Sequencing

How do I begin using the University of Arizona Genetics Core sequencing facility?

  • University of Arizona KFS accounts may be used to pay for services rendered. The account must be current, and contain enough funds to cover the charges.
  • Off-campus users must contact UAGC for a quote before using the UAGC services.
  • The next step is to register your lab group with our Sample Lifecycle Manager (SLM) system.
  • Once your profile has been created, you can begin submitting sample information. Please be sure to complete the online SLM order before bringing the templates to the lab in the Keating Building drop off location or Life Sciences South Room 205.

Where do I take my DNA to be sequenced?

Our facility is located in the Thomas W. Keating BioResearch Building Room 124 (map). Label your submission with the SLM Id, especially the last 4 characters. Please leave your submission in the freezer outside the door. Sequencing staff members will check in your submission on line.

Users at the south side of campus may drop off their templates in the refrigerator in Life Sciences South Room 205. There is a daily pickup from that location.

Remote users can mail their samples to the following address: (Note: If you are sending plates please see our 96-well plate and cap guidelines below)

UAGC - DNA Sequencing
Keating Building
1657 E Helen St. #124
Tucson, AZ 85721-0240

Recommended guidelines for shipping samples to UAGC

Tubes- Snap cap tubes may be secured with parafilm and/or organized in a box to prevent opening during transit. If you are using our DNA extraction or cell line service you can send samples in screw cap tubes with an O-ring to prevent cross contamination.

Plates- Plates sealed with strip caps are preferred since they are most stable during transit. We recommend the following part numbers or similar- USA Scientific plates PN:1402-9600 and USA Scientific Strip caps PN:1400-0800. Temperature and pressure variation can affect adhesive seals and samples may arrive contaminated or displaced.

How do I prepare my templates for sequencing on the low volume service?

Submitting DNA Templates:

Submit all samples in 1.5ml tubes. Write name of template exactly as it appears on the web site work order. On the side of the tube, write your name and the tracking number from the web site. Please note in the comments section of your work order if you anticipate difficult template (e.g. GC rich). If the template is to be used for more than one sequencing reaction, submit only one tube with enough DNA for all of the reactions.

Required Volumes for Submission:
Template- 8 µL/reaction
Primer- 5 µL/reaction

All low volume submissions should be given in 1.5mL eppi tubes. Sample and primer must be submitted in separate tubes for low volume sequencing. 

All high volume plates should be submitted in a v-bottom plate. 

Sample Preparation for PCR products:

The most important factors are concentration and purity . We strongly recommend that all PCR products be prepared with commercially available PCR cleanup kits to remove excess primers, nucleotides, and buffers. The most commonly used PCR purification kit among our users is Qiagen's QIAquick PCR system.

Important! Do not use buffers containing EDTA (such as T.E.) to resuspend your templates. This will cause your sequencing reaction to fail. We recommend submitting samples in sterile ultrapure (MilliQ) water.

What are the ideal concentrations and quantities for templates and primers?

PCR: Optimum concentration depends on length . Use the formula: Length (Base pairs) x 0.02 = Concentration (in ng/µL )

* Templates at optimal concentration use a standard 2.5ul of template per reaction. If template concentration is lower than ideal concentrations, we can use up to 8ul of template. When you submit your sample online, please enter correct concentrations. Our computer will then calculate how much template will be optimal in your reaction.

Primers: 3-5 pmol/µL (3-5 µM)
Note: for a 20-mer primer this is 20ng/µl

How do I prepare my samples for sequencing on the high volume service?

  • Templates are brought to the facility in 96-well format.
  • If using more than one primer on the plate, please provide a plate with primers in the wells corresponding to the proper template locations.
  • DNA must be of high purity, and consistent concentration for best results. The 3730xl sequencer is very sensitive to salts. Users wishing to use the facility's automated sample cleanup, quantification, and normalization services should select these options in SLM at the time of submission.
  • Optimum concentration for PCR products is 10ng/µl & for plasmids 50ng/µl. Please supply at least 8ul of template for each reaction to be performed.
  • Optimum primer concentration 3 pmoles/µl. Please supply at least 4ul of primer for each reaction to be performed.

What instrument do you use to analyze the sequencing products?

DNA sequencing is run on an Applied Biosystems 3730XL DNA Analyzer. The user can expect up to 600 bases of reliable sequence per read in one direction.

How long will it take to get my results?

Average turn around time on the low volume sequencing service is 3 business days. For the high volume service, expect results in 2 to 3 days.

How do I view my sequences?

The files that our automated sequencers generate require specialized software in order to be viewed. As you might expect, these viewers are platform specific. Please see the notes at the bottom of this page on how to configure your computer's file associations so that the files will open when you click on them.

There are several free viewers that are available to look at the electropherograms. Below are a few of the options. These programs are for viewing individual sequence files. If you need assistance with large scale sequencing data management, please feel free to contact us for assistance or recommendations.

Chromas is freely available over the web, and offers many options for working with .AB1 files. It is an excellent trace viewer for the PC platform (all windows OS™s are supported). Follow the link above to download.

A free viewer from ABI, this program works on Windows XP and 2000 platforms. It allows you to view and edit traces. You can get it directly from Applied Biosystems by clicking on the above link.

Finch TV is a great trace viewer with versions available for PC or Mac users. Highly recommended. Follow the link above to download.

Alternately, a text file containing only the sequence of nucleotides can be downloaded from our server. The text file can then be imported into your favorite sequence analysis package for analysis or alignment. Be aware that the UAGC does not trim poor quality sequence from the end of your sequencing data. It is always best to check the quality of your sequence in a program that allows you to evaluate the accuracy of the information.

Configuring your computer to recognize your sequence files:

  • Windows: download the first chromatogram file, and then double-click it. A window will pop up telling you it doesn't know how to open the file. It will offer to let you browse for the correct application. Do so - choose the chromat viewer you downloaded above. Windows should thenceforth remember what program to use. You can continue to download the rest of your files.
  • Mac OS X: download the first chromatogram file, then single-click it and type command-I ('Get Info'). In the subsequent window, click the arrow next to 'Open With:', and in the pop-up menu button underneath, browse for the chromatogram viewer application you downloaded (above). You may wish to click the 'Change All' button, to indicate to your computer that it should always use that application for 'ab1' files.
  • Mac OS 9 (and earlier): Macs have hidden 'File Type' and 'Creator' codes. These codes are embedded invisibly in the file structure, and like the Windows 3-character extension, can identify the appropriate program to use for opening a file. Setting these codes can be a bit difficult: There are many ways to set the File Type/Creator codes (see below).

If you plan to use a web browser (Netscape, Internet Explorer) to download chromatograms, you can configure the browser to set File Type and creator codes:

Netscape: Pull 'Edit' to 'Preferences', select 'Applications' (probably under 'Navigator'). Click 'New...' and fill in the blanks with:

  • Description: ABI Chromatogram
  • Mime Type: application/abi1
  • Suffixes: ab1

Click the 'Application' radio button, then the 'Choose' button. Find your chromatogram viewing program (downloaded as above) and double-click on it. You may also need to select a 'File Type' under 'File Information'. Choose 'ABI1'. Close all windows and Netscape should now be able to automatically open your chromatogram-reading program when you download these files.

Internet Explorer: Pull 'Edit' to 'Preferences', click on 'File Helpers' (under 'Receiving Files'). Click 'Add'. Fill in the blanks as follows:

  • Description: ABI Chromatogram Extension: .ab1
  • Mime Type: application/abi1
  • Application: click the 'Browse' button, find your chromatogram-viewing program and select it.
  • File Type: ABI1 (it may already be filled in correctly from the previous step).
  • File Creator: (this is filled in correctly from the 'Application' step).
  • Binary Data: click this radio button.
  • Use for incoming: click this checkbox.
  • Optional: you can also set the Download Destination, if you wish.
  • Optional: set 'How to Handle' to either 'Save to Disk' or 'Post Process With Application..'.

Close all windows and Internet Explorer should now be able to automatically open your chromatogram-reading program when you download *new* files. It doesn't help you with files you may have already downloaded. If you plan to use an FTP program (or the files are already on your computer), you can use third-party file typers like FileTyper, TypeShuffler, The Associator, DropAttribute or similar. With such a program, set the file type to 'ABI1' and (if you use EditView) set the creator to 'EdVw'.

My sequencing result was not what I expected. How can I tell what went wrong?

The electropherogram is a graphical representation of the fluorescent dye intensity over time. The electropherogram can be used to provide the user with valuable information regarding the quality of their template. Please note, however, when the template does not generate readable results an electropherogram still must be sent for billing purposes. In this case, we truncate the electropherogram to about 30 bases.

Following is a guide developed to make analyzing your electropherograms a little simpler.

Noisy Sequence-

If your template is "noisy"- the electropherogram will show multiple peaks underneath and on top of each other. This is commonly found with PCR products amplified directly from genomic DNA. If several products are amplified from the template, and a single product is not isolated using either gel extraction or another technique, the primers will sequence multiple products. This is the case when you have a normal signal strength, yet there are several peaks in a single location.

  • Is it possible that your primer has more than one binding site and you are getting multiple sequences atop one another?
  • Is your template contaminated by another fragment that generated peaks underneath the desired sequence?

Not Enough DNA Template-

If your sequence has a "weak signal strength"- peaks are low, the signal intensity <100 RFUs for each base, and low signal-tonoise-ratio- your template concentration may be too low for the reaction. The concentrations listed above are for "ideal" templates. Difficult templates may need more DNA to get through certain regions, e.g. long inserts, secondary structures, homopolymers and microsatellites. An increase in template concentration (10X) can often result in a cleaner sequence. Other possibilites include:

  • Primer concentration is too low
  • Mismatch in the primer sequence-A single mismatch can cause the signal to decrease by ten-fold

Homopolymer-

 

 

 


Your template generated a sequence with a "homopolymer region". A homopolymer is a region of repeated bases of varying length.

Often, if you get an email mentioning a homopolymer, this means that the enzyme slipped at the homopolymer and the sequence following is noisy and can cause inaccurate base calling by the computer.

Microsatellite-

A microsatellite region can cause the same problem. There are not too many tricks to get through these regions. The best first step is to run the sample with a primer in the reverse direction so you can obtain sequence on the other side of the region.

Signal Dies Off-

 

 

 

 

Your template generated a sequence that "dies off prematurely"- the sequence will look fine and then either end rapidly before the end of your fragment or slowly towards the end of the fragment.

When the template dies off; there are a few things to think about.

Is it possible that the enzyme is encountering a secondary structure that it cannot get through?
Could there be a contaminant left over from the prep of the sample or the sample itself?

G-C Rich Sequence-

Is the template G-C rich? There are a few things we can do if this is the case. We can try cycling with a higher temperature to help denature the template. We can run the template with 5% DMSO in order to break the GC bonds. Adding DMSO interferes slightly with the reaction and is not recommended for low concentration templates. Please let us know if you know your template is G-C rich.

Clean Vector, Noisy Insert-

Your template appears to have a "noisy insert with a clean vector"- the electropherogram will be good up until about 70-80 bp and then the template appears noisy.

This problem is usually the product of multiple inserts (PCR of clones from colonies- another colony near by).

When a subcloning procedure allows the insert to be oriented in either direction in the vector, positive colonies can have different sequences. If two positive colonies grow extremely close together and have different orientations of insert, the electropherogram will have clean peaks until the site of insertion where sequences get mixed.

No Sequence-

Worst problem of all.your template generated "no readable sequence". This could be caused by a number of different problems.

Some of the more common causes are:

  • Not enough template DNA to generate visible results.
  • Not enough primer to generate visible results.
  • No primer site on the template DNA corresponding to the oligo in the reaction.
  • High enough concentration of contaminant to completely cease enzyme activity


I need my DNA template back. Do you still have it?

  • UAGC will archive DNA templates for 2 months.
  • If you can provide the date when the template was run, and the name of the template, we will be able to return it to you (or use it in another sequencing reaction).
  • After 2 months we will not be able to access your sample.

If you do not find answers to your specific sequencing questions here, please do not hesitate to contact us directly at 520-621-9184 or email uagc@email.arizona.edu.