MOLECULAR GENETICS AND CYTOGENETICS LABORATORY AND METHODS KARLA PLEVOVÁ 20.11.2020 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 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 ¡ 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 laboratory report – interpretation ← a basis for laboratory test request HOW DOES IT LOOK THE MG LAB? Millar et al, J Clin Microbiol 2002 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? CELL CULTURE ¡ Culturing of peripheral blood, bone marrow, tissue sections, … cell culture incubators Sterile hood LIQUID BIOPSIES Very low amount of material ¡ Plasma / serum ¡ Urine ¡ Joint fluid ¡ Cerebrospinal fluid When invasive biopsies are not an option NUCLEIC ACID (RNA, DNA) ISOLATION ¡ pre-PCR area ¡ Manual and automated sample processing NUCLEIC ACID QUANTIFICATION ¡ Spectroscopic and fluorimetric methods NUCLEIC ACID QUALITY CONTROL ¡ Electrophoretic methods ¡ Alternative methods to gel electrophoresis ¡ Lower material input NUCLEIC ACID QUALITY CONTROL 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 ¡ PCR components ¡ Template DNA ¡ Primers ¡ Nucleotides ¡ Polymerase PCR THERMOCYCLER ROOM 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 POST-PCR AREA ¡ Performing QC and downstream analyses ¡ DNA sequencing – Sanger, NGS ¡ Genomic arrays ¡ … SANGER SEQUENCING Modification of PCR ¡ single primer extension ¡ Incorporation of dNTPs and ddNTPs Applications ¡ Basic method for sequence variant detection (mutations, breakpoint localization) SANGER SEQUENCING Applied Biosystems™3130 Genetic Analyzer Fragment analysis – modification of the method Sequencing analysis output mutated allele with insertion wt allele 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 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 AMPLICON SEQUENCING PANEL SEQUENCING ¡ Sets of selected regions of interest ¡ Target enrichment by amplification or hybridization ¡ Why to use gene panels: ¡ One disease can be caused by mutations in different genes ¡ Certain genes are diagnostically relevant for several diseases PANEL SEQUENCING ¡ LYNX panel – diagnostics of molecular markers in lymphoid malignancies 1CLL, 2MCL, 3FL, 4DLBCL, 5ALL, 6Ph-like ALL 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 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 GENOMIC ARRAYS ARRAY-BASED COMPARATIVE GENOMIC HYBRIDIZATION (ACGH) hybridization of labeled DNA analysis of hybridization efectivity fluorescence ratio DNA reference/sample <1 amplification reference DNA tested DNA mix fluorescence ratio DNA reference/sample >1 deletion GENOMIC ARRAYS SINGLE NUCLEOTIDE POLYMORPHISM (SNP) ARRAY DNA hybridization signal intensity capture tested DNA comparison of signal intensity to internal controls labeling EQUIPMENT FOR GENOMIC ARRAYS Hybridization oven Washing & staining system Scanner Affymetrix GeneChip System CYTOGENETICS LAB ¡ Primary sample processing ¡ Cell culturing ¡ Methods not requiring PCR ¡ Imaging methods CLASSICAL CYTOGENETICS – CHROMOSOME BANDING TECHNIQUES 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 THE END… Contact: karla.plevova@mail.muni.cz or plevova.karla@fnbrno.cz Internal Medicine – Hematology and Oncology, University Hospital Brno CEITEC MU Thank you for your attention!