Table 1 Summary of sequencing approaches in hereditary cancer testing (HCT).

Sequencing Approach	Targeted Sequencing / MGPT		ES	GS
Genome coverage	10s–100s genes		All coding genes	Genome-wide
Sequencing Technology	SRS	LRS	SRS	SRS	LRS
Current application level	Clinical (Standard)	Emerging	Clinical	Clinical	Emerging
Relative Cost	+	++	++	+++	++++
Sequencing depth^a	~200–1000x	~50–200x	~100x	~30–40x	~20–40x
Covered regions^b	Exons + splice regions	Exons + Introns + UTR	Exons + splice regions	Genome-wide^f	Whole genome
Primary research potential	Candidate CPG	Candidate CPG, non-coding variants, SV, Epigenetics	Candidate CPG	Candidate CPG, non-coding variants, PRS	Candidate CPG, non-coding variants, PRS, SV, Epigenetics
Variant Detection
SNV/indels ( < 50 bp)	✓	✓	✓	✓	✓
SV/CNV	Limited	✓	Limited	Limited	✓
Challenging HCT genes (PMS2)	Limited	✓	Limited	Limited	✓
Orthogonal confirmation required^c	Yes	No	Yes	Yes	No
Epigenetics^d	X	PCR: X; Native DNA: ✓	X	X	✓
+ RNA Sequencing
Detection of abnormal splicing	✓	✓	✓	✓	✓
Detection of large intronic retentions	Limited	✓	Limited	Limited	✓
Isoform quantification	X	✓	X	X	✓
RNA quality requirements^e	Medium	High	Medium-High	Medium-High	High
+ Somatic Testing
Somatic second hits	✓	✓	✓	✓	✓
Mutational signature inference	X	X	✓	✓	✓
Epigenetics^d	X	PCR: X; Native DNA: ✓	X	X	✓

CPG cancer predisposition genes, ES exome sequencing, GS genome sequencing, HCT hereditary cancer testing, Indel: small insertions and deletions ( < 50 bp), LRS Long-read sequencing, MGPT multigene panel testing, PRS polygenic risk scores, SNV single nucleotide variants, SRS short-read sequencing, SV Structural variants, UTR Untranslated regions.
^aDepends on size of targeted regions and multiplexing.
^bDenotes typically included regions.
^cOrthogonal confirmation methods may include: Sanger sequencing, Multiplex Ligation-dependent Probe Amplification (MLPA), Long-range PCR sequencing or other techniques used by laboratories to validate suspected variants/alterations.
^dExcluding the detection of low-level signals, such as mosaic methylation, where the limited sequencing depth translates into lower detection sensitivity.
^eRIN (RNA Integrity Number) requirements vary by assay, typically RIN ≥ 5 for SRS, and RIN ≥ 7 for LRS applications.
^fCoverage excludes known challenging SRS regions, such as repetitive and segmentally duplicated regions.

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