Table 4.

Core Quality Metrics for Validation and Ongoing Quality Control

Core quality metric	Validation parameters	Ongoing quality control
Nucleic acid quality and quantity	Requirements of DNA quality and quantity differ depending on the tissue source, extraction method, and next-generation sequencing method. Each laboratory must establish: Minimum criteria to ensure accurate variant detection and reproducible results depending on the requirement and established sensitivity of the assay. Standard procedures to adjust DNA input depending on the quality of the sample.	A plan for ongoing monitoring must be established. Any changes on extraction protocols should be followed by close monitoring of all downstream processes to ensure adequate performance of the assay.
Library qualification and quantification	The laboratory must standardize protocols for library qualification and quantification. Library quantification is recommended—each laboratory must validate a method that is suitable for its needs.	The fragment sizes of the library must be measured to ensure they fall within the expected molecular weight narrow range. Ongoing measures should be taken to minimize primer dimers, adapter dimers, and broader bands of higher molecular weight.
Depth of coverage	Requirements vary depending on the platform used and the application. Coverage must be defined to achieve adequate sensitivity and specificity in the regions of interest. Each laboratory must establish the minimum criteria for depth of coverage characteristic of a particular region under standard assay conditions (coverage threshold).	Ongoing measures should be taken to monitor the overall coverage and region coverage in each run. If coverage thresholds are outside the validated range, the samples should be subjected to reanalysis. If only local regions are affected, testing of that region by an alternate method may be performed.
Uniformity of coverage	The required level of coverage across the targeted regions must be defined during the vaLidation stage.	The uniformity of coverage must be monitored and compared to the levels established during the validation. If the coverage uniformity profile falls outside of the expected profile as established by the validation, this may be indicative of errors during the testing process.
GC bias	GC content affects sequencing efficiency and the uniformity of coverage of the targeted regions. The extent of GC bias in all parts of the genome included in the assay should be determined during validation.	GC bias should be monitored with every run to detect changes in test performance or sample quality issues.
Cluster density and alignment rate	The laboratory must define the right balance between overclustering and underclustering and outline the steps to prevent and resolve both instances. As a general guideline, the percentage of clusters passing filter should be >80% and alignment rate should be >95%.	Cluster density and alignment rate should be monitored in every run.
Transition/transversion ratio	Important parameter for whole-exome or whole-genome sequencing. Not required for targeted panels. The ratio of transitions/transversions should be comparable to published values.	The transition/transversion ratio should be monitored with every sample to assess test performance. Ratios lower or higher than expected may indicate that the quality of base calls was Low.
Base call quality scores	The Laboratory must establish acceptable raw base call quality score thresholds for the assay during validation. Preprocessing methods to remove low-quality base calls should be established to reduce the false-positive rate.	Quality scores and quality of signal/noise ratio should be monitored in every run. Low-quality scores can lead to increased false-positive variant calls; thus, result must be interpreted with caution and repeat testing may be indicated.
Mapping quality	Parameters for mapping quality must be established during validation and should demonstrate that the test only analyzes reads that map to the regions targeted by the assay. Steps should be established to filter reads that map to nontargeted regions.	The proportion of reads that do not map to target regions must be monitored during each run. Poor mapping quality may be a result of non-specific amplification, capture of off-target DNA, or contamination.
Duplication rate	Acceptable parameters for maximum duplication rate should be established for each assay. Filtering of duplicate reads by the analysis pipeline should be established to increase the number of usable sequencing data and prevent skewing of allelic fractions.	The duplication rate should be monitored in every run and for each sample independently to monitor library diversity.
Strand bias	Strand bias occurs when the genotype inferred from information presented by the forward strand and the reverse strand disagrees. Each laboratory must define the tolerance level for strand bias and outline specific criteria for when alternate testing should be instituted.	The degree of strand bias must be monitored in all samples.

This is not a comprehensive list.

GC, guanine-cytosine content.