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Next Generation Sequencing

Next generation sequencing service at the Biology Genotyping core

1. Equipment

We have one Illumina MiSeq at the core (http://www.illumina.com/systems/miseq.html). The MiSeq uses one-lane flow cells. As a result, we can start the sequencing quicker in comparison with larger sequencers such as Illumina HiSeq (it uses 8-lane flow cells and often needs time to fill the whole plate).

2. Running options

MiSeq has eight running options in total (http://www.illumina.com/systems/miseq/kits.html). The first five use regular flow cells; the last three use different flow cells and generate less data. Please indicate in the submission form which option you choose.

Since we may not have your kits at the core and need to order it in, we would like you to contact us (sunz@queensu.ca) once you know which one to use. That way we can start the sequencing quicker when your samples are ready.

3. Service provided and sample preparation

The Biology genotyping core can only provide the sequencing service at this moment. All libraries need to be constructed by your own lab. If you have multiple libraries that you want to pool together for the same run, please do so before bring it down to the core.

We will need at least 15ul of the library with concentration > 10nM for sequencing. (If your library was constructed using Illumina TruSeq amplicon or Nextera XT kits and it has already been denatured, please let me know in the submission form).

The quality of the library is critical to sequencing success. Please provide the following readings to us when submitting your sample.

• NanoDrop and Qubit reading.  The OD 260/280 ratio and OD 260/230 ratio will tell us the purity of your DNA. However, don’t rely on NanoDrop for the concentration of your library. It tends to overestimate the DNA concentration and can be as much as 10 folds in some cases. If you have access to a Qubit, it is a much better option for measuring the concentration of your library DNA.
• Bioanalyzer reading. This can give us a reading of the DNA concentration as well as the size distribution of DNA fragments in the library. This reading is essential to evaluate the quality of your library. To process your sample, I need to have a copy of the Bioanalyzer trace accompanying your sample submission form (you can email the Bioanalyzer trace to me or bring the printout to me). If you don’t have your own Bioanalyzer, talk to Dr. Babak (tomas.babak@queensu.ca) in Biology Department or Dr. Feilotter's (hf4@queensu.ca) in Pathology to have the analysis run in their labs.

If you would like to use custom primers for sequencing, they have to be

• HPLC purified
• Reconstituted to 100uM in ddH₂O or 10mM Tris-Cl (pH8.0).
• 10ul volume

You also need to make sure the primers work with your library before bringing them to the core.

4. Price

Note:

• Illumina changes the prices of their kits and reagents when they deem necessary. As a result, the sequencing prices at the core may get adjusted once in a while.
• Theses prices include the price of qPCR quantification of the library which we do for each library.

5. Sample submission forms

You can fill up the on-line submisson form here.

6. Contact and sample delivery

7. Data Management:

We will use the Illumina cloud service--BaseSpace(https://basespace.illumina.com/home/sequence) to store finished sequencing data. You will get an email notice when the data are available. To download data from BaseSpace, you need to set up an account with Illumina—it is free and pretty straightforward to do. Although you can manage the data and use the analysis tools of BaseSpace (with a fee now), it is still strongly recommended that you download a copy of the data to your local hard drive for future reference.

8. Disclaimer:

The quality of the sequences is dependent on the quality of library, which is your own responsibility. Every run will contain some control phiX174 DNA. We determine whether a run meets quality specifications based on the behavior of this commercially available library DNA.  If the internal control fails, we will re-sequence your library for free. If the internal control works well, we will consider the run successful (even if the outcome of your own library is not ideal) and you will be charged.

9. Other useful links:

Both Miseq and Ion PGM are small benchtop NGS sequncers. For high throughput sequencing (HiSeq etc.), you may want to consider the core facilities listed below:

For general information on Genomics research and more: