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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 services for library preparation and high-throughput sequencing using the Illumina HiSeq 2500, HiSeq 4000 and HiSeq X platforms. The facility sequences DNA, RNA, cDNA, immunoprecipitated DNA (ChIP, MeDIP), genomic clones (cosmids, BACs, PACs), PCR amplicons, metagenomic samples, and in-solution or array-captured genomic regions of any organism and helps with project design and data analysis (through the informatics and statistics facilities).

Please see our terms of use.

1. Sanger Sequencing Facility Overview

Create a TCAG (Sanger) sequencing account

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

  • Submit samples using the on-line DNA sequencing Facility GSLE Server (http://genesifter.research.sickkids.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.

4. Sequencing Resources

Electropherogram viewers are freely available for Microsoft Windows, MacOS Classic, and MacOSX from these sites. 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 DNA Sequencing and Synthesis Facility offers high-quality capillary-based DNA sequencing, next-generation sequencing technologies on the Illumina HiSeq platform, and synthesis of conventional and labeled oligonucleotides to researchers in the academic, commercial and pharmaceutical communities.

The Next-Generation Sequencing Facility offers library preparation and high-throughput sequencing using the Illumina HiSeq X, HiSeq 4000 and HiSeq 2500 platforms. Applications include human and non-human whole genome sequencing, targeted sequencing (exomes, custom target, etc), RNA-Seq, small RNA sequencing, sequencing of epigenetic markers (MeDIP, ChIP, RIP, 5mC and 5hMC profiling, whole genome bisulfite sequencing), RAD-Seq, ATAC-Seq, amplicon sequencing, as well as other approaches. Library preparation services include DNA/RNA QC, indexing of samples to allow for multiplexing sequencing, and library QC.

The facility also accepts sequencing-ready libraries made by researchers (consult the facilities for more information and limitations). Turnaround time to generate data usually varies between 4 and 6 weeks and can vary on size of projects and type of sequencing flowcells.

  • Illumina HiSeq 2500:
    The HiSeq 2500 is capable of sequencing flowcells with 2 (Rapid Run Mode flowcells) or 8 lanes (High Output Mode flowcells). The platform generates single end or paired end reads and depending on the type of flowcell read lengths can vary between 50, 100, 125, 150 and 250 bases. Other read length may be available upon consultation. Rapid Run Mode flowcells generate around 150-170 million single reads or pairs of reads per lane for libraries with high sequence diversity. High Output Mode flowcells generate 250-270 million single reads or pairs of reads per lane also for libraries with high sequence diversity. Most NGS applications are performed on this platform.
  • Illumina HiSeq 4000:
    The HiSeq 4000 uses patterned flowcells with 8 lanes and allows the generation of single end and paired end data of 50, 100 or 150 bases. Each lane generates about 260 - 320 million single end reads of 260 - 320 million pairs of reads for high sequence diversity libraries. This platform is more adequate for libraries with more defined size distribution such as exomes, RNA-Seq and small bacterial genomic libraries.
  • Illumina HiSeq X:
    The HiSeq X is used exclusively for whole genome sequencing (human and non-human) and uses patterned flowcells with 8 lanes. The only sequencing option available currently on the HiSeq X is paired end sequencing that generates read length of 150-bases. Each lane generates about 330 - 375 million pairs of reads and each machine is capable of sequencing 16 human genomes every 3 days (complete workflow from DNA QC to analysis is about 2-3 weeks). Genomes are sequenced to a minimum of 30X. For human genomes specifically additional information is required and needs to be provided prior to acceptance of samples.

    Note that the HiSeq platform may not be able to generate as much data as mentioned above if libraries have low diversity sequences (amplicon, ChIP-Seq, WGBS, etc). In these cases is it possible to approach the output range specified by Illumina when a sequence-diverse control library is spiked in the lane and that can take up to 25% of the reads.

6. Next-Generation Sequencing Pricing

Pricing varies according to project, instrument required, depth of coverage and requirements for bioinformatic analysis. We provide free consultation and assistance in project design. Please contact the facility to discuss your needs and pricing. We will happily provide formal quotations or letters of support for grant applications.

7. Next-Generation Sequencing Sample Submission Guidelines

The required amount of starting genetic material needed as recommended by the manufacturer will vary depending on the type of library preparation and sequencing platforms. The facility recommends to measure DNA and RNA concentration using fluorometric methods. Samples should be submitted in aliquots of >10 ul with concentration between 20 and 200 ng /ul. Samples submitted to Bioanalyzer only can be in 3 to 5 ul and we strongly recommend that concentration is provided or additional charges may apply if concentration is outside the chip range and we need to rerun the samples. Below are some typical examples of amounts required for different applications:

  • Genomic DNA for paired end sequencing: 1 - 2 ug.
  • Genomic DNA for mate-paired sequencing: 1 to 30 ug depending on insert size.
  • Small/microRNA: 500 ng to 1 ug.
  • RNA-Seq (poly(A) or rRNA depletion library prep methods): 1 to 2 ug; <200 ng possible for a non-stranded low input protocol.
  • Exome or targeted sequencing: 1-2 ug.
  • ChIP-Seq: 2-20 ng.
  • Amplicon sequencing: ≥ 500 ng.
  • Library-ready aliquots: usually ¼ to half of the elution volume suggested in the manufacter's protocol but no less than 10 ul.

These amounts are typical guidelines only; for specifics, please contact the facility. Quantification of DNA or RNA by spectrophotometric methods may led to overestimation of concentration. If fluorometric or quantitative PCR methods are not available to you, we recommend sending twice the minimum amount required. Sample purity is important for a successful library preparation; please check that your sample has an OD260/280 between 1.8 and 2.0.

We strongly recommend to check DNA samples are not degraded on an agarose gel prior to submitting them to us. For RNA samples, if possible check the integrity using an Agilent Bioanalyzer; the minimum RIN value acceptable for RNA library preparation is 7. If a Bioanalyzer is not available, RNA integrity can be checked on a formaldehyde 1% agarose gel, or we can check on our Bioanalyzer on a fee-for-service basis.

Submit samples using the LabLink server

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

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

Send samples by courier to the address at the bottom of the page.

8. Publications by Next-Generation Sequencing Facility

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

9. Synthesis Facility Overview

The Centre for Applied Genomics Synthesis Platform is dedicated to delivering high-quality primers, probes, and fluorescently labeled oligonucleotides to clients in the academic, industrial and pharmaceutical communities.

The facility offers two ways to order your oligos, an in-house synthesis and a web-portal.

In-house synthesis:

The facility synthesizes oligonucleotides of up to 120 bases in length, using one ABI 3900 high throughput DNA synthesizer. Various specialized chemistries are available, including:

  • flourescein (6-FAM, HEX and TET), Cyanine dyes (Cy5, Cy5.5, Cy3 and Cy3.5) and other dye labeling
  • biotinylation
  • amino alkylation
  • thioalkylation
  • S-oligos (phosphorothioate) used in antisense research

For chemistries not listed here, please contact the facility.

TCAG-IDT web portal:

The facility also offer oligos synthesized by IDT through a web portal. You'll need to set up account with the portal (https://www.idtdna/tcag) in order to place orders.

10. Synthesis Pricing

In-house Synthesis Pricing:
40 nmole 200 nmole 1,000 nmole
Cost per base $0.59 $0.89 $1.89
Cartridge purification $15.00 $15.00 $30.00
Desalting free free free
Yield Guarantee
Scale Guaranteed Yield
40 nmole Minimum 25 µg
200 nmole Minimum 100 µg
1,000 nmole Minimum 500 µg

In-house synthesized oligos are ready for pickup within 48 hours, or can be delivered within the downtown Toronto core at no additional cost. For mail delivery, please contact the facility.

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 $0.40
250 nmole 5-100 bases 15 ODs $0.81
1 umole 5-100 bases 45 ODs $1.61
5 umole 5-100 bases 225 ODs $8.93
10 umole 5-100 bases 450 ODs $16.80

IDT orders are mostly ready in around 2 to 4 business days and they are shipped to TCAG synthesis facility.

11. How to Order Oligonucleotides

To order in-house oligos:

  • Customers within The Hospital for Sick Children can order using our online system: http://tocs.tcag.ca.
  • All other customers can place orders by email. Please fill in the Excel order template and email it as an attachment to Email:

To order web portal oligos:

TCAG-IDT web portal customers can order from http://www.idtdna.com/TCAG/

12. Oligo pick-up locations:

For your convenience, we have 3 (three) pick-up locations for you to get your oligos:

  • MaRS sample pick-up location:
    Users in the MaRS centre can pick up oligos at the UHN Glass Washing and Sterilization Services, Toronto Medical Discovery Tower, Room 4-409.
  • University of Toronto campus sample pick-up location:
    On-campus users can pick up oligos in the laboratory of Dr. Lucy Osborne, room 7360, Medical Sciences Building (1, King's College Circle).
  • SickKids sample pick-up location:
    SickKids users can pick up oligos inside of our administration entrance on the 13th floor of 686 Bay Street, Peter Gilgan Centre for Research and Learning.
  • Mount Sinai sample pick-up location
    Mount Sinai Hospital across from room 10-91. Take the Murray elevators to 10th floor. The drop boxes are on top of wire rack (600 University Ave).

Users at institutions other than the four campuses above will be contacted to make arrangements for pick up or delivery.

13. Paying by Credit Card

Please download this authorization form. (Acrobat 592 KB)

14. Facility Contact

Next-Generation Sequencing Facility Manager

Sergio Pereira, PhD

The Centre for Applied Genomics
The Hospital for Sick Children
Peter Gilgan Centre for Research and Learning
686 Bay Street, Room 139420HH
Toronto, Ontario
M5G 0A4, Canada
Tel.: (416) 813-8642, Internal: 308642
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 139800
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 139310H
Toronto, Ontario
M5G 0A4, Canada
Tel.: (416) 813-8472, Internal: 308472
Fax: (416) 813-8319, Internal: 208319
Email: