Sanger Sequencing Service
The Sanger DNA Sequencing service uses an Applied Biosystems 3130xl Genetic Analyser with a 50cm array and POP-7 polymer installed.
To assess the performance of the electrophoretic analysis, a pGEM sequencing control is included with every plate of samples. Analysis of our pGEM control routinely produces high quality data with read lengths over 850bp (QV20 bases).
There are three service options available. Two post-cycle sequencing reaction services, ‘Capillary separation’ and 'Clean-up & capillary separation', where the customer performs the BDT reaction. Alternatively, there is a ‘Full Sequencing’ service option for customers preferring to supply the template and primer mix and have the GUDSF perform the BDT reaction.
- Full sequencing service
Please submit your single primer and template mix and the GUDSF will perform BDT v3.1 reaction, clean-up, capillary separation & sequencing analysis. Please read the Requirements Guide prior to submission. Optional PCR purification service is also available.
- Clean-up & capillary separation
Please submit your BDT reaction and the GUDSF will perform the clean-up, capillary separation & sequencing analysis.
- Capillary separation
Please perform the BDT reaction and clean-up before submitting your purified BDT reaction products for capillary separation & sequencing analysis.
The GUDSF accepts samples sequenced with Applied Biosystems:
- BigDye Terminator v3.1 Ready Reaction Cycle Sequencing kit
- BigDye Terminator v1.1 Ready Reaction Cycle Sequencing kit.
Sequencing protocols and guides
For customer support and troubleshooting advice please contact GUDSF staff.
Troubleshooting information may also be found in the DNA Sequencing Chemistry Guide under Sequencing Protocols & Guides.
The GUDSF maintains an archive of customer data, however customers are advised to back-up their data. If you require a copy of a past result please contact the GUDSF.
The 3130xl uses Applied Biosystems DNA Sequencing Analysis Software v 5.2 with KB Basecaller. The KB basecaller assigns quality values to each basecall (pure and mixed bases). The QV is calculated using the following equation: QV = -10log10(PE) where PE is the probability that a basecall is erroneous.
QV's range from 1 to 50+. The calculated QV for each base may be visualised on the analysed data as coloured QV bars above each basecall. These QV bars are only visible using the DNA Sequencing Analysis Software or Applied Biosystems SequenceScanner program (see Viewers for sequencing data files above).
A customer may request a sample re-run. Where there is no improvement in the result, a fee may apply. Samples that produce off-scale data will automatically be diluted and re-run at the customer’s expense. Off-scale sequencing samples produce ‘pull-up’ peaks, which affect the quality and reliability of the result.
Occasionally, a sample may exhibit early loss of resolution or other capillary related issue. In these instances, the sample is re-run without charge.