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Sanger Sequencing
System
The Leiden Genome Technology Center has an Applied Biosystems 96-capillary 3730XL system. It is equipped with a 50cm capillary array and uses POP7 polymer as a separation matrix.
Technology
The Sanger Sequencing reaction is performed with the Applied Biosystems BigDyeTerminator v3.1 kit. For the purification we use the Applied Biosystems BigDye XTerminator purification kit, also from Applied Biosystems.
See the Applied Biosystems website for more information on DNA Sequencing by Capillary Electrophoresis by Applied Biosystems.
Sanger Sequencing at the LGTC
First time users
When you have samples for Sanger sequencing and you do not know how to proceed you can make an appointment at the LGTC. We can help you discuss and evaluate the possibilities, costs, how to deliver your samples, and all other issues. For large projects or series of samples it may be wise to start with a few pilot samples to optimize the concentration of template and primer for the sequencing reaction.
Sample submission
To submit your samples, please proceed to the LGTC LIMS system.
Sequencing services
Although Applied Biosystems only guarantees sequence runs up to 500 base pairs on the ABI 3730XL DNA Analyzer, the LGTC Sequence Core has optimized the sequencing protocols to offer long-read sequencing reading up to 800 base pairs and even up to approximately 900 base pairs, provided that reaction circumstances are kept optimal.
At the moment we offer several sequencing services:
- Short run
Both plasmids and PCR products - run up to 600 base pairs.
- Long run
Both plasmids and PCR products - runs as far as possible, sometimes up to approx. 900 base pairs.
- Full 96-well plates
Only when you have 95 samples, a 96-well PCR-plate can be used to deliver your samples. A full plate can be delivered at any time. A 10% price reduction will be given compared to the price of 95 individual samples and will be calculated automatically after selecting the full-plate option in the LGTC LIMS system. Please note that if you decide to deliver strips of 95 individual samples, but choose the full-plate option in the LGTC LIMS system, we will not process your samples.
The samples must be delivered in a 96-well plate (any type of 96 well plate will do) with 15 µl of template and primer mix, see pipetting instructions. It is important to leave position A1 empty on the plate for an LGTC control sample that we use to check the conditions of the run.
- Full 96- and 384-well plates ready to run
We accept two plate formats: full 96-well and 384-well plates. These are full plates for which you already performed the sequencing reaction, so they are 'ready-to-run' in the ABI3730XL sequencer. This means that the ddNTPs (e.g. BigDye v3.1) have already been added, and that the sequencing reaction has already been done and purified. When you have one or more of these plates contact the LGTC before you plan to deliver your plate(s), so we can discuss the delivery details (time, plate type, sample volumes, etc...) with you.
Delivery of samples
Sequence samples can be delivered at any time. When you have less than 95 samples, these must be delivered in 0.2 ml tubes (8-well strips with strip caps). Tubes must be placed in a tray (which can be found on top of the LGTC freezer), and delivered in the small LGTC freezer in room R5-023. Make sure that all the wells are numbered on the side, that we know their correct order when we remove the caps. Label your samples with the submission ID given by LGTC LIMS on a piece of tape. Please put your Order report in the dedicated tray on top of the freezer. Without a completed Order report (including authorization signature) no sequencing reaction will be performed.
For 95 samples or more, samples must be delivered in a 96-well PCR plate. The same pipetting instructions and delivery in the freezer apply.
Pipetting instructions
For all sequencing reaction (individual sequences or full plates) the total volume should be 15 µl, see Sanger sequencing protocol for more information.
Cancellations
Once you have handed in your samples and Order report the requests cannot be canceled.
Availability of sequence data
Sequence results should be available for collection within 5 working days. However, we intend to deliver your results as soon as possible, often this will be within 2 to 3 working days from the moment of delivery. When your results are available for collection you will be notified by email. You can also check the availability of your sequences yourself by logging into the LGTC LIMS System. Always check the LGTC LIMS system before contacting the LGTC to ask if your sequences are available. When technical problems occur that might jeopardize the delivery of your sequence results within 5 working days, the estimated delay of the sequence service will be mentioned on our home page. If such a delay is problematic for you, please contact the LGTC to discuss alternatives. When after 5 days the Status is still "Submitted" or "Checked", please contact us.
The four displayed stages of the sequencing submission are:
- Submitted
Your samples are submitted and still in the freezer.
- Processing
Your samples are being processed.
- Pending
Delay due to technical issues, results are generally delayed for one or two days.
- Checked
Your sequences have been split over different sequencing days.
- Ready
You can download the zip file.
Data and data analysis
You will always receive ab1 files; seq files are only included when indicated in your order. We recommend you to check the raw data since sequences with raw data intensities below 500 are not guaranteed by Applied Biosystems or by the LGTC, and we cannot exclude capillary cross talk.
In all the cases: we will not keep your data for longer than three months, it is your responsibility to make your own back ups. Download your sequence data from the LIMS system within three months after completion; we need to remove older data to prevent the system from crashing due to disk space limitations.
To view or to analyze your sequences there are several options for software:
- To view the ab1 files
Sequence scanner (free to download from the Applied Biosystems website). Good software to view traces and raw data.
- Mutation detection
Mutation Surveyor (Softgenetics): the LGTC has one license, see software tools.
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