ABI Sequencing Service Technical Information

 

 

 

Massey Genome Service Supplied Primers

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.

The Massey Genome Service primer sequences are:

M13 forward:

5' CCC AGT CAC GAC GTT GTA AAA CG 3'

M13 reverse:

5' AGC GGA TAA CAA TTT CAC ACA GG 3'

T7 (Promoter/forward):

5' TAA TAC GAC TCA CTA TAG GG 3'

SP6 (Promoter):

5' ATT TAG GTG ACA CTA TAG 3'

Sequencing Chemistries

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

  • BigDyeTM Terminator Version 3.1 chemistry
  • BigDyeTM Terminator Version 1.1 chemistry
  • dGTP BigDyeTM Terminator Version 3.0 chemistry

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.

 

Below is a table showing the sequencing kits the Massey Genome Service uses, and the types of sequencing applications work best with each chemistry:

Applications

BigDye® TerminatorV3.1 Chemistry

BigDye® TerminatorV1.1 Chemistry

dGTP BigDye® TerminatorV3.0 Chemistry

De novo sequencing

R

S

R

Re-sequencing

R

S

R

Sequencing difficult Templates

R

R

R

Long-read sequencing

R

S

R

Sequencing across all template types (plasmids, PCR products, BACs, cosmids, and bacterial genomic DNA)

R

S

R

Mixed-base detection

R

S

S

Sequencing short PCR Products using rapid Electrophoresis run modules

S

R

S

R= Recommended S= Satisfactory

 

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.

BigDye® Terminator V1.1 Chemistry: This chemistry is designed for specialty applications that require optimal base-calling adjacent to the primer. This chemistry is good for sequencing short PCR products with rapid electrophoresis run modules.

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.

 

Template and Primer Concentration Requirements

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. 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.

Template Type

Template Total Quantity

(in final volume)

Primer Total Quantity

(in final volume)

Final volume required #

PCR product:
100-200bp
200-500bp
500-1000bp
1000-2000bp
>2000bp

Rule: For PCR products use 2.5ng of template for every 100bp.


1.25-5ng
5-12.5ng
12.5-25ng
25-50ng
50-125ng


4pmol

20ul if using your own primer

19ul if using MGS primer

Single-stranded Plasmid

62.5-125ng

4pmol

20ul if using your own primer

19ul if using MGS primer

Double-stranded Plasmid

250-625ng

4pmol

20ul if using your own primer

19ul if using MGS primer

Cosmid, BAC DNA, and Lambda DNA

0.625-1.25ug

6.4pmol

20ul if using your own primer

18ul if using MGS primer

Bacterial Genomic DNA

2.5-3.75ug

6.4pmol

20ul if using your own primer

18ul if using MGS primer

# Make the template/primer premix up to the final volume with filtered molecular grade water.

Example:

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 filter water to the premix to get the final volume of 20 µL.Example:# Make the template/primer premix up to the final volume with filtered molecular grade water.

 

Template and Primer Quality Requirements

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 electrokenetic injection. Capillary electrophoresis is especially susceptible to salt in samples, either from template preparation, from cycle sequencing reactions, or from precipitation methods using salts. The negative ions in salts can be preferentially injected into the capillary array during electrokenetic injection, leading to low signal. In addition, the negative ions compete and interfere with the injection of lager DNA extension fragments, leading to shortened read lengths.

 Methods for Determining DNA Quality

The following methods can be used to examine DNA quality:

  • 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.

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.

 

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.

 

 

Primer Design Recommendations

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.

Sequencing Technical Information

Download the attached Sequencing Technical Bulletin below, which contains information on the following:

  • Quality Values, Mixed Bases and IUB codes.
  • Information included in the Analysis Report.
  • Primer Design Recommendations.
  • Sequencing Reaction Recommendations.

MGS ABI Sequencing Service Technical Bulletin - January 2017.pdf (340 KB)

 QualTrace™ Report Bulletin

Download the attached ‘QualTrace™ Bulletin’ below, which contains information on the following:

  • 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.

MGS QualTrace Report Bulletin - January 2017.pdf (603 KB)

Massey Contact Centre Mon - Fri 8:30am to 5:00pm 0800 MASSEY (+64 6 350 5701) TXT 5222 contact@massey.ac.nz Web chat Staff Alumni News Māori @ Massey