Molecular genetic analysis

Molecular genetic analysis
  • Genome Sequencing
  • Targeted Sequencing (Exome and Panels: Agilent SureSelect; Amplicons)
  • Restriction site associated DNA Sequencing (RAD genotyping)
  • RNA Sequencing (Transcriptome Sequencing, smallRNA Sequencing)
  • Metagenome Sequencing

The diagnostic panels offered by the PRADUS center are developed by the CeGat Institute, a leading expert in the field of molecular genetic analysis in Europe.

The genetics of the CeGat Institute, as groundbreakers, managed to combine genetic diagnostics and high-throughput sequencing into a new method of genetic material analyzing. In 2010, CeGat created Diagnostic Panels, allowing within one research to decode, analyze and interpret all those genes that contain information on particular diseases.

CeGat has been using the technology of “Next-Generation Sequencing” (NGS) since its foundation. Depending on the task and clinical objectives of the study, sequencing is performed on the Illumina HiSeq and MiSeq platforms. There are 3 stages of DNA transformation into a sequence:

1. Preparation of a DNA library.
For the preparation of the DNA library, the DNA must first be fragmented. The ends of the DNA fragments are further repaired, as they are damaged by the fragmentation. The final stage of the preparation of the DNA library is ligation of adapters to both sides. These adapters contain sequence motifs that are required for further stages.

2. Clonal amplification
Illumina technology uses the so-called “Bridge-PCR” for clonal amplification of individual DNA fragments. This PCR is performed on the Flow Cell (a glass side similar to a microscope slide) on which the actual sequencing takes place. DNA fragments are linked via the adapters to the Flow Cell, then the Bridge-PCR is carried out and oligos bound are function as primers. Within this amplification each individual DNA fragment forms a separate cluster of identical DNA fragments.

3. Sequencing
Illumina’s Sequencing-by-Synthesis (SBS) is based on the Cyclic Reversible Termination (CRT) method. Each of the four nucleotides is linked with a different dye and modified by a terminator group. During a reaction cycle all four nucleotides are offered simultaneously to the polymerase for strand synthesis. The four dyes of each nucleotide can be detected using Imaging: the dye and the terminator group are cleaved and a new synthesis cycle starts. The sequence of each cluster is thus simultaneously determined base by base. The individual reads are formed and compared with the reference genome.