PGS-NGS 360°™ – Preimplantation genetic screening with the use of next generation DNA sequencing is based on high-throughput technology of massive parallel sequencing, that allows us to obtain millions of reads for each sample in a single reaction. More reads mean increased depth of sequencing which yields more accurate and reliable results in comparison to conventional Sanger sequencing. One of the parameters determining the accuracy of the sequencing is the value of quality score Q as measured on a Phred scale.
PGS-NGS 360°™ test consists of several steps, all of which affect the overall quality of the results. Sequencing quality parameters provide important information as to the accuracy of each step in the process, including the library construction, basecalling, reads mapping and variant calling. The value of Q on the Phred quality scale is the most common parameter used to assess the accuracy of the sequencing. The value of Q is logarithmically related to the probability of incorrect identification of the base (P). For example, the value of Q20 is the probability of erroneous calling of 1 base per 100 consecutive nucleotides. Other examples of Q are shown in Table 1.
Another parameter describing the sequencing accuracy is the value of AQ. This value determines the quality of reads when compared to the reference sequence after reads mapping. 3 quality levels are commonly used: AQ17, AQ20 and ‘Perfect’.
The AQ17 describes read, for which the read error in comparison to the reference sequence is 2% or less. AQ20 means that the read error is 1% or less. ‘Perfect’ indicates a read that is perfectly aligned with the reference sequence. PGS-NGS 360°™ tests performed at the INVICTA Genetic Laboratory receive the ‘PERFECT’ score for almost 80% of the reads (Figure 1).
NGS method is based on massive parallel sequencing, which provides large amounts of data. Such a large number of reads determines the quality of the sequencing. In addition to the quantity, quality of the sequencing is also very important. Post-light NGS technique used at the INVICTA Genetic Laboratory provides the best quality results, and for this reason we have selected it to perform PGS / PGD for our patients.