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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 Facility), services for DNA or RNA library preparation for most species, and high-throughput sequencing using the Illumina HiSeq 2500, HiSeq 4000, HiSeq X and NovaSeq 6000 platforms and single molecule sequencing on the PacBio Sequel platform. The facility also accepts sequencing-ready libraries. Automated library preparation on an Agilent Bravo automation system is available for limited types of library preparation protocols.

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

  • 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 (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. 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 and high-throughput sequencing using the Illumina HiSeq X (for whole genome sequencing only), HiSeq 2500, HiSeq 4000 and NovaSeq 6000 platforms for any application, and PacBio Sequel single molecule sequencing (mostly for whole genome applications). Library preparation services include human and non-human whole genome sequencing, targeted sequencing (exomes, custom target, etc), RNA-Seq, small RNA sequencing, epigenetics sequencing (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 multiplex sequencing, and library QC.

The facility also accepts sequencing-ready libraries made by researchers (consult the facility 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 flowcell required.

  • Illumina HiSeq 2500:
    The HiSeq 2500 is capable of sequencing flowcells with two (Rapid Run Mode flowcells) or eight 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 lengths 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 have been performed on this platform.
  • Illumina HiSeq 4000:
    The HiSeq 4000 uses patterned flowcells with eight lanes and allows the generation of single end and paired end data of 50, 100 or 150 bases. Each lane generates about 300 - 350 million single end reads or pairs of reads in sequencing runs of libraries of high sequence diversity. This platform is best suited for libraries with defined insert 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 eight lanes. The only sequencing option available currently on the HiSeq X is paired end sequencing that generates read lengths of 150-bases. Each lane generates a minimum of 330 million pairs of reads that corresponds to a human genome read depth of 30X. Each instrument is capable of sequencing 16 human genomes every 3 days. Complete workflow from DNA QC to analysis is about 2-4 weeks. Genomes cannot be sequenced to less than 15X as per Illumina requirements. For human genome sequencing specifically, consult the facility to check requirements for REB/IRB approval and additional paperwork that is required prior to accepting 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 it is possible to approach the output range specified by Illumina when a sequence-diverse control library is spiked in the lane, however this can take up to 25% of the reads.
  • Illumina NovaSeq 6000:
    The NovaSeq 6000 can be used for any application that is compatible with any of the HiSeq instruments. There are four types of NovaSeq flowcells (SP, S1, S2 and S4) that will generate between 325 and 750 million paired end reads per lane. For most applications, projects with more than 50 samples to be sequenced to minimum number of 30 million reads each can be sequenced more cost effectively and quicker on the NovaSeq compared to the HiSeq platforms.
  • PacBio Sequel:
    The PacBio Sequel is a single molecule sequencing platform to be used for applications like whole genome sequencing for de novo assembly and structural variation detection, among other applications. The most current version of the sequencing chemistry usually generates 6-10 Gbases of raw data and about 400 million reads. A large portion of the reads can reach 20-30 Kb in length.

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. Please contact the facility to discuss your needs and pricing. 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 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:

General Considerations for DNA and RNA Sample Submission

When submitting DNA or RNA samples to the NGS Facility, please ensure the guidelines given below are met. Upon sample receiving, the NGS Facility will verify that samples were received in good condition, the number and names of samples submitted to Clarity LabLink LIMS match the number and names received, and that the tubes are clearly labeled. The NGS Facility will contact clients to notify them once samples have been received. This email will make reference to an automatically generated LIMS-ID (ex. ABC1234) specific to the order. For a quicker response time for any questions related to your project, always refer to the LIMS ID each and every time you need to contact the TCAG NGS Facility; preferably, reply to all and to the latest email you have that makes reference to the LIMS ID. Any samples received without a formal order in Clarity LabLink will not be processed.

General Submission Requirements

  • Submit samples in 1.5ml lo-bind tubes or equivalent (for example, Eppendorf DNA LoBind Tube 1.5ml Cat. No. 022431021).
  • Do not send samples in strip-tubes or plates sealed with strip-caps, as these carry a higher risk of sample cross-contamination when opened.
  • If submitting samples in 96-well plates, ensure that the plastic or foil seal is securely closed around all edges and wells.
  • Sample names listed on the sample submission form must match the name label on tubes or plate map.
  • On the sample submission form, please state number of lanes required for project or number of reads per sample (if known).
  • To avoid delays in case samples fail QC steps, we strongly recommend that DNA samples are checked on an agarose gel or automated DNA fragment analyzer prior to submission. 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. For RNA samples that are submitted to us for library preparation, quality check on Bioanalyzer is included in the library preparation service.

DNA Requirements

  • For 10X Genomics long, linked read applications, we recommend to extract DNA using QIAgen MagAttract (as recommended by 10X Genomics); for any other genomic applications any DNA extraction method that generates good quality, pure DNA is acceptable. If using organic methods (phenol-chloroform based methods), please ensure no organic contamination is present in the submitted sample.
  • DNA should be double stranded as most protocols will ligate double-stranded adapters to double-stranded DNA (contact facility manager if sample is single-stranded DNA or a mix of ss-DNA and ds-DNA prior to submission).
  • DNA can be suspended in nuclease-free water or preferably in 10mM Tris-HCl pH 7.5-8.5 (do not add EDTA).
  • Quantify DNA samples using fluorometry-based method (Qubit DNA HS Assay or Picogreen).
  • DNA samples should have OD 260/280 ratio of 1.8 to 2.0.
  • Sample concentration and volume ranges should fall within the categories listed below (Table 1 for samples for library prep and Table 2 for samples submitted for shearing or DNA QC only but not for library preparation).
  • Submit samples with clear ID labels that correspond to the ID provided on the service request form in Clarity LabLink LIMS.
  • Ensure label IDs match names provided in Clarity LabLink and will not rub off or peel easily.

RNA Requirements

  • RNA samples extracted with any method of choice that generates good quality, pure RNA are acceptable. Degraded or partially degraded RNA samples may result in suboptimal libraries and biased sequencing results, or even failed libraries.
  • RNA can be suspended in nuclease-free water (Do not suspend RNA samples in DEPC-treated water or buffers containing detergents).
  • RNA samples should have OD 260/230 ratio of 2-2.2.
  • RNA sample can be treated with DNase if genomic DNA contamination is significant.
  • Samples should have a RIN value of = 7.0 (samples with lower RIN values may be acceptable pending discussion with Facility and method used for library preparation).
  • RNA sample concentration and volume ranges should fall within the categories listed below (Table 1 for library preparation services or Table 2 for samples submitted for QC only).
  • Submit samples with clear ID labels that correspond to the ID provided on the service request form in Clarity LabLink LIMS.
  • Ensure label IDs match names provided in Clarity LabLink and will not rub off or peel easily.
  • RNA samples must be transported/shipped on dry ice at all times.

Table 1: Specifications for DNA and RNA sample submission

Protocols Quantity Range Minimum concentration Minimum volume
Genomic DNA (WGS, WGBS, exome, targeted capture, metagenomics, etc) 1500 ng to 2,000 ng 20 ng/ul 20 ul
Genomic DNA for PacBio Sequel 5,000 ng to 15,000 ng 20 ng/ul 20 ul
Genomic DNA for 10X Genomics linked reads 400 ng to 1,200 ng 20 ng/ul 20 ul
Low Input DNA (ChIP-Seq, MeDIP, etc) 10 ng to 300 ng 10 ng/ul 20 ul
Total RNA - standard input protocols 200 ng to 2,000 ng 20 ng/ul 20 ul
Low Input total RNA 10 pg to 10 ng 5 ng/ul 5 ul
10X Genomics - Single Cell 3' end RNA-Seq 200 cells/ul 2000 cells/ul 20 ul
Total RNA for small RNA 300 ng to 2,000 ng 50 ng/ul 20 ul
Customer-made libraries 2 nM to 10 nM 2 nM 20 ul

Table 2: Specifications for Shearing and Bioanalyzer/TapeStation sample submission. For samples submitted for library prep (refer to Table 1).

Shearing & QC only Quantity Range Volume
Covaris S2 - DNA 00 ng to 5,000 ng 50 ul
Covaris LE220 - DNA 100 ng to 10,000 ng 50 ul
Bioanalyzer - DNA > 0.005 ng > 3 ul
TapeStation DNA 10 ng/ul to 50ng/ul 5 ul
Bioanalyzer - RNA > 0.05 ng > 3 ul

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 samples 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 Service Request Form with your shipment.
  • Send your samples to the shipping address below.
  • For shipment starting outside Canada, a commercial invoice may be required for customs clearance (check with courier).

Shipping Address:

Sergio Pereira/Karen Ho/ Travis Dawson
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. Oligo pick-up (and Sanger Sequencing sample drop off) locations:

For your convenience, we have four locations for you to pick up 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. T. Mallevaey, room 7308, Immunology hallway, Medical Sciences Building (1, King's College Circle).
  • SickKids sample pick-up location:
    SickKids users can pick up oligos inside 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 users can pick up oligos across from room 10-91. Take the Murray St. elevators to the 10th floor. The drop boxes are on top of the 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: