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Difficult Template Sequencing

Difficult templates are often challenging to sequence, due to secondary structures and other inherent factors. While the standard DNA sequencing protocol may perform satisfactorily for borderline cases, templates within the specifications noted below typically perform best with GENEWIZ's optimized proprietary protocols.

To save time and cost we recommend that you select an "alternative protocol" by choosing either difficult template, GC-rich, Hairpin or RNAi at the Special Request pull-down menu during the online ordering process.

Template examples that may benefit from the alternative protocols include:


  • GC rich: a template of > 60% GC content or high GC-content concentrated in a small region;
  • Hairpin structures: sequence containing two inverted repeats, separated by at least three nucleotides;
  • Various repeats: certain di- or tri-nucleotide repeats;
  • Motifs causing band compressions: mainly 5'-YGN1-2AR motifs, (Y: pyrimidine; R: purine; N: any base)

  • Features
  • Case Studies
  • Advanced Technology: GENEWIZ provides our difficult template sequencing service (see above) using modified protocols, developed in-house, specifically for success with difficult templates. Years of use have confirmed that these alternative protocols deliver best-in-class results.


  • Individual Consultation: GENEWIZ provides individualized scientific consultation and customized solutions for your specific DNA sequencing needs, through our Technical Support team.


  • Award-Winning Technical Support: Our Technical Support team, staffed by multiple Ph.D.'s with a friendly, problem-solving spirit, is available to advise and assist you at no charge Monday - Friday, 8AM - 8 PM Eastern. GENEWIZ Technical Support also posts results on Saturdays, so you can work through the weekend.

Case 1: Small Hairpin RNA (shRNA) Template Sequencing

The small hairpin RNA templates used in RNAi studies frequently present challenges in sequencing such as early termination or ambiguity, caused by the double-stranded or hairpin structures. Our proprietary RNAi Protocol produces superior results for our customers, saving time and cost. For a comparison, see the chromatogram below:

Case 2: Repeat / GC rich Template Sequencing

Repeat/GC-rich templates have special secondary structures and melting temperatures. DNA containing high GC content and hairpin structures are relatively more difficult to denature and very challenging to sequence.

Our proprietary Hairpin and GC-rich protocols produce superior results, with strong signals and uniform peak heights at the GC region.

How To Order

Create an account and place orders for difficult template sequencing through our online system. For best results, prepare your samples according to the sample submission guidelines. When placing your sequencing order for difficult templates, select one of our proprietary protocols listed in the "Special Request" column. Additional charges apply.

Visit Getting Started for more details. If you have any questions, feel free to contact GENEWIZ Technical Support by phone at 1-877-436-3949 option 2, or by email at dnaseq@genewiz.com.