Next generation DNA Sequencing has rapidly become the state of the art for high-throughput genomic analyses. This robust technology provides single platform access to applications ranging from genomic (re)sequencing, whole-transcriptome analysis, small RNA, miRNA, non-coding RNA, as well as the analysis of transcription factor binding and epigenetic modifications present at specific genomic loci through the use of ChIP-seq technology. Using a massively parallel sequencing approach, the Illumina GAIIx Genome Analyzer is capable of generating gigabases of high quality sequence in a single run maximizing throughput while minimizing the cost and time taken to generate this data.
StemCore Laboratories is pleased to offer this research enabling technology to the scientific community. Funding for this facility has been made possible through the TIMEx Grant awarded through the Canadian Foundation for Innovation through its Large Scale Institutional Endeavours Program. This grant has been jointly awarded to the Ottawa Hospital Research Institute, The Ottawa Hospital, the Children's Hospital of Eastern Ontario, the University of Ottawa, and the University of Ottawa Heart Institute to provide for the expansion of research infrastructure.
StemCore Illumina facility is equipped with a state of the art Illumina Sequencing System which consists of:
- cBot Cluster Generation System. Hybridization of prepared libraries to the flow cell in preparation for DNA sequencing is performed on the cBot Cluster Generation System. Following hybridization the library undergoes sequential bridging amplification with repeated denaturation and extension of the original library fragments to create localized amplification of single molecules which are subsequently sequenced by the GAIIx.
- GAIIx Genome Analyzer. Each flow cell contains millions of unique clusters. The Illumina GAIIx performs sequencing by a proprietary process chemistry referred to as "Sequencing by Synthesis". In this process fluorescent nucleotides are flowed over the flow cell, the excess is washed off, and sophisticated high resolution imaging technology, coupled to analysis software allows base calls to be performed at each cycle of sequencing. Thirty-six cycles of sequencing is the norm which is accomplished over a three day period. Modifications are also possible which will allow basecalling of up to 76 nucleotides.
- Paired-end Module. Paired end reads are useful to help assign the genomic location of individual reads by sequencing an individual cluster from its 3' and 5' end. This application has been used extensively for ChIP-Seq applications.
Sequencing/Analysis Requests and Sample Submission
- Samples should be provided with sample submission form and other documentation as required for specific samples to be analyzed (see below). We are able to offer the complete range of NGS applications as provided by Illumina. If you require sequencing of species other than mouse or human, or require bioinformatics support above what is provided by the Illumina processing pipeline please contact Dr. Ted Perkins, the lead scientist heading up our bioinformatics group.
- Samples should be assayed by PicoGreen, RiboGreen, or Qubit (Invitrogen) for accurate quantitation. Nanodrop readings are not accurate at the very dilute concentrations utilized in next generation sequencing protocols, especially for ChIP-Seq applications.
- Picogreen Assay can be done on site by request
- Genomic DNA for (re)sequencing applications should be provided as follows:
- 50 ul of 200 ng/ul genomic DNA in 10 mM Tris pH 7.4
- Total RNA for whole transcriptome analysis should be provided as follows:
- A total of 2 micrograms of total RNA in maximum of 50 ul of nuclease free water is required.(Life technologies, Cat#10977-015)
- Prior to library construction, QA of the total RNA will be performed on the Agilent BioAnalyzer.
- ChIP-Seq samples should be provided as follows:
- 5-10 ng chromatin fragments in max. 50ul of nuclease free water(Life technologies, Cat#10977-015)
- Input chromatin for each ChIP should be sheared (sonicated, digested, etc) to fragment sizes of between 200-800 bases for optimal results
- Demonstration of optimal fragment size is required by submission of gel image of input chromatin along with the sample
- Library construction may also be performed by the user with cluster generation and sequencing performed by StemCore. If interested, information regarding applications and kits available can be found at Illumina.
- For information on pricing please email email@example.com.
- Please note that Bioanalyzer and Q-PCR assays are included for Qc of library construction.
- Samples from local researchers should be dropped off at StemCore Laboratories on the 5th Floor of the Critical Care Wing Monday-Friday between the hours of 9:00 AM and 4:00 PM. A fully completed Request Form should be prepared at the time of sample submission. Please ensure that the label on the tube exactly matches the template name given on the request form.
- Samples from external sources should be shipped on ice to below address.
- In advance of shipment of the samples please fax the completed request form to Kathy Sheikheleslamy at (613) 739-6294. Please also include a copy of the previously faxed request form with the shipped samples.
Analysis Time Frame
Every effort to coordinate rapid sample processing and throughput within the facility will be made. However, next generation sequencing is a lengthy process (see below) and a single 51 base read with library construction and processing takes approximately 2 weeks to complete. Additionally, each flowcell contains 8 channels. One channel of the flow cell is dedicated to the internal PhiX control sample on every run and therefore constructed libraries will be processed in batches of 7, or less if it is desired that the sample be run over more channels to increase sequencing depth. Batch processing will ensure that prices for this service are kept in check.
- Library Construction generally takes 2-4 days to perform.
- Cluster Generation and 51-cycle Single-read Sequencing runs are performed over 4 days.
- Pipeline Processing and QA checks will add another 2-3 days to overall time-frame.
Deliverables and Results Retrieval
Illumina GAII pipeline analysis concludes with mapping of sequence reads to a reference genome. The results delivered will comprise sequence data with associated quality scores in fastq format, and mapping results generated with the Eland algorithm. These files will be made available for download; an email will be sent with details of how to download the files will be sent once analysis has been completed.
StemCore will not archive analysis files once the data have been downloaded.
Please enquire for pricing of services.
Next Generation Sequencing Resources
A wealth of information regarding next generation sequencing is available on the internet. While this list is by no means meant to be exhaustive it provides links to some helpful sites for next generation sequencing applications, protocols, and analysis tools:
Multiple Software Packages for Next Generation Sequencing Analysis
613.737.8899 x73251 (lab phone)
Ottawa Hospital Research Institute
Room 5132, CCW
501 Smyth Road
Canada K1H 8L6