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 and MiniSeq. 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

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.
  • All NGS service requests must be entered in iLab and reference the Service Agreement number provided when the project is initiated. Samples should be provided with a copy of the sample submission form and a reference to the iLab request ID
  • 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.
  • Input samples and NGS libraries will be stored for a limited period after which the PI will be notified to retrieve the samples if desired, otherwise samples will be discarded.
Library Construction

Preparation MethodApplicationSamples Per Run Cost
(per sample)
++
Sample Submission InformationAdditional Comments
ManualTruSeq RNA v2As required, up to 96 $332.00 0.1 - 1.0 ug Total RNA in 40 ul nuclease free ultrapure water For mRNA using poly-A selection.
Protocol has been optimized using 300 ng input and quality RNA as assessed on the Fragment Analyzer. RIN ≥ 8.
 TruSeq Stranded mRNAAs required, up to 96 $301.00 0.1 - 1.0 ug Total RNA in 40 ul nuclease free ultrapure water For mRNA using poly-A selection.
Protocol has been optimized using 300 ng input and quality RNA as assessed on the Fragment Analyzer. RIN = 8.
 TruSeq Stranded Total RNAAs required, up to 96 $393.00 0.1 - 1.0 ug Total RNA in 40 ul nuclease free ultrapure water For mRNA and ncRNA, excluding miRNA using Ribo-depletion.
Protocol has been optimized using 300 ng input and quality RNA as assessed on the Fragment Analyzer. RIN = 8.
 NEB Next Ultra II RNAAs required, up to 96 $260.00 5 ng – 1 µg Total RNA (poly(A) mRNA workflow) in 40 ul nuclease free ultrapure water For mRNA using poly-A selection.
Low input
 NEB Next Ultra II Directional RNAAs required, up to 96 $262.00 5 ng – 1 µg Total RNA (poly(A) mRNA workflow) in 40 ul nuclease free ultrapure water For mRNA using poly-A selection.
Low input
 NEB Next Small RNAAs required $230.00 100 ng–1 µg Total RNA in max 6 ul nuclease free ultrapure water For small RNA, magnetic size-selection targets 21nt miRNA and 30nt piRNA.
 Nextera XT (Illumina)Maximum of 96 $211.00 1 ng gDNA in 5 ul nuclease free ultrapure water DNA is simultaneously fragmented and tagged with sequencing adapters in a single tube enzymatic reaction. Nextera XT supports ultra-low DNA input of only 1 ng. It enables a wide range of input samples, including small genomes, PCR amplicons greater than 300 bp, plasmids, microbial genomes, concatenated amplicons, and double-stranded cDNA.
 NextFlex Rapid DNA (Perkin Elmer)As required $297.00 1-10 ng DNA in 40 ul nuclease free ultrapure water For gDNA or amplicons, low input requirement for gDNA don to 1 ng
 NextFlex ChIP-Seq (Perkin Elmer)As required $291.00 1-10 ng DNA in 40 ul nuclease free ultrapure water For use with ChIP or genomic DNA samples. Optimized for down to 1 ng of DNA input. Enhanced Adapter Ligation Technology offers optimal coverage and unique reads. Optional gel-free, bead-based size selection step.
++ 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$1,668.00
150 Cycle High Output25M$2,057.00
300 Cycle High Output25M$3,010.00
300 Cycle Mid Output8M$1,376.00
NextSeq 50075 Cycle High Output400M$2,744.00
150 Cycle High Output400M$5,134.00
300 Cycle High Output400M$7,940.00
150 Cycle Mid Output130M$2,243.00
300 Cycle Mid Output130M$3,270.00
* Runs may be multiplexed to obtain required read depth and minimize per sample cost.

Deliverables and Results Retrieval

In most cases, BaseSpace will be utilized to facilitate transfer ownership of data. When sequencing is complete, you will be provided a link to facilitate this transfer. More information regarding BaseSpace and data transfer can be found here: https://support.illumina.com/help/BaseSpace_Sequence_Hub/Source/Informatics/BS/TransferOwner_swBS.htm

In cases where data transfer cannot occur within BaseSpace, you will be provided directions to obtain your data electronically.

StemCore will not archive analysis files once the data has been provided.

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)