Next generation sequencing (NGS) technologies produce millions of sequences in a single sequencing run. Prior to analyzing this huge amount of data, it is crucial to check its quality. Implementation of NGS in a clinical laboratory requires quality control procedures for each steps in the sample’s processing work flow. For INVICTA’s PGS/PGD procedure one of the QC checkpoints of the sequencing run is the Test Fragment Accuracy. As each test fragment is typically sequenced over 1000 times it is a very good measure of sequencing quality that ensures high reliability of tests performed in our laboratory.
Introduction of new procedures for routine diagnostics should be performed conservatively, with an emphasis on precision, effectiveness and efficiency of the method and explicitness in interpreting the results. Quality control and standardization of the massively parallel sequencing experiments and data reporting are important issues in clinical laboratory. The internal QC involves procedures for continuous monitoring of operations and systematic day-to-day examination of the produced data. Quality control is essential for raw NGS data analysis. There are several quality controls you can look at to verify if your sequence data are high-quality.
One element of quality control of data obtained at the INVICTA Genetic Laboratory is the Test Fragment Accuracy. The Test Fragment is oligonucleotide of known sequence attached to the control beads, spiked into the sequencing reaction before loading the sequencing chip. Ourcurrently used sequencing kit uses Test Fragments which are slightly less than 100 bp in length. Once sequencing and analysis are complete, the test fragment metrics are available in the summary run report. The test fragment metrics in report include: sequence of the test fragment, number of reads identified for this Test Fragment, Average Read Length with Q17 score or better and 50AQ17 value.
The example of read length histogram of Test fragment sequence from one of our sequencing run is presented below.
Q17 is a quality score, corresponding to 1 base error allowed per 50 bases. Given the sequence length almost 100 nucleotides, this corresponds to 1 error at most for entire sequence. Thus, for a given read the Q17 Average Read Length would be one base before the second error in Test Fragment.The second parameter 50AQ17 is the number of reads for this Test Fragment with a minimum of 50 base pairs in length and an error rate of 1 in 50, Phred-like 17, or better (after alignment with Test Fragment sequence).
Analysis results for a Test Fragment are displayed on condition there are at least 1000 high-quality Test Fragments reads with at least 85% match against the appropriate template. Therefore, test fragments plays an important role in reporting sequencing quality for each and every PGS/PGS NGS based run.