NGS - Next Generation Sequencing
NGS is the newest method of molecular genetic testing that enables effective "reading” of many genetic (DNA) sequences at the same time. The NGS is a method with great potential for further development and wide use for all types of genetic testing (both targeted sequences/genes and the whole genome), including the possibility of combining them in a single experiment. NGS method (with so called low-coverage) can be used for preimplantation genetic screening of numerical chromosomal changes (PGS (PGT-A)) as well as for preimplantation genetic testing of unbalanced forms of familial rearrangements (PGT-SR) associated with the screening of numerical changes of other chromosomes.
If analysis is done on trophectoderm sample, NGS due to its higher sensitivity of quantification, improved dynamic range of changes and better determination of signal vs noise can more reliably assess mosaic chromosomal findings (than previously used analysis by array comparative genomic hybridization (aCGH)). Finding of chromosomal aberration/s in mosaic signifies simultaneous presence of euploid (normal) and aneuploid (abnormal) cell lines in the corresponding embryo. Such findings are always related with de novo arisen (sporadic) chromosomal aberration in frame of PGS (PGT-A) analysis. In situation when there is no (left) embryo with normal finding and after appropriate genetic counselling and upon written patient consent, transfer of embryo with mosaic aberration/s might be considered.
Principle of the NGS method
Before the examination by NGS method, DNA is isolated from the collected cells and amplified by whole genome amplification (WGA). From successfully amplified samples are enzymatically prepared so-called "libraries", suitable for simultaneous "reading" of many DNA sequences. Each sample / library has a unique label which allows to analyze in one experiment DNA of more subjects (embryos) at the same time. Samples / libraries are mixed in the same ratio and the resulting library is then ready for final sequencing. Proper sequence reading (sequencing) is carried out by stepwise synthesis of new DNA strands complementary to the read fragments. By special software, read sequences are compared with normal human genome and their genomic position is determined. After allocating the sequences to the individual samples, final quantitative assessment of all chromosomes is performed.
Limitation of the NGS method
The used NGS method with low-pass whole genome sequencing is limited by the size of the chromosomal rearrangements. Small chromosomal losses or gains cannot be detected and at the same time any other disease or development defects of the foetus which are not caused by the change of the number of examined chromosomes or their major parts cannot be excluded.
Genetic analysis and consultation
2019/08/27 IVF Zentren Prof. Zech – Pilsen, RNDr. Martina Hrubá, Ph.D.