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Amplicon Sequencing: Request a Quote

Questions? Please contact our Next Generation Sequencing (NGS) Team at ngs.europe@genewiz.com, +49 (0)341 520 122-0.


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Frequently Asked Questions

1. What is contiguous amplicon sequencing?

Contiguous amplicon sequencing involves sequencing the in-tact amplicon without fragmentation.  NGS adapters are ligated directly to the ends of your amplicons.  Paired-end sequencing will start at the 5’ and 3’ ends of your amplicons.  This is employed with smaller amplicon sizes, typically <550bp.  This is helpful for variant phasing and library diversity studies. 

2.  When does GENEWIZ need to fragment amplicons for sequencing and what does this impact?

GENEWIZ recommends fragmentation with amplicons >570bp.  Amplicons are randomly fragmented, with NGS adapters ligated to each individual fragment.  Once fragmented, phasing information is lost and library diversity studies are not possible.  Studies are focused solely on variant frequency detection across the full reference sequence.

3. What is “PhiX” and why is it needed for amplicon sequencing?
PhiX is a well-studied bacteriophage that has balanced genomic content.  Most amplicons have low diversity at the 5’ and 3’ ends, which inherently reduces Illumina data quality based on current base calling algorithms.  PhiX is routinely spiked in, at various concentrations depending on the library diversity, as a control increase diversity and maintain high data quality.

4. How much PhiX is typically spiked-in to a sequencing run and how does this affect my data?

We recommend spiking in ~10-30% PhiX, depending on the original library diversity.  The PhiX sequences are removed from the data reported to the Cleint and are not used for downstream analysis.

5. What does the “unique sequence identification and abundance calculation” bioinformatics option entail?

We have created a bioinformatics pipeline that reports every unique sequence and their relative abundance at the molecular level.  This is particularly effective for library diversity studies.  Please ask our NGS team for an example report.

Amplicon Length Library Preparation Method MiSeq Configuration Recommended
100-270bp Contiguous 2x150bp
270-470bp Contiguous 2x250bp
470-550bp Contiguous 2x300bp
>550bp Fragmentation 2x150bp

6. How do I contact GENEWIZ for a technical consultation for my project?

GENEWIZ's NGS Team is composed of Ph.D. scientists who can help you optimize your project design and provide consultation to answer any questions. For any form questions that require support, please contact the NGS team at ngs.europe@genewiz.com, +49 (0)341 520 122-0.

7. Can GENEWIZ recommend the best data delivery option?
Yes, GENEWIZ will recommend the best data delivery option based on the platform and project details. Data delivery options include:

  • Secure File Transfer Protocol (SFTP)
  • Customer Cloud Account
  • Hard Drive (additional charges may apply)
  • Other (please explain in additional comments section of quote request form)