ABI Sequencing Service technical information

The Massey Genome Service supplies the following primers for customers using the Full Sequencing Service:

• M13 forward
• M13 reverse
• T7 Promoter/Forward
• SP6 Promoter

Customers can request to use these primers by ticking the appropriate box or boxes on the online sequencing request form in the section called MGS primers and by indicating in the column called Primer Name which primer to use with each template.

Massey Genome Service primer sequences

The Massey Genome Service uses the following sequencing chemistries for the Full Sequencing Service:

• BigDyeTM Terminator Version 3.1 chemistry
• dGTP BigDyeTM Terminator Version 3.0 chemistry (available upon request, please email MGS before request this for Full Sequencing Service)

Although all of the chemistries are relatively versatile, some are better than others for specific types of templates.

The Massey Genome Service uses the BigDye™Terminator Version 3.1 chemistry for most sequencing unless the customer specifies the use of one of the other two chemistries.

Please indicate which type of sequencing chemistry you want the Massey Genome Service to use if requesting the Full Sequencing Service, by ticking the appropriate box on your online request form.

BigDye® Terminator V3.1 Chemistry

This chemistry is designed for the majority of applications, and produces data with uniform peak heights and optimized signal balance to produce long reads. Improved peak patterns also contribute to more accurate base assignments for heterozygote and mutation detection.

dGTP BigDye® Terminator V3.0 Chemistry

This chemistry is designed to sequence through GT and GA repeats, and G homopolymers that the BigDye® Terminator V 3.1 chemistry has not been able to get through. But the BigDye® Terminator V 3.1 chemistry will generally sequence through most repeats.

The Massey Genome Service requires customers who are using the Full Sequencing Service to send the template and primers premixed in 0.2ml individual PCR tubes or 0.2ml strip tubes. F

For the Sequencing Capillary Separation Service and Sequencing Capillary Separation Service with reaction cleanup please send the sequencing reactions in 0.2ml individual PCR tubes or 0.2ml strip tubes and follow the guideline below for setting up your sequencing reactions.

For the Sequencing Plate Service and Sequencing Plate Service with reaction cleanup, please send the sequencing reactions in a 96 well plate, sealed with strip tubes, plastic seal or foil seal. The strip tubes provide the best result.

Example of requirements

You have a 500bp PCR product at a concentration of 6.25 ng/µL and your primer is at a concentration of 2 pmol/µL.

For PCR products use 2.5ng of template for every 100bp. So for a 500bp product you will need to add 12.5ng to the template/primer premix. At a concentration of 6.25 ng/µL you will need to add 2 µL template to the premix.

For the primer you need 4pmol total amount in the template/primer premix. So at a concentration of 2 pmol/µL you will need to add 2 µL primer to the premix. You then need to make the premix up to a final volume of 20 µL. So you will need to add 16 µL of filtered molecular grade water to the premix to get the final volume of 20 µL.

DNA quality

Poor template quality is the most common cause of sequencing problems. The following are characteristics of poor quality templates:

• noisy data or peaks under peaks
• no usable sequencing data
• weak signal

The quality of DNA in a reaction can affect the performance of the ABI3730 DNA Analyzer. Potential contaminants include:

• proteins
• RNA
• chromosomal DNA
• excess PCR primers, dNTPs, enzyme, & buffer components (from a PCR amplification used to generate the sequencing template)
• residual salts
• residual organic chemicals, e.g., phenol, chloroform, & ethanol
• residual detergents.

The presence of residual salts, proteins, RNA and detergents can interfere with capillary electrophoresis and electrokinetic injection.

Capillary electrophoresis is especially susceptible to salt in samples, from:

• template preparation
• cycle sequencing reactions
• precipitation methods using salts.

The negative ions in salts can be preferentially injected into the capillary array during electrokinetic injection, leading to low signal. The negative ions also compete and interfere with the injection of lager DNA extension fragments, leading to shortened read lengths.

Please QC all DNA templates; PCR products/plasmids, cosmids, BACs or bacterial genomic DNA, by spectrophotometry or fluorometry. The OD260/280 for DNA should be 1.8 to – 2.0.

High GC content and/or secondary structures

If you think any of your samples have either a high GC content and/or secondary structures, please request in the Customer Notes section on your online request form, to have 5% DMSO added to your samples.

Difficult to sequence repeat and homopolymer regions

If you suspect your templates have difficult to sequence repeat and homopolymer regions, please select in the Reaction Type section on your online request form, the dGTP BigDye Terminator v 3.0 chemistry.

Note that if the repeat or homopolymer regions in your templates are long and complex, the dGTP BigDye Terminator v 3.0 chemistry still may have difficulty sequencing through this difficult region.

Cosmids, BACs or bacterial genomic DNA

If you have either cosmids, BACs or bacterial genomic DNA, please select in the Procedures section on your online request form, full sequencing service for BAC templates or bacterial genomic DNA. This is required to run your samples using the correct protocol.

The Capillary Separation service is a more cost effective option for sequencing. If you would like more information please don't hesitate to contact us.

Agarose gel electrophoresis and Spectrophotometry can be used to examine DNA quality.

Agarose gels reveal the presence of contaminating DNAs & RNAs, but not proteins. Spectrophotometry can indicate the presence of protein contamination, but not DNA & RNA contamination. These two methods should be used together to get the most information about your DNA template before sequencing.

Agarose gel electrophoresis

Purified DNA should run as a single band on a 1-2% agarose gel. Uncut plasmid DNA can run as three bands: supercoiled, nicked, and linear.

Spectrophotometry

The A260/A280 ratio should be 1.8-2.0. Smaller ratios usually indicate contamination by protein or organic chemicals, and larger ratios contamination from RNA. Example: Nanodrop.

Detecting contaminating salts

Neither of these methods shows the presence of contaminating salts that can cause noisy data. If you suspect that your DNA is contaminated with salt, remove the salt before sequencing. The most efficient method for salt removal is ultrafiltration with a Centricon-100 column. Spin columns & ethanol precipitation can also be used.

The following recommendations are provided to help optimize primer selection.

• Avoid >2 GC’s in last 5 nucleotides at 3’ end of primer.
• Primers should be at least 18 bases long to ensure good hybridization.
• Avoid runs of an identical nucleotide, especially runs of four or more Gs.
• Keep the G-C content in the range 30-80%, preferably 50-55%.
  For cycle sequencing, primers with TM>45°C produce better results than primers with lower TM.
• For primers with G-C content less than 50%, it may be necessary to extend the primer sequence beyond 18 bases to keep the TM>45°C.
• Use of primers longer than 18 bases also minimizes the chances of having a secondary hybridization site on the target DNA.
• Avoid primers that hybridize to form dimers.
• Avoid palindromes because they can form secondary structures.
• The primer should be as pure as possible, preferably purified by HPLC.
• To ensure specificity of primer (BLAST search) to the target.
• Dissolve primer stocks in 10mM TE buffer, pH8.0. But dilute working primer solutions in water because salt can affect primer extension.

Download our Sequencing Technical Bulletin which contains information on:

• quality values, mixed bases and IUB codes
• information included in the Analysis Report
• primer design recommendations
• sequencing reaction recommendations.

Download the QualTrace™ Bulletin which contains:

• background information on the QualTrace™ Software and what the software does
• what information is contained in the QualTrace™ Report, which is downloaded with each sequencing request
• solutions to failed and noisy sequencing data and the identification of other sequencing data problems.

All ABI sequencing data is uploaded to our server for customers to download. For each sequencing request you will receive an .ab1 file and a .txt sequence file for each sample, and also an ‘Analysis Report’ file, if you ticked to receive this on your online sequencing submission.

To open the .ab1 files, you need specific software. There are two free download software packages available which allows you to open, view and edit the ABI sequencing .ab1 files:

• Chromas
• Sequence Scanner v1.0

Download Chromas
Download Sequence Scanner v1.0