Sanger Sequencing at Eurofins Genomics

Sequence Verification under ISO 17025 & GLP

Sanger Sequencing for Sequence Verification and Primer Walking

Certified to ISO 17025 & GLP

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Depending on your project requirements, we offer the following options for verifying the identity of your DNA sample:

Sequence Verification

Sequence Verification is a service designed to confirm the exact nucleotide sequence of a known DNA construct. It is the ideal solution when you need to verify the identity and integrity of an already amplified or synthesized DNA construct (e.g., plasmids, BACs, etc.). Using high-quality Sanger sequencing, we accurately determine the nucleotide sequence on both strands and detect possible variants, such as point mutations, insertions/deletions, or recombination events.

Primer Walking

Primer Walking enables de novo sequencing of DNA constructs or amplicons by sequentially designing primers based on newly obtained sequence information. The process begins with an initial primer (typically targeting known regions such as PCR primer sites or plasmid backbone elements). As each new sequence segment is obtained, a subsequent primer is designed to extend the sequence further. This iterative process continues until the entire target region has been fully sequenced.

Whether you require precise sequence confirmation or the analysis of a complex DNA region, we recommend bi-directional (2-fold) sequencing. This approach improves variant detection and provides a reliable view of both DNA strands. To further enhance result accuracy, sequencing should be performed from at least two independent reactions (4-fold coverage). This not only increases overall confidence in the data but also allows for the identification of strand-specific features such as nicks or hairpin structures. In cases where ambiguities arise, additional primers are designed and applied to resolve them.

Our service includes:

Quality control of incoming DNA
Design and synthesis of specific primers
Bioinformatic analysis using the appropriate software:
- Assembly of the sequence data to generate a consensus sequence
- Quality check of the consensus sequence incl. quality report
Free storage of template DNA and all primers for 3 months
Verification accuracy up to >99,99999 %

Your benefits at a glance:

Tailored support from our Sanger Sequencing Team
Design and synthesis of required primers included
Wide variety of bioinformatics solutions and reporting options available (see below)
Certificate of Analysis (CoA) for each sample
Comprehensive Report (including one review) available as an alternative to the CoA
ISO 17025 accredited production process
Service available in full compliance with GLP
Customer audits can be arranged upon request

Related Information

Data Format & Delivery

You will receive the following original data to your online account:

*.ab1: electropherograms in AB1 format
*.cons: consensus sequence in FASTA format (MS-DOS TXT)
*.qual: quality report for each consensus sequence (MS-DOS TXT)
*.align: alignment of single reads to build a consensus (MS-DOS TXT)
*.comp: alignment comparison to a reference sequence (MS-DOS TXT)

Delivery Times:

Sequence Verification: 10 working days / 10 kb
Primer Walking: 8 working days / 2 kb

Sanger Sequencing under GLP

Sanger Sequencing in Compliance with Good Laboratory Practice (GLP)

In addition to the services described above, the following components are included when sequencing is performed under GLP:

Preparation of Study Plan incl. description of materials and methods (incl. one revision after sponsor´s review)
Evaluation and documentation of sequencing results.
Provision of an overview table (embedded in the Study Report, if feasible) where possible sequence variations are listed
Preparation of Study Report incl. GLP compliance statement (incl. one revision after sponsor´s review)
Study Inspection by QA and Quality Assurance Statement
Archiving the study data / documentation for 15 years

To ensure the quality of our GLP sequencing service, we are routinely assessed by first and second external auditors. Additionally, we perform regularly facility and process audits.

References:

Sequencing is performed according to standard methods and in compliance with

CLSI Guideline MM09-A; Vol. 34 No. 4, 2014; Nucleic Acid Sequencing Methods in Diagnostic Laboratory Medicine, Approved Guideline-Second Edition

Conduct of sequencing studies is performed according to

OECD Guideline ENV/MC/CHEM(98)17 (rev.1999): The Principles of Good Laboratory Praxis (Monograph No. 01) and OECD Guideline ENV/JM/MONO(1999)23 (rev. 2002): Short Term Study and GLP (Monograph No. 07)

Quality assurance (QA) activities related to sequencing studies (review of the study plan, inspection of study, study report and raw data) are conducted according to

OECD Guideline ENV/JM/MONO (1999) 20 (03-October-2002): Quality Assurance and GLP

Individual Projects

Individual projects are also possible to meet further specific requirements under ISO 17025. Additional services can be offered across departments, e.g. with upstream extraction including pre PCR. Please ask us for an individual service and we will check to what extent this can be implemented.

Request a Quote for your individual project

Literature

Sequencing result guide

Primer Walking sample submission guide

Quality is important for us at Eurofins

Our products and services are produced and performed under strict quality management and quality assurance systems.

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