Science

Sanger sequencing

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illustration of a method of dna sequencing

The Ramaciotti Centre for Genomics is committed to providing a quality Sanger DNA sequencing service with a fast turnaround time. Our services are flexible and can be customised to meet your needs. All sequencing performed by the Ramaciotti Centre for Genomics is carried out in our ISO/IEC 17025 accredited laboratories.

If the Sanger sequencing service you require is not listed on the service menu on the right or you need assistance with project design please contact us.

If you are a returning customer please login to our Sanger Sequencing Portal to submit your order. 

  • Our Core Prep services are an easy option for researchers who would like to fast track their project or for those who are new to Sanger sequencing.

    Core prep

    You submit a mix of purified PCR product and primer, we perform the sequencing reaction and capillary separation.
    Turnaround time: 3-5 days.*

    Core prep with clean up

    You submit unpurified PCR product and primer, we perform the PCR clean up, sequencing reaction and capillary separation. Clean up options include ExoSAp-IT and magnetic bead clean up (plate submissions only).  
    Turnaround time: 3-5 days.*

    Returning customers

    Please log in to our Sanger Sequencing Portal to submit your samples.

    New customers

    Please download our user guides for detailed instructions on how to use our Sanger sequencing services:

    Data delivery

    You will receive an email notification once processing is complete, you will then be able to access your data via your dnaLIMS account.  The following files are available to download:

    • Raw trace (.ab1 file)
    • Raw, unfiltered basecall sequence (.seq file)
    • Text file of you sequence
    • FASTA Sequence
    • System QC score of the sequence

    Quality guarantee

    • We include internal controls (pGem) on all sequencing runs to ensure quality data.
    • All traces are manually inspected before data is released.
    • Free re-run service if results do not meet our quality standards.

     

    *  Working days are Monday – Friday (excluding Public Holidays). Samples must be received by 12 noon to be included in the day's sequencing run.

  • Our User Prep services are useful for researchers who are hands-on and have access to the required sequencing reagents.

    User prep 

    You submit clean, pelleted sequencing product, we perform the capillary electrophoresis. Turnaround time: 1-3 days.*

    User prep plus

    You submit the product of the sequencing reaction, we perform the clean up and the capillary electrophoresis. This service is only available for plate submissions. 
    Turnaround time: 1-3 days.*

    Returning customers

    Please log in to our Sanger Sequencing Portal to submit your samples.

    New customers

    Please download our user guides for detailed instructions on how to use our Sanger sequencing services:

    Data delivery

    You will receive an email notification once processing is complete, you will then be able to access your data via your dnaLIMS account.  The following files are available to download:

    • Raw trace (.ab1 file)
    • Raw, unfiltered basecall sequence (.seq file)
    • Text file of you sequence
    • FASTA Sequence
    • System QC score of the sequence

    Quality guarantee

    • We include internal controls (pGem) on all sequencing runs to ensure quality data.
    • All traces are manually inspected before data is released.
    • Free re-run service if results do not meet our quality standards.

    *  Working days are Monday – Friday (excluding Public Holidays). Samples must be received by noon to be included in the day's sequencing run.

  • Fragment analysis measures the size of DNA fragments by comparing the unknown fragment to DNA fragments with known lengths (size standards). Size standard are combined with the sample and co-injected for capillary electrophoresis. Common applications include: DNA fingerprinting, parentage analysis, microsatellites analysis, AFLP and ARISA. 

    For this service you submit 1-3ul of cleaned/diluted product and we perform the capillary electrophoresis with your choice of LIZ size standard. Plate submissions only.
    Turnaround time: 1-2 days.*

    Available size standards

    • LIZ500
    • LIZ600
    • LIZ1200

    Returning customers

    Please log in to our Sanger Sequencing Portal to submit your samples. 

    New customers

    Please download our user guides for detailed instructions on how to use our Sanger sequencing services:

    Data delivery

    You will receive an email notification once processing is complete, you will then be able to access your data via your dnaLIMS account.

    Quality guarantee

    Free re-run service if results do not meet our quality standards.
     


    *  Working days are Monday – Friday (excluding Public Holidays). Samples must be received by 12 noon to be included in the day's processing list.

  • 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. 

    Template Recommended quantity
    PCR - 100 - 200bp
    200 - 500bp
    500 - 1000bp
    1000 - 2000bp
    >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:

    Ramaciotti Centre Sanger Sequencing Guide

    DNA Sequencing by Capillary Electrophoresis by Applied Biosystems

    Qiagen Guide to Template Purification and DNA Sequencing

    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 of a range of sequencing data. Trial and student discounts are 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.
  • Service type Submission format Sample number Price/sample (ex GST)
    Core Prep

    Tube

    Plate

    Any

    40 - 96

    $10.00

    $8.00
    Core Prep & ExoSAP-IT

    Tube

    Plate

    Any

    40 - 96

    $12.00

    $10.00
    Core Prep & Bead Clean Up Plate 40 - 96 $12.00
    User Prep

    Tube

    Plate

    Any

    40 - 96

    $5.60

    $3.60
    User Prep Plus Plate 40 - 96 $6.60
    Fragment Analysis Plate 40 - 96 $4.50

    Included in our services:

    • Routine quality checking of all traces.
    • Easy access to your sequencing data.
    • Technical support.
    • Troubleshooting advice.

  • Software for viewing and editing chromatograms

    There are a number of analysis program options available:

    • Applied Biosystems Sequencing Analysis 5.1.1 for Windows XP and SeqScape (Sequencing Analysis and alignment) from Applied Biosystems.
    • Genemapper 3.7 for Windows XP is available for Microsatellite, AFLP and RFLP analysis from Applied Biosystems.

    The Centre does not provide software for analysis of sequencing data but can provide an analysis service if required (conditions and fees apply). Please contact us for further information.

    Other analysis programs (including freeware)

    • Sequence Scanner (Sequencing Analysis) and Peak Scanner (Fragment Analysis) Applied Biosystems for PC.
    • Free EditView software from Applied Biosystems for viewing and editing chromatograms (Macintosh OS 9.x, 1.5 Mbytes).
    • Free FinchTV software for viewing and editing chromatograms (Macintosh OS X, Windows, Linux).
    • Free 4Peaks software for viewing and editing chromatograms (Macintosh OS X).
    • BioEdit – Biological sequence alignment editor for Windows 95/98/NT.
    • Genedoc – a multiple sequence alignment editor, analyser, and shading utility for Windows.
    • STRand – http://www.vgl.ucdavis.edu/STRand (Fragment Analysis) for PC.

    Other helpful links and guides

    • Genbank – sequence submission support and software.
    • BLAST – a set of similarity search programs designed to explore all of the available sequence databases regardless of whether the query is protein or DNA.
    • Entrez – an integrated search and retrieval system that takes information from all of the databases at NCBI.
    • Genographer – The MSU Sequencer Users’ Group, EPSCoR Plant Biotechnology Group, Barley Genetics, Genographer and Barley Production Homepages.
    • The Association of Biomolecular Resource Facilities