1. Sanger Sequencing
Successful Sanger sequencing is achieved by good template-primer complementary and template-primer ratio. The table below shows the recommended quantity of template to use in a sequencing reaction base on the size of the targeted template . The AB3730 DNA Analyzer is a highly sensitive instrument, consequently you may not need as much template as indicated in the table. Input amounts may have to be optimised as over-saturation of signal will cause a reduction in data quality.
As a starting point we suggest that clients read our Sanger Sequencing Guide.
|PCR - 100 - 200bp
200 - 500bp
500 - 1000bp
1000 - 2000bp
|1 - 3 ng
3 - 10ng
5 - 20ng
10 - 40ng
40 - 100ng
|Single-Stranded DNA||50 - 100ng*|
|Double-Stranded DNA (Plasmid DNA)||200 - 500ng*|
|Large DNA (e.g. BACs,PACs,YACs,cosmids and fosmids)||0.5 - 1.0ug|
|Bacterial genomic DNA||2 - 3ug|
Table referenced from DNA Sequencing by Capillary Electrophoresis by Applied Biosystems.
2. Fragment Analysis
The most common issue with fragment analysis is that the size standard does not run properly e.g.no size data or under/over-saturation of the fluorescence signals. Good sample preparation for fragment analysis requires a fine balance between the quantity of the sample and the size standard, and if you are multiplexing, a fine balance between all the fluorescence labels and the size standard. Proper optimisation will ensure that you have clean peaks for subsequent analysis.
To obtain high quality data it is essential that you have a robust PCR reaction that amplifies the target consistently across your samples. PCR products should be concentrated and require considerable dilution before sample submission (e.g. 1 in 100). The dilution is intended to avoid over-saturation of signals and to dilute out common contaminants.
Excess reagents and salt can interfere with the electrokinetic injection of the machine and lower the quality of the data. Other contaminants, such as residual protein or detergents carried over from DNA extraction can adhere to the capillaries of the array thereby adversely reducing the quality of the data. If a considerable dilution of the sample is not possible, we require our customers to perform a cleanup step (e.g. ethanol precipitation) prior to submission.
Very strong signals are difficult to analyse and have a similar negative impact to contaminants. We suggest that you perform a serial dilution (e.g. 1:20 to 1:150 ) on a subset of samples to optimise input. If the size range and fluorescence label do not overlap, multiple targets with the same fluorescence label may be pooled or a multiplex PCR can be performed.
3. Sequencing Guides
Below are links to resources for general information on Sanger sequencing and troubleshooting your data:
4. Sequencing Clinic
The Centre runs a sequencing clinic whereby clients can book one on one time with a Sanger sequencing expert. Please contact us for further information or to make an appointment.
5. Software for Viewing and Editing Chromatograms
We advise our customers to check the chromatograms manually to ensure correct base calling and genotype calling. There are a large number of programs available, each having its own specific advantage.
- Applied Biosystems Sequencing Analysis 5.1.1 for Windows XP and SeqScape (Sequencing Analysis and alignment) from Applied Biosystems.
- FinchTV software for viewing and editing chromatograms (Macintosh OS X, Windows, Linux). Free access.
- Geneious is a comprehensive suite of tools for analysis a range of sequencing data. Trial and student discounts available.
- Genemapper 5.0 for Windows 7 is available for Microsatellite, AFLP and RFLP analysis from Applied Biosystems.
- Sequence Scanner (Sequencing Analysis) and Peak Scanner (Fragment Analysis) Applied Biosystems for PC. Free access.
The Centre does not provide software for analysis of sequencing data but can provide an analysis service if required (conditions and fees apply). Contact us for further information.