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TCAG Facilities

DNA Sequencing/Synthesis

The DNA Sequencing and Synthesis Facility offers high-quality capillary-based DNA sequencing, three next-generation sequencing technologies , and synthesis of conventional and labeled oligonucleotides to researchers in the academic, commercial and pharmaceutical communities.

The Next-Generation Sequencing Facility offers consultation for project design (including involvement with the Informatics and Statistics Facilities), services for DNA or RNA library preparation for most species, and high-throughput sequencing using the Illumina NovaSeq 6000 and MiSeq platforms, as well as single molecule sequencing on the PacBio Sequel IIe and Oxford Nanopore PromethION platforms. The facility also accepts sequencing-ready libraries under certain conditions. Automated library preparation on an Agilent Bravo automation system is available for some types of library preparation protocols.

Please see our terms of use.

1. Sanger Sequencing Facility Overview

To create a new account, please email Beverly .

The Sanger Sequencing Facility provides high-quality capillary-based fluorescent sequencing on dual ABI 3730XL instruments. For large scale products, a third ABI 3730XL can be used. This instrument is usually reserved for SNP and microsatellite genotyping.

The facility routinely sequences:

  • plasmids
  • PCR products
  • genomic clone ends (cosmids, BACs, PACs, phage)
  • whole genomes

Sanger CE read lengths are typically 700 bases, with a usual turnaround of two days. The facility currently averages about 3,500 sequence reads per week.

2. Sanger Sequencing Pricing

The facility charges CDN $3.50 per read. BAC, LAMBDA, Cosmid and Fosmid reactions are $12.00 each. ALL submitted samples will be billed, unless the DNA sequencing facility is at fault as determined by our control run on each plate. We accept Visa/MasterCard/AMEX or institutional purchase orders for payment.

3. Sanger Sequencing Sample Submission Guidelines

  • If you are planning to submit 20 or more samples/reactions for sequencing, please consider sending them in PCR strips or a PCR plate. You can label the tubes as the sample number or well. This helps move things through the pipeline faster for all users. Thanks for your consideration.
  • Submit samples using the on-line DNA sequencing Facility GSLE Server (https://genesifter.tcag.ca/gsle/mainPage). Instructions for creating an account, placing orders, etc. can be downloaded. Please note that the Finch Server is unavailable from 2:30 - 3 a.m. every day for backup and maintenance.
  • Samples must be submitted containing 7.0 µL purified template DNA (at the appropriate concentration - see "DNA Template Concentration" instructions below). The entire sample volume will be used for the sequencing reaction. For the best results, please prepare your DNA in WATER, not TE or buffer. EDTA and salts can interfere with the sequencing enzyme.
  • If you are using a custom primer, samples must be premixed with template and (one) primer in one tube in order to be processed. 0.7µL to 1µL of primer at a concentration of ~50ng/µL (~5µM or ~5pmol/µL) must be added, for a total volume submitted to the sequencing facility of 7.7µL to 8µL.
  • If you are using a standard primer (see www.tcag.ca/primerList.html), the facility will add the primer to your samples. In this case, the volume submitted to the sequencing facility should be 7.0 µL.
  • NOTE: all reactions are done at an annealing temperature of 50C. Try to keep the temperature of your custom primer below 65C. Anything higher may cause inconsistencies with the sequencing results. We cannot adjust this unless you are submitting orders of full (~95 samples) plates.

DNA Template Concentration
The quantity of DNA template depends on the size of the template you are sequencing.

  • Plasmids - 200-300ng in 7µL
  • PCR product (>4kb) - 150-200ng in 7µL
  • PCR products (2-4kb) - 100-150ng in 7µL
  • PCR products (1-2kb) - 50-100ng in 7µL
  • PCR products (<1kb) - 50ng in 7µL
  • PCR products (<500bp) - 20ng in 7µL
  • PCR products (<200bp) - 10ng in 7µL
  • BAC/Cosmid DNA/Phage DNA - 600-800 ng in 12 ul with 2 ul primer for a total volume of 14 ul
  • Genomic DNA - Please do not submit this

The quality of your sequencing reaction is directly related to the quality of your template DNA. Please follow our guidelines (Acrobat 10 KB)carefully to maximize the chance of success of your sequencing reaction.

Mail samples to:

Tara Paton

DNA Sanger Sequencing Facility
The Centre for Applied Genomics
The Hospital for Sick Children
Peter Gilgan Centre for Research and Learning
686 Bay Street, Rm 13-9800
Toronto, ON, Canada M5G 1H3

4. Sequencing Resources

Electropherogram viewers are freely available for Microsoft Windows, MacOS Classic, and MacOSX from these sites. Users may be required to register to access the freeware. TCAG does not endorse or support any of this software.

Other useful tools. TCAG does not endorse or support these tools.

5. Next-Generation Sequencing Facility Overview

The Next-Generation Sequencing Facility offers library preparation services for human and non-human whole genome sequencing, targeted sequencing (exomes, gene panels, etc), RNA-Seq, small RNA sequencing, single cell RNA-Seq, epigenetics sequencing (MeDIP, ChIP, RIP, 5mC and 5hMC profiling, whole genome bisulfite sequencing), single cell ATAC-Seq, amplicon sequencing, as well as other approaches. Library preparation services include DNA/RNA QC, indexing of samples to allow for multiplex sequencing, and library QC. The facility also accepts sequencing-ready libraries made by researchers. Please consult the facility for more information. Turnaround time for data generation varies between 4 to 8 weeks and is dependent on the size of projects and type of sequencing flowcell required.

Current sequencing platforms in operation are:

  • Illumina NovaSeq 6000:
    The NovaSeq 6000 is a short-read technology platform that can be used for a multitude of medium and large sequencing projects. The four types of NovaSeq flowcells (SP, S1, S2 and S4) can perform single end or paired end sequencing to generate between 325 million to 2.5 billion clusters per lane, or between 32 to 750 Gbases per lane depending on the flowcell and read length. Most common applications on the NovaSeq are whole genome sequencing of complex organisms, RNA-Seq, and epigenomics.
  • Illumina MiSeq:
    The Illumina MiSeq system allows a wide-range of sequencing applications and due to its lower data output per run, the MiSeq is adequate for small scale projects requiring one to 25 million reads in total or about 510 Mbases to 15 Gbases of raw data. MiSeq read lengths can vary between single end reads of 36 bases to paired end runs of 2x300 bases. This is a popular platform for sequencing amplicons, small genomes and gene panels, and microbial RNA-Seq of bacteria for few samples, among others.
  • PacBio Sequel IIe:
    The PacBio Sequel IIe is a single molecule sequencing platform commonly used for whole genome sequencing for de novo assembly and structural variation detection, de novo transcriptome assembly, and other applications. The platform can generate between 80-100 Gbases of raw data and reads can reach 2-30 Kb in length depending on original DNA quality and library yield.
  • Oxford Nanopore PromethION-24:
    The PromethION-24 instrument is a sequencing platform for short or ultra-long molecules of DNA or RNA, and offers the flexibility to generate small or large amounts of data. The instrument can run up to 24 PromethION flowcells simultaneously. The facility offers whole-genome library prep using the LSK-110 kit, and will consider other applications upon request.

6. Next-Generation Sequencing Pricing

Pricing varies according to project, instrument required, depth of coverage and requirements for bioinformatics analysis. We provide free consultation and assistance in project design and cost estimate. We will happily provide formal quotations or letters of support for grant applications. Prices are available in this table; although we will keep this list updated as often as we can, prices are subject to change without notice at any time.

7. Next-Generation Sequencing Sample Submission Guidelines

The required amount of starting genetic material needed as recommended by the library preparation and/or sequencing manufacturer will vary depending on the sequencing application, type of library and sequencing platforms to be used. More information is given in this link; Please contact the facility before sending samples if your samples fall outside the requirements.

Shipping Instructions

In order to ensure your samples do not get degraded, damaged, lost or misplaced please follow the instructions below:

  • Ensure RNA samples are sent on dry-ice, unless otherwise stated
  • DNA samples can be shipped/transported at room temperature, but ice packs or dry ice is preferred
  • DO NOT ship any type of sample in wet ice under any circumstance. Melted ice may damage the package and courier may refuse to handle or deliver it
  • Ensure samples are clearly labelled and organized in racks or boxes
  • Seal the samples inside a plastic bag
  • Place order in Clarity LabLink. Sample names listed on sample submission form must match name label on tubes
  • Include a printed and signed copy of the completed Clarity Service Request Form with your shipment
  • Send your samples to the shipping address below.
  • For shipment originating outside Canada, a commercial invoice may be required for customs clearance (check requirements with your courier)

Shipping Address:

Valerie Crowley/Karen Ho
Sequencing Facility - The Centre for Applied Genomics
The Hospital for Sick Children
Peter Gilgan Centre for Research and Learning, Rm. 139420HH
72 Elm Street, Toronto, ON, Canada M5G 1H3

Sample/Library Return

Once QC/Sequencing has been completed and data has been analyzed, samples and libraries may be returned to you at cost. You will be contacted by the TCAG NGS facility stating that your samples are ready to be returned, should you so desire. Please be diligent in replying to this email as samples older than three (3) months from their sequencing/analysis completion date will be discarded.

Submit samples using the LabLink server

For instructions on creating an account, placing an order, etc., download the following document.

Next Generation Sequencing - Sample Submission forms (Microsoft Excel format)

8. Publications by Next-Generation Sequencing Facility

Selected publications by Next-Generation Sequencing Facility users (Co-authored or Acknowledged)

9. Synthesis Facility Overview

The facility offers DNA, RNA synthesis and synthetic biology reagents through an arrangement with IDT. An account with the IDT-TCAG web portal (https://www.idtdna/tcag) is needed in order to place orders.

10. Synthesis Pricing

TCAG-IDT web portal pricing:
Scale Base length Min.Yield Price per base
25 nmole 15-60 bases 3 ODs $0.23
100 nmole 10-90 bases 6 ODs $0.43
250 nmole 5-100 bases 15 ODs $0.88
1 umole 5-100 bases 50 ODs $1.75

IDT orders are usually ready in around 2 to 4 business days and are shipped to TCAG.

11. How to Order Oligonucleotides

IDT-TCAG web portal customers can place orders at http://www.idtdna.com/TCAG/

Please contact the facility if you need assistance.

12. Paying by Credit Card

Payment can be made through the secure SickKids payment gateway.

13. Facility Contact

Next-Generation Sequencing Facility Manager

Valerie Crowley, PhD

The Centre for Applied Genomics
The Hospital for Sick Children
Peter Gilgan Centre for Research and Learning
686 Bay Street, Room 139703
Toronto, Ontario
M5G 0A4, Canada
Tel.: (416) 813-7654 ext. 309036
Fax: (416) 813-8319 (Internal: 208319)
Email:

DNA Sequencing

Tara Paton, PhD
DNA Sequencing Facility

The Centre for Applied Genomics
The Hospital for Sick Children
Peter Gilgan Centre for Research and Learning
686 Bay Street, Room 139702
Toronto, Ontario
M5G 0A4, Canada
Tel.: (416) 813-7654 ext. 303579
Fax: (416) 813-8319, Internal: 208319
Email:

DNA Synthesis

Zhizhou Hu

The Centre for Applied Genomics
The Hospital for Sick Children
Peter Gilgan Centre for Research and Learning
686 Bay Street, Room 139410
Toronto, Ontario
M5G 0A4, Canada
Tel.: (416) 813-8472, Internal: 308472
Fax: (416) 813-8319, Internal: 208319
Email:

For questions or comments contact the TCAG Manager,

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