An Introduction to the DNA Sequencing Core

The DNA Sequencing Core provides University of Michigan investigators access to high-quality automated DNA Sequencing technology on a recharge basis. The cost of this service compares favorably with similar cores at other institutions, as does the quality of the data obtained. Turnaround time is very short; results are usually returned by email within 48 hours of sample submission. This document provides basic information needed by prospective clients of the Core.
Last updated 23-Jul-07

Description of Service

The Core accepts DNA samples (pure plasmids, mini-prep plasmids, M13 clones, cosmids, lambda clones, PCR products, gel isolated fragments, bacterial genomic) and processes them according to protocols for Applied Biosystems DNA Sequencers (Model 3730 XL sequencer). One sequencing run (one template, one primer) typically produces 900 nucleotides of high-quality sequence data or more, for good-quality template and a well-designed primer. The results are returned in two forms: a raw sequence file and a chromatogram file. The sequence file (a flat ASCII text file) is returned to the researcher by electronic mail. The chromatogram file is available on our data server (password protected to prevent unauthorized distribution of your data).

Standard Primers Provided

The Core provides five standard primers free of charge: T7, T3, SP6, M13FORW and M13REV. Please see the table of primers and their sequence for reference. We will also sequence using custom primers submitted by the individual investigators. See Design of Primers for Automated Sequencing for suggestions on how to design your own primers.

Controls Included

Each sequencing gel includes at least one lane of standard DNA known to generate good sequence. In addition, if any of the customers' samples utilize one of the Core's five standard primers, we will also include a positive control lane for that primer. The gel, sample handling and data analysis are extremely reliable and the Core personnel have a very low rate of error. If any problems are indicated in our sample processing, we repeat the affected samples free of charge.

Turnaround Time

We typically process samples within two business days after receipt. Check the current backlog for up-to-date turnaround information. Samples are processed Monday through Friday for overnight instrument runs. Processing typically begins between 6:00 and 10:00 on that day's reactions, and the gels are loaded into the sequencers for their overnight run at about 4:00 PM. The data are processed the following day and returned by email to the customers, usually between 11:00 AM and 4:00 PM. Some additional notes:
  • If the current backlog is more than 2 business days, you may wish to consider requesting Rush Processing
  • If we have no backlog, you may get the faster turnaround time by bringing the samples to the core before 9:00 AM so they may be selected for that day's processing. We cannot guarantee this, however, unless you request RUSH service.
  • In the event the numbers of samples exceeds our ability to process them, on-campus investigators take precedence over off-campus clients, and bulk-rate samples (96-well plates) take lower priority than normal samples.

Cost of Service

The basic cost of DNA sequencing is $3.00 for processing one template with one primer for all clients affiliated with the University of Michigan. Non-University clients are accommodated as time permits. Clients who submit entire 96-well plates of samples are eligible for a discount ( final price of $2.40 per lane). Discounts are also offered to affiliates of certain research units or of specific center grants. Please consult with the PI of your center grant to see if you are eligible for such a discount. The best discounts can be obtained via high-volume submission. Contact the Core Director for more information.

Results

When your samples have been completed, you will receive an email with the DNA sequence of your sample. This is what's known as a "flat ASCII file", and can be transferred into most standard DNA sequence analysis programs by cut/paste or other methods. We also make the actual chromatogram available for you. Certain sequence assembly packages will import these files, allowing you to make critical judgements on sequence quality while you are assembling the final sequence - this is highly recommended. The chromatograms are available on the Download Server. You will need your PI's Login and password to connect. Chromatograms are available for downlowad for 2 weeks, after which the data is archived. We also provide links to several programs capable of opening and viewing chromatograms.

Quality of Data

The sequence returned to investigators may be up to 1100 nucleotides long, but in general only some of that is reliable. The amount depends on a variety of factors, but the primary determinant is the quality of the template DNA. Beyond that high-quality sequence, the remainder of the file is useful in specific experiments where sequence accuracy is unimportant. It is crucial for each customer to evaluate their own data and to determine the extent of reliable data. Because data evaluation is highly dependent on your individual needs, we cannot perform this step for you. See our page on Interpretation of Sequencing Chromatograms if you are unsure how to evaluate the results.

Typical Problems

Automated sequencing is very reliable in the hands of our experienced technicians. As a precaution, we always check that control samples sequenced properly, that excess terminators were removed, that the gel resolution was adequate and that the computerized data extraction proceeded normally. If a problem is detected in any of these, customer samples are repeated free of charge. Extensive information is available on our web site to help you design effective sequencing experiments, and to aid you in troubleshooting the results of your sequencing. Please see our Frequently Asked Questions page for more information on these topics. The Core has no control over the quality of template, the design of the primer or the presence of 'problem' spots on the template. We will do our best to help you to avoid such problems, but we must charge for sequencing reactions that fail for such reasons.

Contact the Core

The Core Location and Hours of Operation are posted online and e-mailed to you when you submit samples. Please feel free to call the Core Director, Dr. Robert Lyons, at 764-8531. For general problems, including using the website and checking your samples you can contact the DNA Sequencing Core Main Office at 764-1461. We also have an LSI Submission Desk which can be reached at 765-9168 You can contact the DNA Sequencing Core Lab at 764-8067, but remember, technicians are kept very busy processing samples, most issues can best be handled by contacting the office or the Core Director
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Back to Sequencing Core Home Page Please address any comment, questions, or suggestions to Robert H. Lyons(boblyons@umich.edu)