Re-sequencing projects are offered in different project phases based on your requirements.
Phase 0: Extraction of genomic DNA / RNA from tissue / bacteria / fungi
The isolation of genomic DNA from starting material is performed using a commercial kit. The kit works on the basis of sample lysis under highly denaturing conditions in combination with selective and reversible DNA binding to silica membranes. Quality and quantity of the eluted DNA will be checked by agarose gel electrophoresis.
Phase I: Establishment
PCR primer design and synthesis
The supplied sequence information is used to design the appropriate primers required for the PCR reactions. All primers are optimised and their physical properties such as GC content, Tm and self-annealing are verified by our expert team. Primers are subsequently synthesised in-house.
PCR and purification
PCR reactions are established based on a standard genomic DNA (when available) or on one or two genomic DNA samples provided by you. The PCR products are later purified and quality controlled using agarose gel electrophoresis. All PCR products with unsatisfactory results are re-generated a second or third time if necessary. Re-generation includes a new primer design for each PCR product, primer synthesis, PCR amplification and purification of the PCR products.
Sequencing of the test PCR
To ensure the quality of the PCR process all established PCR reactions are then sequenced from both sides
Phase II: High throughput PCR & sequencing
High throughput PCR and purification
After successful PCR primer design, PCR reactions with all genomic DNA samples are set up and run in high throughput. For 16S and ITS sequencing specific primers are used. The PCR products are subsequently purified and quality checked via agarose gel.
High throughput sequencing
All PCR products are sequenced in double strand quality. This step includes one repetition of the sequencing reaction, if a reaction fails. If both reactions fail, the PCR amplification will be repeated for this sample
Phase III: Bioinformatic analysis
Bioinformatics includes analysis of the single reads, quality clipping and assembly to create a consensus sequence of each sample / gene. On request, we use the obtained consensus sequences to perform a database search (BLAST) to accomplish the biological classification of each sample.
Each sequence can be compared against a reference sequence, supplied by the customer and can be analyse to find potential sequence differences like heterozygotes, insertions/deletions or codon changes.
In order to generate a complete mutation report, we can run an analysis using appropriate software.
Sequencing by primer walking allows precise sequence identification of long fragments with highest accuracy
De novo sequencing typically requires sequencing of both strands.
It starts using standard or specific primers starting from common elements (e.g. origin of replication or resistance gene) to sequence the first 750 - 1000 bases of the delivered PCR product, plasmid or BAC, PAC, Cosmid, Fosmid DNA from both sides. Based on the resulting sequence data, we define and synthesise new primers which are used to sequence further bases thus “walking” along the sequence. The region of interest is sequenced bi-directional until one or both strands are completely deciphered (2fold or 4fold coverage).
If there are any ambiguities in the sequence, further primers are designed to resolve them. This process ensures the highest data accuracy of 99.9% with single-strand sequencing and 99.999% with double-strand sequencing which represents less than one error in 100,000 base pairs.
Our Primer Walking service includes:
- Plasmid DNA extraction in single tube format (optional)
- PCR product purification in single tube format (optional)
- Quality control of incoming DNA
- Verification of the provided reference sequence
- Design and synthesis of specific primers
- Bioinformatic analysis using the appropriate software incl.:
- Assembly of the sequence data to generate a consensus sequence
- Quality check of the consensus sequence incl. quality report
- Verification accuracy of >99,9% by single-strand sequencing
- Verification accuracy of >99,999% by double-strand sequencing
- Free storage of template DNA and all primers for 3 months
- Delivery time: 2 kb within 6-8 working days
TOPO-TA cloning of PCR products with subsequent sequencing is offered in different project phases based on your requirements.
Phase 0: Purification of PCR products
On request, we will perform purification of unpurified PCR products by performing an ethanol precipitation step applying polyethyleneglycol (PEG).
Phase I: Cloning in vector and plating
The supplied samples are cloned into an appropriate vector using a commercial cloning kit. Chemically competent E.coli cells will be used for transformation. Cells will be plated and grown on LB-Agarplates containing appropriate antibiotics. In case of unsatisfactory results (e.g. no clones at all or only empty vector clones) the cloning reaction will be repeated once.
Phase II: Clone picking, DNA preparation and high throughput sequencing
After plating and growing the specified number of single cell clones per PCR product will be picked, grown and plasmid mini preparation will be done using a commercial kit. The purified plasmid DNA will be used as template to sequence the respective insert using two (double strand) standard primers (e.g. M13for and M13rev). In case of failure the sequencing will be repeated once using alternative primers.
Phase III: Bioinformatic analysis
On request, bioinformatics includes the clipping of vector sequence and the alignment of single reads to create a consensus sequence of the insert.
On request, we also compare each sequence against a reference sequence(s), supplied by the customer and analyse it to find potential sequence differences like heterozygotes, insertions/deletions or codon changes.
In order to generate a complete mutation report, we will run an analysis using appropriate software.
All Sanger sequencing projects can be performed in compliance with Good Laboratory Practice (GLP)
In addition to the service descriptions for Re-Sequencing and Primer Walking projects, the following services for sequencing under GLP are included:
- 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.
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
Re-Sequencing Projects incl. 16S / ITS Sequencing Projects:
You receive sequence data as FASTA databases, as well as the traces of all sequence runs in *ab1 file format.
If bioinformatic analysis is ordered, consensus sequences of each sample respectively a complete mutation report using specialised software will be provided.
If BLAST analysis is ordered, we will report the species corresponding to the BLAST search results with the highest score for each sample. If too many ambiguities are occurring in the taxonomic ranking we will report the corresponding genus or at least the family if possible. A tabular BLAST report will be generated.
Data will be provided to your online account.
Primer Walking Projects:
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)
- *.mutation: tabular summary of all observed differences to the reference sequence (XLS or MS-DOS TXT)