CE Sequencing

Contact: Elizabeth Jensen
Location: Thomas building, DE-302
Contact phone: (206) 667-4470
Contact fax: (206) 667-2825
Contact e-mail: genomics@fredhutch.org

Lab Introduction and Consulting

The Genomics Resource's Genetic Analysis lab provides capillary electrophoresis-based sequencing services. Two options, "full-service" and "ready-to-go" for this service are described below. Sequencing is performed using two staff operated Applied Biosystems (ABI) 3730xl DNA Analyzers. An overview of ABI's platform and applications is available at their website.

Option A: Full-service Sequencing

You provide a pre-mix of DNA template + primer and we perform the sequencing PCR, sample clean-up, and analysis.

Sample Preparation and Submission

The quality of the sequence is dependent on the quality and concentration of the DNA template and primer. All DNA templates and primers should be resuspended in water or Tris buffer. Please ensure the DNA templates are free of salts, proteins, and organics. PCR products should be free of extra primers, dNTPs, and should not contain any fluorescent tags. Details for sample submission are available in the full-service submission guidelines document.

In House Primers: We have M13Reverse, M13Forward, T7, and SP6 available in the DE-302 refrigerator at no additional charge. All primers are checked using pGEM before being distributed.

Samples should be delivered to DE-302 along with a completed sample request form (see below).

Process and QC

Turnaround time: One can expect to receive results within 1-2 business days from sample submission.  Please note, samples submitted after noon will be considered received the following business day.

Quality control: A pGEM/primer control is added to every Full-Service sequencing run to ensure all reagents and instruments are working correctly.

Sequencing Failures: If you think we made an error we will do everything we can to correct the situation. We will be glad to repeat the SAME sample a second time to aid in troubleshooting under the following conditions:

  • We need to be notified of the problem within 3 business days.
  • If we cannot explain the results, we will resequence the original sample from the original tube.
  • If the repeat works you will not be charged for the repeat.
  • If your sample fails a second time we will assume the problem was with the sample and you will be charged for the original run AND the repeat run.

Option B: Ready-to-Go Sequencing

In this option, the investigator runs the reactions before bringing them to the Genetic Analysis lab.

Sample Preparation and Submission

Genetic Analysis lab staff will perform a BigDye XTerminator clean up step to remove unincorporated BigDye terminators and salts and stabilize the post-purification reactions. General recommendations and protocols can be found here. You can contact the lab for additional suggestions and troubleshooting assistance. The Genomics Resource sells aliquots of ABI's BigDye Terminator Cycle Sequencing Reagent in DE-302. Reagents are sold in whole reaction quantities, however many find they can get quality results using ¼ reaction amounts. For more details, contact the laboratory staff.

Please note, samples must be delivered in strip tubes with strip lids or in 96-well plates. If preparing a 96 well plate, load samples in a vertical orientation (A-H).

Following sample preparation, samples should be delivered along with a completed sample request form (see below) to DE-302. Samples should be placed in the refrigerator labeled "Sequencing Samples", on Ready-to-Go shelf. Genetic Analysis Lab staff will load onto an ABI-3730xl instrument.

Researchers who deliver samples, accompanied by a completed submission form, by 4 PM Mon-Thur or by the Fri deadline of 2PM will typically receive results the following business day (M-F). Please note that the stated turnaround estimates will be affected by holidays, equipment malfunctions, and staffing issues.

ABI ready-to-go sequencing request forms:
If you are using a Fred Hutch project ID for billing, please fill out the Internal submission form at the Hutchbase site, print the completed form and bring in with your sample.

If billing is to be submitted to the University of Washington or other external institution, fill out the External submission form.

Results and Downstream Analysis

For either the full-service or ready-to-go options, data generated in the resource are transferred to servers supported by the Hutchinson Center's IT department.. Data is transferred to the user's Homelink account in the researcher's geneticanalysis folder and/or can be sent to investigator via email and/or as a hard-copy printout. Please indicate on the request form which method(s) you would prefer. If you do not have a Homelink account, please Data Storage and Archiving for more information.

Results sent electronically will include a text file (.seq) a chromatograph (.AB1), and a phred (.phd) file.

Available software to visualize and analyze your sequencing data can be found at the Genomics IT website (License agreements dictate that some of the software is available only from Fred Hutch based computers. If you are not at Fred Hutch and need access to the software, please contact Genomics and we will arrange access for you.) For more information, please contact Ryan Basom at rbasom@fredhutch.org or (206) 667-2747.