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• TCAG-IDT web portal for ordering oligos

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 2000 and HiScan-SQ, Applied Biosystems SOLiD 5500xl 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
2. Sanger Sequencing Pricing
3. Sanger Sequencing Sample Submission Guidelines
4. Sanger Sequencing Resources
5. Next-Generation Sequencing Facility Overview
6. Next-Generation Sequencing Pricing
7. Next-Generation Sequencing Sample Submission Guidelines
8. Publications by Next-Generation Sequencing Facility
9. Synthesis Facility Overview
10. Synthesis Pricing
11. How to Order Oligonucleotides
12. Paying by Credit Card
13. Facility Contacts

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.

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.

• Submit samples using the on-line DNA sequencing Facility Finch Server (http://tcag-2.ccb.sickkids.ca/Finch). Instructions for creating an account, placing orders, etc. can be downloaded. Please note that the Finch Server is unavailable from 2:30am-3:00am every day for backup and maintenance.
• 96 sample submission
• Creating an account
• Placing an order
• Retrieving results
• 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 20-33 ng (3-5 pmols) in 0.7 µl 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

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

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.

• FINCHTV TRACE VIEWER - Microsoft Windows, MacOSX, Linux, Solaris
• CHROMAS LITE CHROMATOGRAM VIEWER - Microsoft Windows
• APPLIED BIOSYSTEMS SEQUENCE SCANNER SOFTWARE - Microsoft Windows

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

• UCSC In-Silico PCR
• Primer3 for design of PCR Primers
• BioEdit Sequence Alignment Editor for Windows

The Next-Generation Sequencing Facility provides high-throughput sequencing using three different instrument platforms. Based on the project needs and type of material to be sequenced we will help the customer to decide on the most appropriate platform. The following sequencing capabilities are available in our facility: de novo or re-sequencing of whole or partial genomes, ChIP-Seq, MeDIP-Seq, RNA-Seq, micro RNA sequencing, exome sequencing, target resequencing, and many others.

The three platforms require the genetic material to be processed into a DNA library. Libraries are instrument-specific and can be constructed in-house on a fee-for-service basis, or by the customer using the manufacturer's recommended protocol. Each instrument allows multiple libraries to be sequenced at the same time as indicated below. The use of barcodes or tags allows more than one source of DNA to be prepared in the same library and run at once on the same instrument. The number of reads varies from tens of thousands to hundreds of millions depending on the platform, number of partitions and type of library.

Whole Genome Sequencing with Complete Genomics
Contact Joanne Herbrick for details. ()

Illumina HiSeq 2000:
Number of partitions for a single run: The HiSeq 2000 flowcell is divided into 8 lanes. Up to 8 different libraries (or the same library loaded in up to 8 lanes) can be run at a single time, if one lane is not required for a control DNA. Up to 12 samples can be barcoded and sequenced in the same lane, for a total of 96 samples per flowcell.

Read lengths and average number of reads: The HiSeq 2000 is capable of producing 60 to 100 million single reads 50- or 100-bases long, and paired-end reads of 2 x 50 or 2 x 100 bases per lane, in 5 to 14 days.

Applied Biosystems SOLiD System 4.0 and 5500xl:
Number of partitions for a single run: The SOLiD 4 is able to run two sequencing slides at once. Each slide can be partitioned into 1, 4 or 8 regions, allowing up to 16 libraries or 16 pools of libraries to be run at the same time.

Read lengths and average number of reads: SOLiD 4 reads up to 50 bases in 5 days. Mate-paired libraries are sequenced for 25 bases at each end of the fragment in 10 days. SOLiD 4 is capable of generating 300-500 million reads per full slide. Runtime for SOLiD 4 sequencing varies between 5 to 16 days, depending on the experiment.

During the summer and fall of 2011, the SOLiD 4 instruments are being upgraded to SOLiD 5500xl. 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. 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 7 to 21 days depending on read length.

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.

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.

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.

• TCAG LIMS placing an order
• Requesting a user ID

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

• Next Generation Sequencing
• Bioanalyzer for Next Generation Sequencing

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

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

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 synthesizes oligonucleotides of up to 120 bases in length, using one ABI 3900 high throughput DNA synthesizer and two ABI 3400 synthesizers.

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.

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

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.

Customers within The Hospital for Sick Children can order using our online system: http://tocs.tcag.ca. Online ordering for other customers will be available soon. Questions regarding the online customer service system can be sent to: tocs-info@sickkids.ca.

All other customers can place orders in two ways:

• by email, here is how.
• by fax at (416) 813-8319. Please use the order forms below:

Oligo Order Form (for oligos up to 42 Bases)
Oligo Order Form (for oligos up to 105 Bases)

TCAG-IDT web portal for ordering oligos

Please download this authorization form.

DNA Sequencing

DNA Synthesis