Illumina Next Generation Sequencing Facility

Overview

Next generation sequencing (NGS) has rapidly become the state of the art for high-throughput genomic analyses. This robust technology provides access to numerous applications ranging from genomic (re)sequencing, whole-transcriptome analysis, small RNA, miRNA, non-coding RNA, targeted sequencing, ChIP-Seq and ATAC-Seq. Using a massively parallel sequencing approach, NGS is capable of generating gigabases of high quality sequence, maximizing throughput while minimizing the cost and time to generate this data. Infrastructure funding for this facility has been provided through a partnership with The Ottawa Hospital to provide research capacity to the Eastern Ontario Regional Laboratory Association (EORLA) personalized medicine initiative and by the Canadian Foundation for Innovation.

Facility

NextSeq 500, MiniSeq, and NeoPrep System

StemCore's Next Generation Sequencing Facility is equipped with Illumina's NextSeq 500, MiniSeq, and NeoPrep System. The NextSeq 500 and the MiniSeq employ the latest enhancements in two-channel, sequencing by synthesis (SBS) technology. With these instruments StemCore can provide access to a wide variety of scalable next generation sequencing (NGS) applications with data ranging from 7.5 Gb up to 120 Gb generated per sequencing run. Both platforms deliver high quality data with reduced cycle and data processing times due to the two colour sequencing approach. The recent addition of the MiniSeq (May, 2016) will permit greater flexibility in experimental design and faster turn-around-times for smaller scale projects.

Sequencing Platform Comparison

 MiniSeqNextSeq
ApplicationsAmplicon, targeted RNA, small RNA, targeted gene panels, small genomeExome, transcriptome, ChIP-Seq, targeted resequencing
Max Output7.5 Gb120 Gb
Max Read Length2x150 bases2x150 bases
Max Reads per Run25 million400 million
Run Time4-24 hours12-30 hours
Chemistry2 colour2 colour

The NeoPrep System uses digital microfluidics to prepare sequencing-ready libraries with less sample input and more cost effectively than manual library preparation methods. Each library card employed during a single run can produce libraries from 1-16 samples and includes all reagents for library construction, quantitation, and normalization*. The TruSeq Nano DNA Kit and TruSeq Stranded mRNA Kit are currently supported. The NeoPrep provides a more cost-effective solution for mid to high throughput projects.

Services
  • Nucleic Acid Quality Assessment and Quantitation
  • Library Construction
  • Library Sequencing
    • StemCore or User Prepared
  • Consultation
    • Experimental Design
    • Pilot Projects
    • Proof of Concept Experiments
    • Grant Support
    • Manuscript Preparation
    • We will provide all support necessary to bring your project to successful completion.
    • Please contact Pearl Campbell to discuss
NGS Services, Pricing and Sample Submission Guidelines
  • A service agreement must be established and signed by the PI and Institutional Signing Authority (external clients) for each project prior to processing samples. The agreement will include a statement of work and a quotation.
  • Samples should be provided with a Sample Submission Form and other documentation as required for samples to be analyzed (see below). Each submission form should reference the Service Agreement number provided when the project is initiated.
  • Samples should be accurately quantitated using a fluorometric method (PicoGreen, RiboGreen, or Qubit). StemCore Laboratories is able to perform these assays for a nominal fee. NanoDrop readings are not accurate at the very dilute concentrations utilized in next generation sequencing protocols, especially for ChIP-Seq applications.
  • StemCore is experienced in a wide variety of library preparation protocols. If your application is not listed below please contact us to discuss.
Library Construction

Preparation MethodApplicationSamples Per Run Cost
(per run or sample)
+, ++
Sample Submission InformationAdditional Comments
ManualTruSeq RNA Library v2As required $325.00 0.1 - 1.0 ug Total RNA Protocol has been optimized using high quality RNA as assessed on the Fragment Analyzer. RIN ≥ 8.
 TruSeq DNA NanoAs required $250.00 User Sheared:
Shear DNA as per protocol given in TruSeq Nano DNA Library Prep for Neoprep (Illumina Document # # 15041110 Rev. D)
Provide sheared gDNA at the following concentrations:
350 bp inserts - 100 ng in 52.5 ul RSB;
550 bp inserts- 200 ng in 52.5 ul RSB
StemCore will shear upon request (additional charges apply#) Please provide DNA at the following concentrations:
350 bp inserts -100 ng in 50 ul;
550 bp inserts -200 ng in 50 ul
 
 TruSeq Stranded mRNAAs required $325.00 0.1 - 1.0 ug Total RNA Protocol has been optimized using high quality RNA as assessed on the Fragment Analyzer. RIN ≥ 8.
 TruSeq Small RNA (Illumina)As required $350.00 1 ug total RNA in 5 ul nuclease free ultrapure water Protocol has been optimized using high quality RNA as assessed on the Fragment Analyzer. RIN ≥ 8.
 Nextera XT (Illumina)Maximum of 96 $65.00 1 ng gDNA in 5 ul nuclease free ultrapure water The Nextera XT DNA Library Preparation Kit allows the preparation of sequencing-ready libraries for small genomes (bacteria, archaea, viruses), amplicons, and plasmids in less than 90 minutes with only 15 minutes of hands-on time.
 TruSeq RNA Access (Illumina)Maximum of 16 $440.00 10 - 100 ng Total Human RNA Capture-based assay of human coding transcriptome. Can be used on fresh, frozen, or FFPE samples. Input amount is RNA quality-dependent. DV200 > 70%; 20 ng DV200 from 50-70%; 20-40 ng DV200 from 30-50%; 40-100 ng DV200<30%; not recommended
 NextFlex ChIP-Seq (BIOO)As required $300.00 1-10 ng DNA in 40 ul nuclease free ultrapure water The NEXTflex ChIP-Seq Kit also features Bioo Scientific’s proprietary Enhanced Adapter Ligation Technology, resulting in library preps with a larger number of unique sequencing reads and increased yields from minimal quantities of starting material. The NEXTflex ChIP-Seq Kit now incorporates a bead-based size selection protocol, which completely eliminates the gel purification step. Additionally, there is an optional protocol which eliminates the size selection step entirely.
 NextFlex qRNA-Seq Kit v2As required $300.00 10 ng - 1 ug Total RNA in 14 ul nuclease free ultrapure water or 500 pg - 100 ng fragmented genomic of ChIP DNA. Fragments must be ≤ 800 nt. 300 nt average size is ideal for ChIP-Seq. Can be used for RNA-Seq or ChIP-Seq. Molecular indices allows discrimination of biological duplicate reads from PCR artifact.
 CleanTag Small RNA (TriLink)As required $250.00 1-1000 ng Total RNA in 2 ul nuclease free ultrapure water The kit includes CleanTag chemically modied adapters that greatly reduce adapter dimers.
+ NeoPrep costs are per run. All other costs are per sample.
++ Please contact StemCore to discuss additional discounts available for project-related pricing.
# Cost for Covaris shearing is $10.00 per sample.

Library Sequencing

Next Generation SequencingRun ModuleApproximate Reads per RunCost Per Run *
MiniSeq75 Cycle High Output25M$1,400.00
150 Cycle High Output25M$1,750.00
300 Cycle High Output25M$2,400.00
300 Cycle Mid Output8M$1,200.00
NextSeq 50075 Cycle High Output400M$2,200.00
150 Cycle High Output400M$4,200.00
300 Cycle High Output400M$6,100.00
150 Cycle Mid Output130M$1,825.00
300 Cycle Mid Output130M$2,725.00
* Runs may be multiplexed to obtain required read depth and minimize per sample cost.

Deliverables and Results Retrieval

Illumina NextSeq 500 pipeline analysis concludes with mapping of sequence reads to a reference genome. Samples from a supported reference organism (human, mouse, rat) are mapped to the genome using Bowtie. The results delivered will comprise sequence data with associated quality scores in fastq format, and mapping results. 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. Mapping to other organisms can be performed on request if a reference sequence is available.

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 Gareth Palidwor of the Bioinformatics group.

StemCore will not archive analysis files once the data have been downloaded.

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, guidelines, and analysis tools:

Mailing Address

Ottawa Hospital Research Institute
Sprott Center for Stem Cell Research
StemCore Laboratories
Room 5132, Critical Care Wing
501 Smyth Road
Ottawa, Ontario, Canada K1H 8L6

Contact Information
Caroline Vergette
cvergette@ohri.ca
613.737.8899 x73251 (lab phone)
613.739.6294 (fax)

Pearl Campbell
pcampbell@ohri.ca
613.737.8899 x73110 (office)