INSTRUMENTATION

The Donnelly Sequencing Centre is a full-service, Illumina CSPro core sequencing provider. Our facility utilizes the latest technology including Illumina HiSeq2500s (v4 high-output, and v2 rapid run chemistry), NextSeq500s (v2), MiSeq (v2 & v3), and NeoPrep instruments. Each instrument and chemistry has unique sequencing capabilities and sequencing yields. For more information on each platform please explore Illumina's Sequencing Platform Comparison Tool and/ or email or call us if you are unsure of your project-specific platform requirements.


SERVICES

The Donnelly Sequencing Centre is the only Illumina CSPro certified NGS full-service provider in Toronto, Canada.
We offer a full suite of library preparation services for Illumina platforms, quality control services, library-preparation training, and sequencing services. We also specialize in sequencing of high-throughput CRISPR screens (adapted to HiSeq2500 and NextSeq500 platforms), high-throughput phage display screens, and custom-prepared libraries.

Use Illumina's Methods Explorer Tool to find the NGS library preparation method most compatible with your research goals.

Let us help you! Consultations are free, and help ensure your libraries achieve maximum performance across our platforms. Please contact the DSC Team at dSeqCentre@gmail.com or at +1 416.978.8579 for further inquiries.

Bioinformatics:
Currently, the DSC does not offer sequencing analysis services beyond bcl to fastq conversion and sample de-multiplexting. We highly encourage our customers new to NGS and bioinformatics to give Illumina's BaseSpace application a try. Upon request, we upload your data onto BaseSpace. To request that your data be uploaded onto Basespace, please fill out the relevant section in the Sample Submission Form (see 'SAMPLE SUBMISSION AND SHIPMENT' section below).
Basespace contains many applications for sequencing analysis including BWA, Cufflinks, TopHat, Velvet, Issac, RNAexpress, DNAstar, FASTQC and many others. These services operate in the cloud and do not require you to download your data locally. Illumina account is required for access. Sign up at Illumina.com and explore sample data sets, and application instructions. Illumina terms and conditions apply and many applications are free.

Data distribution:
De-multiplexed FASTQ TAR files are distributed across our servers, and may be downloaded to your local servers.

Turn-around times:
This is a general guide:
Project-specific turn-around times reflect the number of samples submitted and the type of library preparation required, as well as the requested sequencing platform.
Standard library preparation protocols: up to 3 weeks (1-30 samples, scale up by 1 week for each additional 30 samples).
Custom protocol development: up to 6 months.
Newly published protocol optimization: up to 4 months.

Sequencing-platform specific turn-around times for full-flowcell requests on all our platforms (MiSeq, HiSeq2500, NextSeq500) is less than 1 week. This assumes pooled, high-quality user-prepared submission that passes all our internal QCs on the first go.
For partial FC requests:
NextSeq500: it can take up to 4 weeks to find a compatible matching project.
HiSeq2500: it can take up to up to 8 weeks depending on the current submission volume to fill up a full flowcell.

BUDGETS, QUOTES, & PROCUREMENT

Budgets: Many factors affect pricing. Having a framework budget for your project will ensure that your request matches your budget expectations. We recognise that some projects have budgetary constraints. Sharing your budgetary consideration with us can help inform the best sequencing design for your project. It will help us help you get the most out of your data.
Quotes: all quotes expire 3 months from date of issue. If your quote is expired at the time of submission, current pricing will apply.
Procurement: Clients external to the University of Toronto require a purchase order number (PO) from your Institute's procurement department. To generate a PO, your procurement department will require a quote. Our quotes contain all relevant information for vendor registration. The Donnelly Sequencing Centre is registered with many institutes including SickKids: 0000052641 and UHN: 1004064. After sequencing and data distribution is complete, you will receive an official invoice from the University of Toronto. Invoice documentation must be forwarded to your procurement department upon receipt. Payment status is monitored.

Quick turnaround on quotations:
For quick turnaround on quotations, this is the minimum information you need to provide:
1. Lead PI's full name and affiliation (i.e., the name of the lead investigator with the funds to cover the project) - email signature will do;
2. Sequencing application (i.e., CRISPR, ChIP, CLIP, RIP, mRNA, total RNA, small RNA, etc);
3. Model organism;
4. Number of samples that need library processing (if library construction services are not required, please indicate user-prepared library type);
5. For samples that need library construction, please indicate your preferred library preparation kit (by default the DSC uses lot-controlled NEB kits);
6. Preferred instrument (see list above), sequencing length/ strategy; for custom sequencing please send us an illustration of your library construction approach;
7. Desired depth of coverage or number of reads per sample (in Millions);
8. GC content and sample complexity; for challenging samples (GC/AT content >65% and low-complexity), GC content and complexity may determine the platform most suitable for your application;
9. Budgetary considerations can help inform the best sequencing design for your project. It will help us help you get the most out of your data.


SAMPLE SUBMISSION AND SHIPMENT

Sample submission is a simple process. Fill out the Sample Submission Form, including shipment tracking ID, quote #, institutional purchase order and financial officer information and e-mail the completed form to dSeqCentre@gmail.com; a digital copy is required! Submission of this form confirms your acceptance of our terms and conditions (found in 'Terms & Conditions' excel tab). Please follow our samples sheet naming convention: yy_mm_dd_PILastName_PIFirstName_NumOfSamples_seqApplication (eg., 16_07_23_Blencowe_Ben_18_totalRNASeq).
For external requests we require an official purchase order (PO) from your home institution. Research institutes will not process payment without one! DSC does not accept projects without the official PO.
SHIPMENT: Please use standard size 1.5 ml tubes, labelled with institute/Professor's name, and date. Allow 3 days for shipments originating from the US, 4 days outside of North America. Please ship your samples no later than Tuesday AM to allow for delays at customs. Include all relevant documents for customs (commercial invoice, dry ice label, sample sheet). Email a copy of your completed sample submission form and we will track your package. All shipments should contain plenty of dry ice and be addressed as follows:

University of Toronto
Donnelly Sequencing Centre
160 College Street, Room 643
Toronto, Ontario, M5S 3E1
+1 416.978.8579
Attention: Tanja Durbic/ Graham O'Hanlon


INPUT MATERIAL SUBMISSION REQUIREMENTS

We offer complete library construction services for your experimental needs. All submitted material should be suspended in molecular grade water, or 10 mM Tris pH 8.0, never in TE (EDTA inhibits downstream enzymatic reactions). RNA samples should be DNase-treated (we highly recommend kits that have DNase-inhibition reagents that do not contain EDTA, such as Ambion DNA-free DNA removal kit (cat # AM1906)). Likewise DNA samples should always be RNase-treated. We ask for at least 30uL volume per sample regardless of sample type. Please see the required input material recommendations for sample submissions, and suggested quality control procedures listed below.

Genomic DNA Submissions
We require the following minimum DNA amounts (RNase-treated) for each of the listed library preparation methods:

  • NeoPrep: automated Nano gDNA sample preparation (max. plexity/ pool: 16-plex)- 25-75ng required, 150ng recommended
  • Nextera XT kit sample preparation- 2 ng required, recommended minimum 100 ng
  • Nextera DNA kit sample preparation- 50 ng required, recommended minimum 100 ng
  • Nextera Mate Pair sample preparation- 1-4 ug required, recommended minimum 2-8 ug
  • Nextera Rapid Capture Exome sample preparation- 50 ng required, recommended minimum 100 ng
  • Illumina TruSeq Exome enrichment protocol- 1 ug required, recommended minimum 2 ug
  • Mate-Pair - 20 ug recommended
  • ChIP-Seq - 10 ng required, recommended minimum 20 ng
  • Nano gDNA sample preparation- 100 ng required, recommended minimum 500 ng
  • PCR-free gDNA sample preparation- 1ug required, recommended minimum 2 ug
  • Amplicon sample preparation- 250 ng required, recommended minimum 500 ng

RNA Submissions
We require the following minimum RNA amounts(DNase-treated) for each of the listed library preparation methods:

  • NeoPrep: automated stranded mRNA sample preparation (max. plexity/ pool: 16-plex)- 25-100ng required, 200ng recommended
  • Standard and stranded mRNA and total RNA sample preparation- 100ng-1ug total RNA required, recommended minimum 4 ug total RNA
  • Small RNA sample preparation-1ug total RNA required, recommended minimum 4 ug total RNA
  • Illumina TruSeq RNA Access sample preparation (FFPE)- 10ng total RNA required, recommended minimum 20 ng total RNA

User-Prepared Library Submissions & Challenging Samples:
We require a minimum of 40 nmoles (in 30 uL at a minimum) of adapter/primer-free library suspended in molecular grade water, or 10 mM Tris pH 8.0, never in TE. We will conduct internal quality controls prior to sequencing your library. We recommend KAPA Library quantification kits (D-Mark) for yield assessment. Please note that the DSC does not guarantee library performance of customer prepared libraries, nor do we guarantee performance of challenging samples (high GC/AT content), those generally considered a challenge for the current Illumina sequencing technology.


All submissions are subject to internal quality controls before library preparation begins:

Quality control ensures NGS libraries perform according to Illumina standards. The Donnelly Sequencing Centre verifies the integrity of all submitted samples. DNA samples are checked on a gel, and assessed for intact, high molecular weight material. RNA samples will be subject to Agilent bioanalyzer quality assessment. Illumina recommends RNA integrity numbers (RIN) greater than 8. Please follow submission guideline amounts for individual libraries. We recommend quantitating input material using fluorescent methods for both DNA and RNA (Qubit Quant-it dsDNA HS Assay, and Quant-it RNA assay, Invitrogen). Nanodrop quantitation is not accurate. If using this method, please submit recommended minimum material. Clients will be contacted if samples do not meet specifications.