How is the probe uniformity of a targeted sequencing panel for genetic diseases evaluated? Is additional experimentation needed?

Introducing a panel cannot be directly applied to clinical use. A phased validation approach is recommended: first, use standards and known samples for analytical performance evaluation, then perform orthogonal validation against existing methods or Sanger/MLPA, especially for genes associated with pseudogenes or homologous regions. Manufacturer reference material data cannot be used as the sole basis for acceptance. Insufficient coverage of certain regions may occur in real samples, so thresholds should be set and supplementary testing plans prepared, such as using supplemental probes or orthogonal methods. If a domestic panel shows stable performance in preliminary data, it can serve as a subsequent alternative or replacement, but it is best to first conduct a parallel comparison with imported panels before making a decision. This way, spending a few extra weeks in the early stage is more prudent for long-term application.

There is a large price gap between domestic and imported products, but domestic does not necessarily mean inconsistent batch-to-batch performance; the key lies in production quality control. It is recommended to ask the manufacturer to provide COAs for 2-3 consecutive batches, batch-to-batch coverage data, and probe synthesis QC documents, and to request a trial Panel. Conducting a small parallel comparison is very necessary: select 8-16 samples and use imported and domestic panels separately for parallel library preparation and sequencing, focusing on comparing uniformity, on-target reads ratio, pseudogene/homologous region off-target rates, and variant concordance. Domestic brands may respond faster in service and replenishment, but long-term stable supply capability should be evaluated. In the contract, clearly specify delivery timelines, COA standards, allowable batch-to-batch variation ranges, and compensation mechanisms to make procurement more reliable.

Manufacturer reference material data can only be used for preliminary screening; real clinical samples will vary due to factors such as DNA quality, input amount, and library preparation batch. It is recommended to first run multiple real samples after arrival to calculate the target region coverage ratio, fold-80, uniformity, and reproducibility. Pseudogenes/homologous regions are key points, as they are prone to off-target capture or misalignment; the capture efficiency and specificity of these regions should be statistically analyzed separately, and validation with known variant samples is best. In addition to uniformity, 20x and 50x coverage rates should also be examined; in genetic disease testing, insufficient depth in certain exons can affect pathogenicity determination. These metrics can be used as acceptance criteria; if they do not meet the standards, the manufacturer should be required to optimize or provide replacement products.