Table 2.

Implementation of NGS test in the diagnostics.

		Commercial IVD/IVDR/FDA assaya		Laboratory-developed test (in-house/custom)
Process	Tools, material	Steps to be fulfilled		Steps to be fulfilled		Specifications and TP53-CLL-specific notes
Test familiarization	Explore the clinical and analytical needs	Define turnaround time, costs, number of samples, instrumentation, personnel, etc.				Standalone or panel assay
Test development	Study available technologies	Choose a commercial method		Test design		Regions to be sequenced Library preparation method Sequencing technology Bioinformatics
				Determine required test performance metrics and sequencing settings		Aim to detect low-VAF variants Consider: DNA input, coverage (each base of TP53 gene has to meet the minimum read depth)
				Prepare SOP
Optimization	Perform pilot run(s) Reference material Non-DNA controls	General assessment of method applicability for the purpose		Identify and solve errors and weaknesses
				Check if the required parameters are met
				Set the critical values (pass/fail criteria)		Output library quality and quantity Sequencing performance (level of background noise/overall error rate, coverage non-uniformity, low/high coverage, base call quality metrics etc.)
Verification/validation	Reference material (different from the material used during optimization)	Verification	Prepare verification protocol	Validation	Prepare validation protocol
			Test the ranges provided by the manufacturer and technical variables in the laboratory		Include variables influencing assay performance	Amount of input DNA Number, type of samples in sequencing run Sequencing machine and other instruments Personnel
			Verify the values provided by the manufacturer		Assess parameters describing the test performance	Limit of blank, limit of detection Repeatability, reproducibility Proportion of true/false positives/negatives, etc. (See Supplementary Table 3)
Quality control (QC) and continuous monitoring	Diagnostic samples Regularly analyzed positive and negative controls	Monitor of specimen parameters				Sample purity, DNA quality, etc.
		Check variant profile				Proportion of TP53-mut/del(17p) and low-burden TP53 mutations, variant profile (Figs. 1, 4, 6)
	Samples supplied by EQA provider	External quality assessment (EQA)
	Samples provided by independent laboratory	Interlaboratory comparison

^aWhen using commercial research-use-only tests, the steps of test development and optimization are the same as for IVD/IVDR assays, but full validation must be performed.