The journey of your sample from collection to the "opening" of your DNA
In this section, you will learn about the journey of your sample from collection to the final "deciphering" of your DNA. Thanks to the state-of-the-art technology we use in our molecular genetics laboratory, many of the processes are fully automated. This significantly increases the speed of obtaining the necessary data from your DNA.
The whole process consists of many steps that build on each other and there is a control mechanism in each step to eliminate possible errors. By standardising all parts of the analysis, it is possible to deliver the results of the required tests in the time we allow for each type of test.
Set of patients to be sequenced
Initially, we identified a set of patients for sequencing in our laboratory. Modern instruments do not perform the analysis and associated processes one sample at a time but in bulk.
Preparing the DNA for analysis
DNA isolation is essential before any molecular analysis of the genome can be performed.
- The purity of this DNA, i.e., the minimal presence of other types of molecules in the isolate, is crucial to the analytical methods used.
- The first step in isolation is to homogenize the tissue and break down the cell membranes to reveal the cell nuclei containing the desired DNA.
- This is followed by the removal of the unwanted cellular contents, either chemically or physically.
- The next process involves either precipitating the DNA molecules with a suitable alcohol solvent, or passing them through a "mesh" on which only the DNA is trapped while other, contaminating substances flow through.
The patients' isolated DNA is then prepared to the desired concentration.
Automated preparation of the DNA library
Automated preparation of the DNA library is then performed, which is a DNA fragmentation process.
In layman's terms, this can be thought of as cutting long DNA molecules into shorter sections of the desired length, modifying the ends of the resulting fragments and purifying them using magnetic beads.
Measurement of the concentration of the prepared DNA library
In order to work further with DNA, we need to make sure that we have DNA in sufficient concentration and that the majority of the DNA fragments produced are of the required size.
After that, the entire library is checked and the samples are pooled.
Pooling of samples
Pooling of the entire DNA library = the DNA of all patients to be sequenced is pooled into one tube.
Hybridization
At this stage, the desired genomic regions are captured using probes mounted on the fragmented DNA sample. Next, the hybridised DNA library is purified on magnetic beads.
Loading a finished DNA library into sequencing cartridges
The finished DNA library is then transferred from the tube to the sequencing cassette, which is loaded into the sequencer.
Sequencing
The sequencing itself can then be started using the sequencer. Sequencing is the final key point of the whole process and serves - simply put - to decipher the genetic code, which means to find out the primary order of nucleotide bases in a molecule.
- The newest sequencing method, which we also use in our lab, is NGS sequencing (Next Generation Sequencing), which allows the parallel sequencing of many DNA fragments simultaneously.
- Each fragment is read and then the software assembles the overall sequence.
- It's easy to think of it as reading multiple books at once.
Evaluation
We then evaluate the sequencing data to see if the gene or multiple genes we are targeting have a mutation.