Table 2.
Characteristics of Assays Discussed in this Guideline
| Method | SNVs/InDels | CNV | Rearrangements | Methyl. class | MGMT | Comment | Throughput samples | Throughput markers | Duration** | Cost |
|---|---|---|---|---|---|---|---|---|---|---|
| NGS DNA | Yes | Yes | Yes | No | No | Sole method for efficient comprehensive variant detection, up to 10-fold lower LOD than Sanger seq, larger target size and higher multiplexing than ddPCR | High and flexible, potentially scalable > 96 samples | High (select targeted genes as panel up to exome/genome as target region); CNV calling depending on target region and unbiased library generation | Depending on exact method and target size 2–5 d from extracted analyte to raw result; pooling of samples for cost-efficiency must be considered | CapEx $$$ Reagents $$ Marker $ Interpr $$$ |
| NGS RNA | (Yes) | No | Yes | No | No | Most efficient method for detection of gene fusions with unknown partners or screening for variety of possible fusions; may allow for detection of activating SNVs and InDels | High and flexible, potentially scalable > 96 samples | High (select gene fusions up to complete coding RNA or transcriptome) | Depending on exact method and target size 2–5 d from extracted analyte to raw result; pooling of samples for cost-efficiency must be considered | CapEx $$$ Reagents $$ Marker $ Interpr $$$ |
| Methylome profiling (“850k”) | No* | Yes | (Yes) | Yes | Yes | Sole method for DNA methylation-based classification, efficient genome-wide CNV analysis (besides DNA-based NGS) | High, array design allows only multiples of 8 samples | High: Coverage of methylation classes depends on subsequent algorithm (classifier), not on assay setup; covers genome-wide CNVs with pre-selection; suggests (but not proves or excludes) several rearrangements depending on their exact configuration (BRAF, FGFR1, FGFR3, ZFTA, YAP1 and others) | 3 d from analyte to raw result; pooling of samples for cost-efficiency must be considered due to chip design | CapEx $$$ Reagents $$ Marker $ Interpr. $$ |
| FISH | No | Yes | Yes | No | No | Efficient assay for interrogating suspected single genomic event like specific copy-number alteration, specific single rearrangements; more prone to artificial false results in CNV analysis than 850k | Typically, one marker (may consist of two loci, eg, break apart FISH) per test | within 1 d | CapEx $ Reagents $$ Marker $$$ Interpr $ |
|
| Sanger | Yes | No | No | No | No | Typically, one marker (hotspot variant) or small region with few markers (eg, H3-3A K28 and G35) per test | Within 1 d | CapEx $ Reagents $ Marker $$ Interpr $ |
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| Pyro-Seq | Yes | No | No | No | Yes | Typically, one marker (eg, hotspot variant) or small region (MGMT CpG sites) with one test | Within 1 d | CapEx $$ Reagents $$ Marker $$ Interpr $ |
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| (MS-) MLPA |
Yes | Yes | No | No | Yes | Typically, one or two combined markers per test (eg, 1p/19q and IDH1/IDH2, or IDH1/IDH2 and MGMT promoter) | Within 1 d | CapEx $ Reagents $$ Marker $$ Interpr $ |
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| qPCR | No | No | Yes | No | No | Typically, one marker per test | Within 1 d | CapEx $ Reagents $ Marker $ Interpr $ |
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| RT-PCR | No | No | Yes | No | No | Typically, one marker per test | Within 1 d | CapEx $ Reagents $$ Marker $$ Interpr $ |
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| MSP | No | No | No | No | Yes | Typically, one marker per test | Within 1 d | CapEx $ Reagents $$ Marker $$ Interpr $ |
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| MSA (LOH) | No | Yes | No | No | No | Within 1 d | CapEx $ Reagents $$ Marker $$ Interpr $ |
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| ddPCR | Yes | No | (Yes) | No | No | Requires low amount of DNA; Very high sensitivity and allows quantitative analysis |
Typically, one or few markers per test (eg, FGFR1 mutation and duplication or BRAFV600E mutation and duplication) | Within 1 d | CapEx $$ Reagents $ Marker $ Interpr $ |
Some methods could potentially be employed for other information as indicated here (eg, RNA-seq could be screened for indication of SNVs), but these are use cases not typically employed and validated in diagnostic settings.
Bold: essential criterion.
Abbreviations: NGS, next-generation-sequencing; FISH, fluorescence in-situ hybridization; (MS-)MLPA, (methylation-specific) multiplex ligation-dependent probe amplification; qPCR, quatitative polymerase chain reaction; RT, real time; MSP, methylation-specific PCR; MSA, microsatellite analysis; LOH, loss of heterozygosity; dd, droplet digital; $–$$$ rough categorization of cost for the items “CapEx”, “Reagents”, “Marker”, "Interpr'; CapEx, Capital Expense for equipment required to run assay; Reagents, reagents for assay; Marker, cost per marker covered per run; Interpr., interpretation cost, from $ (low, marker can be readily evaluated) to $$$ (needs extensive bionformatics pipeline and expertise); LOD, limit of detection.
*850k analysis does not identify SNVs or small insertions/deletions, but the methylation prediction can suggest presence of a certain genomic alteration, for example, if an astrocytoma, IDH-mutant is predicted. Still, it does not discriminate between the different variants of oncogenic IDH alterations.
**Duration applies to mere lab workflow from already extracted analyte to raw results; some assays require pooling samples over several days or even weeks depending on laboratory size, multiplying the duration. In turn, pooling samples in order to streamline lab processes applies for most assays. However, this aspect is only specifically mentioned for those assays which cannot run efficiently for single/low number of samples.