One of the most important attributes for a DNA sequencing system is accuracy. The Ion Personal Genome Machine™ sequencer has brought scalability, simplicity and speed to the high throughput sequencing field. This platform, using the concept of massively parallel sequencing, produces from 10 Mb to more than 1 Gb of sequence, highly accurate read sequences. Using this technology in Invicta Laboratory to perform PGS-NGS 360°™ we obtain the highest data and reproducible data in comparison to all other technologies used in preimplantation diagnosis.
Accuracy metrics are calculated either through prediction algorithms or through actual alignment to a known reference genome. Predicted quality Q is useful to filter and remove lower quality reads prior to downstream alignment.
After mapping to a reference sequence, the quality control report provides information about: total aligned bases, % aligned bases and the reference sequence coverage (Figure 1).
Measurement of accuracy based on alignment performed with a given reference sequence is referred to as ‘mean raw accuracy’. This is single pass accuracy implying that we are measuring the true per base error associated with a single read. Mean raw accuracy 1x signifies average raw accuracy of 1-mers plotted by their position in the read. Figure 2 shows the raw accuracy rate across the length of the sequence reads.
In order to get the actual observed measure of raw read accuracy we take individual reads, map them to the known sequence and count the number of mismatches. The raw read accuracy of 99% indicates an error rate of 1%. It means that one in one hundred bases is a mismatch to the reference. In this parameter all biological variety of sequence is also reflected (e.g. single nucleotide polymorphism – SNPs), therefore a value very close to 100 % would be possible only in situation of alignment against perfectly the same sequence that is present in sequencing reaction. To determine true variants from base calling errors you have to pile these reads up together along the reference. The powerful feature of NGS technology, which is the ability to produce huge amounts of sequencing reads, allows us to filter out mismatches that are likely to have been base calling errors and keep those that are likely to be real variants using statistical inference. In the end we get a consensus sequence and its corresponding consensus accuracy with respect to the reference.
The overall quality of semiconductor sequencing data can be examined by monitoring the raw accuracy rate across the length of the sequence reads. All the improvements and innovations, that we made at INVICTA Genetic Laboratory, have resulted in excellent sequencing accuracy to ensure the best possible service provided with INVICTA PGS-NGS 360°™.