. Samples may be submitted as:
b. Nucleic acid (DNA or RNA): Please contact Julianna Orlando for details regarding sample concentration and quantity requirements.
The Core will perform a QC of the submitted samples. We do not assume responsibility for the data output if the investigator refuses the Core’s QC step. Alternatively, the investigator may provide their QC data for each sample (examples include spec readings, Agilent traces (BioAnalyzer or TapeStation), real time sample data and standard curve, etc.) The investigator will be contacted if a sample does not meet the minimum quality and quantity standards.
- User-built libraries. The investigator builds the libraries using kits that are compatible with the Illumina HiSeq 2500 platform. Please submit at least 20ul of 2nM of your pooled samples in 1.5 ml centrifuge tubes.
To download the NGS sample submission form, click here.
Multiplexing is available and varies depending on the kit and the investigator’s scientific question. Typically, the different library types are multiplexed as follows:
- TruSeq RNA libraries: multiplex six in one lane
- TruSeq DNA libraries: multiplex six in one lane
- TruSeq small RNA libraries: multiplex 48 in one lane
- TruSeq exome libraries: multiplex six in one lane
- TruSeq ChIP libraries: multiplex six in one lane
Please discuss the desired depth of coverage with a bioinformatician or statistican. Please contact Katherine Gurdziel, PhD. to inquire about the Core’s bioinformatics services.
The core will contact the investigator to take back any unused sample at the end of the run.
The HiSeq 2500 can accommodate the following six run types:
- single-read, non-indexed (sequencing in one direction, one sample per lane)
- single-read, single-indexed (sequencing in one direction, pool of samples per lane, each with one index)
- single-read, dual-indexed(sequencing in one direction, pool of samples per lane, each with two indices)
- paired-end, non-indexed (sequencing in both directions, one sample per lane)
- paired-end, single-indexed (sequencing in both directions, pool of samples per lane, each with one index)
- paired-end, dual-indexed (sequencing in both directions, pool of samples per lane, each with two indices)
We perform 50 and 100 cycles (base pairs) per read in the High Output mode. In addition to the 50 and 100 cycles per read, the Rapid mode also allows us to perform 150 cycles per read.
Each flow cell must contain a DNA lane as a control. This can consist of DNA-Seq samples or exome-Seq samples. If neither is available, one lane can be chosen to have a 30% PhiX spike in to act as the control lane for the entire flow cell. This will be discussed with the investigator prior to the run. All lanes will have a 1% PhiX spike in as an internal control. Please note that we cannot guarantee the number of clusters per lane as clustering is based on random binding to the flow cell.
Please complete the NGS Project Info Form, and we will send you a price estimate for your project.
Demultiplexed data will be provided to the investigator in fastq format. The data will be transferred to the investigator’s server or onto an external hard drive provided by the investigator. The Core will attempt to maintain a copy of the data; however, this depends on storage availability on the Core’s servers.
For information regarding bioinformatics services, please contact Katherine Gurdziel, PhD.
If needed, please contact Julianna Orlando for further information regarding the Illumina HiSeq 2500 Sequencer.