--- module: 092-03 language: en chapter: 92 title: "Chromosomes, Human Variation, Population Genetics, and Genomic Methods" module_title: "Cytogenetics, sequencing, genomic methods, variant interpretation, and clinical limits" source_sha256: 69044f1433461384e4416c7bc89d4b71ecdcc37921f9a0bf241841d75bc4cce6 --- # Genomic methods, variant interpretation, and limits ## Choosing a test ### Genomic testing is not one technology ### Each method has blind spots ### Select by phenotype and suspected mechanism ### Integrate quality, frequency, function, context ## Cytogenetic methods ### Karyotype: aneuploidy and large rearrangements #### Detects balanced translocations and inversions #### Misses small copy and nucleotide changes #### Culture can select against cell populations ### FISH: labelled probes on selected regions #### Break-apart shows disruption, fusion juxtaposition #### Normal result does not exclude other abnormalities ### Microarray measures genome-wide copy number #### Submicroscopic deletions and duplications #### Long regions of homozygosity #### Misses balanced changes, repeats, small variants ## Arrays and PCR ### SNP arrays assay predefined markers #### Imputation depends on LD and ancestry #### Rare and structural variants imputed poorly ### PCR amplifies a selected region #### Quantitative, reverse-transcription, digital #### Allele dropout, contamination, primer-site variants ## Sequencing technologies ### Sanger: accurate reading of a targeted fragment #### Confirms variants and familial sites #### Misses copy number and low-level mosaicism ### Short-read sequencing reads millions of molecules #### Panel, exome, genome breadth #### Align, call, filter, annotate, review ### Coverage is uneven #### Repeats, pseudogenes, high GC are difficult #### Mean depth does not prove every base #### Orthogonal confirmation for hard findings ### Short reads struggle with phasing and long repeats ### Long reads span structure, repeats, haplotypes ### No platform is comprehensive alone ## RNA and methylation ### RNA sequencing: expression, splicing, fusion #### Depends on tissue expression and stability #### Nonsense-mediated decay may erase transcript #### Complements, not replaces, DNA analysis ### Methylation: imprinting, silencing, episignatures #### Bisulfite is indirect and damages DNA #### Varies by tissue, age, cell composition ## Nomenclature and classification ### Specify reference, coordinate, alleles, protein #### Transcripts change exon numbers and effects ### Five germline classification categories #### Evidence of association, not personal certainty ### Loss of function needs an established mechanism #### Late stop, exon skipping, in-frame splice #### Gene may tolerate haploinsufficiency ### Missense predictions are supporting evidence ### Segregation strengthens evidence #### Unaffected carrier weakens, not refutes ### De novo strong when parentage confirmed ## Uncertain and somatic results ### Uncertain variants should not drive irreversible acts #### Reclassification as evidence grows #### Patients need a plan for review ### Negative result does not exclude genetic disease ### Tumour allele fraction reflects purity, clonality ### Tumour variant may reveal germline predisposition ### Clonal haematopoiesis mimics findings ## Reproductive and population testing ### Cell-free DNA is screening, not diagnosis #### Placental mosaicism, vanished twin, low fraction #### Predictive value depends on prior probability ### Newborn screening targets treatable conditions #### Screen-positive result needs confirmation ### Preimplantation testing of trophectoderm cells #### Not an absolute guarantee ## Validity, equity, iteration ### Secondary findings affect relatives ### Analytical, clinical validity and utility distinct ### Underrepresented groups get more uncertainty ### Avoid race as a shortcut for genotype ### Negative result reflects current knowledge