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.
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 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.