Table 2.

Comparison of methods used to identify rearrangements.

METHOD	ADVANTAGES	DISADVANTAGES
Cytogenetics/karyotyping	• Whole genome coverage • Can detect large rearrangements	• Cannot resolve rearrangements that occur within the same chromosomal band
Fluorescence in situ hybridization (FISH)	• Common fusion partners can be identified if partners known a priori	• Fusions with at least one unknown partner result in false negatives • Cannot resolve small intrachromosomal rearrangements less than 3 Mb
Break-apart FISH	Common gene involved in fusions can be identified if gene is known a priori	• Does not identify gene fusion partner • may result in false negatives • Cannot identify exact breakpoints if non-canonical
Array-based comparative genomic hybridization (array CGH)	• Detects large inter- and intrachromosomal rearrangements	• Does not detect intragenic rearrangements
PCR-based techniques	• Can detect known partners in fusion if partners known a priori • Can be used to detect such “intra-band” and intron events only when breakpoints are known	• May miss small insertions/deletions if allele-specific primers are not used
Whole-genome sequencing (WGS)	• Detects small and large rearrangements	• Higher false-negative rate due to lower depth of coverage
Exome only sequencing	• Improved false-negative rate as compared to WGS	• Does not detect intronic rearrangements
Exome sequencing with targeted intron capture	• Improved false-negative rate as compared to WGS • Detects rearrangements with introns or other regions of genome, e.g. promoter	• Requires additional coverage of introns known to be involved in fusion breakpoints • Introns have to be “well behaved” (not too big; not too many internal repeats) • May miss less common breakpoints involving other introns • Cannot infer frame of read for all intronic fusions
Transcriptome sequencing	• Detects presence and abundance of fusion events (but will require high sequencing depth)	• Higher fail rate with FFPE* tissue • Will miss rearrangements that only affect promoter elements (ie IGH-CCND1 rearrangement)
Hybrid capture enriched RNA sequencing	• High sensitivity for fusions involving targeted genes	• Difficult to generate good cDNA libraries from FFPE* • Only detects events involving target genes • Will miss rearrangements involving only promoter regions
Single primer enrichment technology (SPET)	• Detects low abundance gene fusion transcripts from fresh or FFPE* tissues • Requires only one partner of the fusion to detect novel fusions	• Only provides targeted enrichment for desired genes

Abbreviation:

^*FFPE, formalin-fixed, paraffin-embedded.