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DNA Sequencing

Introduction

DNA sequencing is performed on the Applied Biosystems (ABI) Model 3730, a capillary based electrophoresis system that uses ABI fluorescent dye terminator chemistry (BigDye V3.1). For details on the features of the instrument and chemistry, refer to the ABI website. Our standard running conditions are capable of sequencing 48 samples every 3.0 hours, up to 384 samples (over 384Kb) in 24 hours. We take care of the sample set-up, cycling, cleaning and sequencing. You provide the template and primer already mixed together following the recommended concentrations. This way all samples are treated uniformly by our robotic sample set-up and processed with control samples. This helps us keep the cost down and the quality high using efficient sequencing methods.

Sequencing orders are placed through the Core Server. Instructions for using the server are available here and in the download section of this website. A copy of your order form with your sample(s) can be dropped off 24/7 in refrigerators located in the laboratory of 241 Remsen, or the Borwell loading dock (next to the Rubin elevator). Data from samples submitted Monday through Friday before 10:30 AM at the Borwell loading dock or 11:00 AM in 241 Remsen will be posted to your account by early morning the next working day. However we do usually post data from the first run of the day before we leave. So if your samples are submitted early enough to make the fist run you can receive you data the same day.

Data Distribution and Quality

When sequencing is completed for each run the data will be posted on our web site. Each sample will have two files associated with it. The first file is a .ab1 and will contain the electropherogram (sequence trace) for your sample(s) along with the run and analysis conditions. You will also receive a file with a .seq extension. This file is a text file with raw sequence called by the computer. This file can be viewed using Microsoft Word. If there is no .seq file for a sample that means it was unsuccessful. The ABI 3730 Genetic Analyzer is a Windows based platform therefore the data files are in Windows format. While the .seq file does tell you the sequence information it does not give you any information about quality of that sequence information. We highly recommend reviewing the electropherogram and the associated run data for each of your samples. There are free software programs that can be used for this.

We like FinchTV for easy viewing of electropherograms. The data files can be edited and you can access good Blast sites directly from the viewer. FinchTV is available for free from the Geospiza website and it can operate on both Mac and PC’s. There is free software for PC’s from ABI’s web site called Sequence Scanner. Sequence Scanner is also easy to use and is very good for evaluating the quality of your sequences. 4Peaks is Mac based software that is very good also. See “General Information” for the URL of these programs.

Look at the data file(s) and evaluate the quality of your results. Check your sample(s) signal strength (< 50), proper spacing between the bases and that the appropriate base caller was used. A Quality Value score (QV) is calculated for every base and ranges from 1 to 50. Reliable data should have a QV score of at least 20, which is equivalent to a 99.0% level of confidence for a correct call. QV scores below 20 should be reviewed on the electropherogram.

If you have sufficient non-problematic template, a good primer, and clean extension products, it is not unusual to get well over a 1000 bases or more of unambiguous data from our standard run. Once you develop some proficiency at editing the computer-called sequence, you can often get another 50 bases. We cannot emphasize strongly enough that the quality of you sequence data depends on the purity and quantity of your template and the quality of your primer.

If you are experiencing problems with your sequencing please contact us. We are here to help you and will often repeat your or re-run your samples at no charge.

Cost

The cost of running a DNA sample of sequence can very and depends on multiple factors. We do have discounts for larger numbers of samples (~40 or more) submitted in plates. Please contact us before you submit anything in a plate for details of how to set a plate up. A complete price list can be found on you MB Core account under the Core Rates tab.

Turnaround Time

Turnaround time is usually less than 24 hours for all standard reactions. Some of the data from our samples is turnaround on the same day we receive them. Turnaround time is dependent on the number of samples in the queue and the time of day the sample arrives at the laboratory. Generally, we start processing samples at 11:00 AM Monday through Friday. However, we will start a run anytime of the day if there are sufficient numbers of samples to run (47 samples + our control). Because of this we encourage you to bring your samples to the core when they are ready for processing. You never know when we will be starting an early or a late run during the day.

While Saturdays are not normal working hours for the MB Core we do try to make it a point to post the data from Friday’s runs at some point in the day. There will be a few Saturdays through out the year when we will not be able to post the data.

If you find yourself in a "true" crises situation and must get your data back as fast as possible please contact us and we will do are very best to accommodate you.