Table 3.

Factors to consider with respect to the analytical validity required for genomic analysis using NGS

Category	Recommendation level
	Required	Recommended	Optional
Sample preparation	Tumor cell percentage DNA* concentration DNA fragment size Library concentration
Sequence related	Cluster density BQ* score ≥ specified threshold Percentage of valid reads Percentage of reads ≥ specified threshold
Analysis related	Mapping quality Mean read depth in analysis range Proportion with base depth ≥ specified threshold Percentage of bases with quality value ≥ specified threshold Mean insert size PCR* duplication percentage	Percentage of bases that differ from reference sequence AT/GC* bias
Mutation related	Depth of mutation loci Mutation quality Allele frequency Strand bias Number of mutations at same locus Number of mutations in specified threshold range		Number of germline mutations Haplotype bias
QC related	Determination of sex in analysis	Estimated contamination percentage	Presence or absence of genotype match Base percentage for mutation loci SNP/indel* ratio Ti/Tv* ratio Homo/hetero ratio •CNV profile

Definitions of abbreviations with an asterisk: DNA deoxyribonucleic acid, BQ base quality, i.e., value expressing the reliability of bases detected by the sequencer, PCR polymerase chain reaction, AT/CG adenine (A) and thymine (T) or guanine (G) and cytosine (C), SNP/indel refers to a ingle nucleotide polymorphism (SNP) and/or a base sequence insertion or deletion (indel), Ti/Tv transversion (Ti), i.e., a mutation between a pyrimidine (C, T/U) and a purine (A, G); transition (Tv), i.e., a mutation between pyrimidines or purines (Modified from Table 4 in Reference [18])