Molecular genetics and cytogenetics laboratory and methods KARLA PLEVOVÁ 21.2.2023 Statement This presentation is intended exclusively for educational purposes. Every form of misuse, including copying, distribution and sharing on public online platforms or social media is strictly prohibited and can be punished. Outline of the presentation What to expect from a molecular genetics and cytogenetics laboratory (MGC lab)? How does it look in the MGC lab? What methods are available in the MGC lab? What to expect from a MGC lab? MGC LAB = PARTNER Specifics of a MG lab – a need for assays designed for individual families or individual patients → a high proportion of laboratory developed tests compared to other diagnostic labs in hospitals Discuss with the staff, learn what methods they use, know what the methods can be good for Application of results Establishing and refining diagnosis Prenatal and preimplantation testing Hereditary predisposition assessment Disease prognostication Treatment optimization Disease activity monitoring Disease complication diagnostics Prochazkova et al, BMC Medical Education 2019 Technical aspects of the laboratory methods Target regions, analytes Specificity and sensitivity, limit of detection, … Tools for data analysis and their limitations Time for processing – few hours or few days? Standardization and validation Regular quality assessment Compliance with legislation regulations → a basis for laboratory test request → expectations and outcomes Laboratory manual! Practical example BRONCO diagnostic panel for hereditary cancer syndromes • What genetic syndrome am I looking for? • Are suspect genes included in the panel? • What defects could I expect? • How quickly do I need the result? • … • Is the method suitable for answering my questions? Result of a laboratory test The report is an essential part of any laboratory test Content – concise but comprehensive: What exactly was tested What method was used What results were obtained Who reports the results Who reviewed the results What preanalytic, analytic, and postanalytic factors could influence the results What cannot be identified (e.g. failed or low covered regions) Comply with ISO 15189 technical requirements for medical laboratories How does it look the MG lab? Sample reception Primary processing Pre-PCR area Post-PCR area Other post-PCR methods Cultivation room Analytical space Pre-PCR Post-PCR Sample biobank Computation, data storage Quality control requirements Internal QC External QC national international Needed for method (and laboratory) accreditation Compliance with international regulations for in vitro diagnostic methods https://ukneqas.org.uk/about-us/ Sample processing and separation Diverse input material (peripheral blood, tissue specimens etc.) Sterile hoods (esp. in connection with cell cultivation and biobanking of samples) Cell separation needed in specific contexts (e.g. analysis of somatic changes Materials used Peripheral blood Bone marrow Liquid biopsies Aspirates Fine-needle biopsies Fresh tissue Formalin-fixed paraffin-embedded (FFPE) tissue Swabs (e.g. buccal) Postnatal genetics X Prenatal testing X Oncology Peripheral blood processing Different cell population used according to the application: Leukocytes Mononuclear cells Granulocytes Lymphocytes Specific cell subpopulations Why to perform cell separation? Example of TP53 gene testing in chronic lymphocytic leukemia Polymerase chain reaction (PCR) Fundamental reaction of molecular biology and genetics Amplification of regions of interests PCR assembling in pre-PCR area Carried out in thermocycler Various modifications real-time PCR Quantitative method – fluorescent detection of generated products Need for specific primers and probes Relative and absolute quantification test sample Droplet digital PCR (ddPCR) Alternative method for marker absolute quantification Highly precise Need for specific instrumentation 1 2 3 4 Next-generation sequencing (NGS) ~ massively parallel sequencing (MPS) PCR amplification of DNA fragments or direct sequencing of individual fragments (single molecule sequencing) The most common approach – sequencing by synthesis (Illumina sequencers) Millions of fragments are amplified simultaneously (vs capillary sequencer max 96 reactions) Short reads (tens to hundreds base pairs) NGS - targeted regions Illumina machines and their capacity HiSeq 4000 12 genomes/run, 1.5 TB/run NextSeq 500 1 genome/run, 120 GB/run MiSeq 0.15 genome/run, 15 GB/run NovaSeq 48 genomes/run, 6 TB/run MiniSeq 0.07 genome/run, 7.5 GB/run iSeq 0.01 genome/rin, 1.2 GB/run New sequencing machines on the market Oxford Nanopore Technologies Element Biosystems Complete Genomics Singular Genomics PacBio Short-read vs. long-read sequencing Kraft & Kurth, Medizinische Genetik 2019 Schwarz et al, Medizinische Genetik 2021 NGS – regions of interest genome exome selected genes or loci 3 200 000 000 bp 30 x read depth 20 000 genes 100 x read depth < 100 genes ≥ 1000 x read depth Practical example Limit of detection (LoD) of various NGS methods McNulty et al, J Mol Diagn 2020 In theory… (binomial sampling statistics) method Amplicon Panel WES WGS coverage 10 000 x 1000 x 100 x 30 x LoD ~ 1 % ~ 5 % 15–20 % ~ 30 % NGS – diverse targeted markers BCR- ABL AGTAATATGCCT ACCAATATAACT AG-ACCATATG-CT Panel NGS Sets of selected regions of interest Target enrichment by amplification or hybridization Why to use gene panels: One disease can be associated with variants in different genes Certain gene is diagnostically relevant for several diseases Jennings et al, J Mol Diagn 2017 Practical example LYNX panel – diagnostics of molecular markers in lymphoid malignancies 1CLL, 2MCL, 3FL, 4DLBCL, 5ALL, 6Ph-like ALL Navrkalova et al, J Mol Diagn 2021 Whole exome sequencing (WES) Identification of causative variants Discovery of novel genetic markers Searching for treatment targets Whole genome sequencing (WGS) Mainly experimental method for exploring unknown variants Applications similar to WES, additional information about non-coding regions and chromosomal abnormalities Typical sequencing coverage ~ 30–100x – detection of somatic clonal or germline mutations Shallow sequencing (~ 0.5-10x coverage) – genome-wide detection of chromosomal abnormalities, low yield of mutation detection In clinical practice a potential benefit of combination of shallow and panel sequencing 8 11 13 18 Practical example Benefits of NGS in cancer diagnostics and monitoring Reconstruction of clonal architecture and cancer evolution Need for multidisciplinary team – biologists, geneticists, computational scientists, bioinformaticians, statisticians Single-cell technologies Experimental methods Applications in cancer research, immunology, developmental biology, … Analysis of DNA variants, chromatin activity, RNA expression profiles, protein expression, … Coexistence of cellular features in single cells diagnosis pretreatment (run1) pretreatment (run2) 2nd relapse 3rd relapse https://www.10xgenomics.com/ Cytogenomics lab Sample reception Primary processing Pre-PCR area Post-PCR area Microscopic techniques Cultivation Analytical space Sample biobank Schoumans J et al, 2016 Aneu- ploidy CNA Poly- ploidy Clonal heterogeneity Focal amplification Balanced rearrangements Unbalanced rearrangements cn-LOH Classical cytogenetics +++ + +++ +++ ++ +++ +++ Interphase FISH +++ ++ + +++ +++ +++ ++ ArrayCGH +++ ++ - + +++ - ++ - CGH+SNP array +++ +++ + + +++ - ++ ++ SNP array +++ +++ ++ ++ +++ - ++ +++ CNA – copy number alteration, gains or lossess of genetic material cn-LOH – copy neutral loss of heterozygozity Cytogenomics methods Comparison of sensitivity of the techniques Classical cytogenetics chromosome banding techniques Methods not requiring PCR Imaging methods Cheap Clonal composition assessment Molecular cytogenetics Fluorescent in situ hybridization (FISH) Targets specific regions based on DNA sequence Detection of chromosomal abnormalities with diagnostic, prognostic and predictive value Probe types: centromeric genewhole chromosome Molecular cytogenetics FISH methods for genome-wide analysis mFISH mBAND Genomic arrays Molecular cytogenetic technique for detection of genomic gains and losses Detection of copy-neutral loss of heterozygosity Not possible to detect balanced rearrangements Precise breakpoint localization, identification of affected genes High resolution, genome-wide Working with DNA, no need for viable cells Equipment for genomic arrays hybridization oven washing & staining system scanner Affymetrix GeneChip System Practical example Muscular dystrophy Panel NGS – imbalance of SNPs on chr2 Genomic array – UPD chr2 in 80% of cells – germline mosaicism Take-home messages • Discuss • Standardize • Integrate The end… Contact: karla.plevova@mail.muni.cz or plevova.karla@fnbrno.cz Thank you for your attention!