Targeted Resequencing of DNA : Capture method
In humans, a large number of diseases are associated with genetic mutations in specific regions. In addition, multifactorial diseases present specific genomic profiles. Therefore, studying sequence variation at individual level could be a major tool for clinical research. The most popular application of our times is notably the study of the coding regions of the genome, known as exomes.
Thanks to the development of cutting-edge sequencing technologies, the France Génomique platforms are able to offer targeted resequencing for effective identification of variants that may appear in candidate regions.
Two approaches are used : 1) amplicon sequencing (sequencing of PCR-amplified fragments), and 2) sequencing by hybridisation (SBH). For the latter, the target sequences are captured by hybridisation with specific probes (using either solid-phase capture or liquid-phase capture methods).
First, the extracted DNA is fragmented (with enzymes or mechanically).
Next, adapter sequences are ligated to both ends of the fragments.
After denaturing, the fragments are hybridised to specific probes for/in the region of interest.
With solid based capture, the probes are fixed to a DNA chip, whereas with the solution-based method, they are fixed to magnetic beads.
The non-hybridised fragments are eliminated and the target sequences are recovered.
Amplification is then performed (to increase the quantity of material to be sequenced) using adapter-specific primers.
Finally, the amplified fragments are passed through quality control and then sequenced using next generation sequencers.
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