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TCAG Facilities

DNA Sequencing/Synthesis

Next-Gen sequencing samples are submitted to TCAG using LabLink

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 high-throughput sequencing using the Illumina HiSeq 2500 and HiScan-SQ, Life Technologies SOLiD 5500xl and Ion Torrent Proton and Roche 454 GS FLX Titanium. 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.

Please see our terms of use.

1. Sanger Sequencing Facility Overview

Create a TCAG 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 successful read. BAC, LAMBDA, Cosmid and Fosmid reactions will remain at $12.00 each. Failed reads will be charged provided the control from the same sequencing run passes our QC metrics. 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 primer in one tube in order to be processed. The primer must be added at a concentration of ~50ng (~5 pmols) in 0.7-1µl volume, for a total sample volume submitted to the sequencing facility of 7.7 µl. Be sure to only add ONE primer to each tube.
  • 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.

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, 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 high-throughput sequencing using the Illumina HiSeq 2500, Life Technologies SOLiD 5500xl and Ion Torrent Proton, and Roche 454 GS FLX Titanium technologies. The facility sequences genomic DNA, cDNA, RNA, immunoprecipitated DNA (ChIP, MeDIP, etc.), genomic clones (cosmids, BACs, PACs, etc.), large PCR amplicons, and array-captured regions of any organism.

  • Whole Genome Sequencing with Complete Genomics
    Contact Jo-anne Herbrick for details. ()
  • Illumina HiSeq 2500:
    Number of partitions for a single run: The HiSeq 2500 is capable of sequencing flowcells with 2 or 8 lanes. Multiple samples can be sequenced in the same lane reducing the total costs of the experiment per sample.

    Read lengths and average number of reads: The HiSeq 2500 is capable of producing 150 to 200 million single reads of 50- or 100-bases long, and paired-end reads of 2 x 50 or 2 x 100 bases per lane, in 1 to 12 days, depending on type of sequencing and flowcell used.

  • Life Technologies Ion Torrent Proton:
    Number of partitions for a single run: The Proton PI chip doesn't contain any partition, but multiple samples can be sequenced in the same chip depending on type of experiment and throughput desired per sample.

    Read lengths and average number of reads: A single PI chip generates up to 160 million reads, with average size of 120-130 bp.

  • Life Technologies SOLiD 5500xl:
    Number of partitions for a single run: The 5500xl flowchip is divided in 6 lanes, allowing 1 sample or pool of barcodes samples to be loaded per lane or in multiple lanes. The SOLiD barcode system allows up to 96 samples to be sequenced simultaneously per partition on both SOLiD 4 and 5500xl.

    Read lengths and average number of reads: SOLiD 5500xl provides reads up to 75 bp for fragment, 75 x 35 bp for paired end and 60 x 60 bp for mate-paired sequencing runs. Runtime on the 5500xl instrument varies from 2 to 21 days depending on read length and number of lanes used.

  • Roche 454 FLX Titanium:
    Number of partitions for a single run: The 454 libraries are deposited in a Pico-Titer Plate (PTP). Each PTP can be divided in 2 (minimum), 4, 8 or 16 regions. The system has 16 barcodes, allowing for up to 256 samples to be sequenced simultaneously.

    Read lengths and average number of reads: Read lengths for 454 FLX are 400 bases on average using the Titanium chemistry, in 12 to 16 hours. A full sequencing plate can generate between 800 thousand and 1.2 million reads depending on the sample.

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 libraries and sequencing platforms. Below are some examples.

  • Genomic DNA for paired end sequencing: 500 ng to 5 ug.
  • Genomic DNA for mate-paired sequencing: 1 to 30 ug depending on insert size.
  • RNA-Seq or microRNA: 100 ng to 10 ug.
  • Exome sequencing: 6 ug.
  • ChIP-Seq: ≥ 20 ng.
  • Amplicon sequencing: ≥ 500 ng.
  • Library-ready aliquots: usually ¼ to half of the elution volume suggested in the manufacter's protocol.

These amounts are guidelines; for specifics, please contact the facility. Quantification of DNA or RNA by spectrophotometric methods may lead to overestimation of the concentration. If other methods such as Picogreen and qPCR are not available, 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. For RNA samples, if possible check the integrity using an Agilent Bioanalyzer; the minimum RIN value acceptable for RNA library preparation is 8. 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 documents.

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. Users at institutions other than the 3 campuses above will be contacted to make arrangements for pick up or delivery.

Users at institutions other than the 3 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: