Nanopore Sequencing – New Service

PLASMID

• Concentration: 50 ng/µL in 10 mM Tris (pH 8.0) or H₂O
  It is important to accurately measure the concentration of your DNA. If the concentration is high and dilution is required, it is recommended to re-quantify the sample after dilution to 50 ng/µL in order to report the exact concentration on the submission form.
• Ratio: A260/280: between 1.8 and 2.0 and A260/230: between 2.0 and 2.2
  These ratios are good indicators of DNA purity. Residual RNA, solvents, salts, or proteins in your sample may negatively affect sequencing quality.
• Volume: Minimum of 15 µL per sample. If the concentration does not reach 50 ng/µL, please double the volume per sample.
• Warning! The main cause of Nanopore sequencing failure is DNA degradation. Do not vortex your plasmids. To ensure DNA quality, it is recommended to run your samples on an agarose gel prior to submission.

Please contact us by email (sequences@crchudequebec.ulaval.ca) to submit your request for Nanopore sequencing services. We will provide you with the procedure and the necessary documents to complete your service request.

• An assembly report (wf-clone-validation-report.html)
• A consensus sequence (FASTA)
• An annotated map generated by pLannotate (.gbk)

Please contact us at sequences@crchudequebec.ulaval.ca to schedule the submission of your samples.

• Always label your tubes with the sample name, your name, and the mention “Nanopore” or “ONT” to distinguish them from samples intended for Sanger sequencing.
• Purification methods compatible with Nanopore sequencing include magnetic beads or high-quality commercial spin columns.
• We strongly recommend performing a second wash during miniprep-style purifications, as well as a dry spin before elution to remove any residual contaminants from the column.
• The use of phenol–chloroform negatively affects Nanopore sequencing flow cells by damaging their physical components and inhibiting the sequencing process. Do NOT send samples purified using this method.
• All samples must be submitted in 1.5 mL microcentrifuge tubes (Eppendorf-style). When submitting multiple samples (24 or more), plates or tube strips are easier for us to handle. This also reduces labeling effort and minimizes handling errors.
• We recommend protecting your samples in a 50 mL plastic tube (a tip box can also be suitable). The use of bubble wrap is also recommended.

• If you are using parafilm to seal your tubes, use the minimum amount.
• Do not use adhesive tape to label your tubes, as it tends to come off at −20 ºC.
• Do not use adhesive tape to bundle your samples together, as it is difficult to separate and may smear the ink on the tubes. Instead, use an envelope, a resealable plastic bag (e.g., Ziploc®), or any other easy-to-handle container.
• It is not necessary to place your DNA on ice or dry ice if it is not contaminated with endonucleases.
• It is recommended to protect your samples during shipping by mail or courier: use a padded envelope with bubble wrap or a rigid container such as a 50 mL screw-cap Falcon tube.

Warning: please ensure that your samples do not arrive over the weekend or on a public holiday.

• Samples can be dropped off at the reception desk of Block T, room TR-72, or at the entrance of the Infectious Disease Research Centre, room RC-709.
• You may also deliver them directly to room R-4756 if you have access.
• Samples can be sent via internal mail; however, please allow an additional day for delivery.

To learn more about the technology used for Nanopore sequencing, the following video provides a clear overview of how it works: https://www.youtube.com/watch?v=RcP85JHLmnI.