|
Week - 1 |
Introduction to Molecular Diagnostics
Content:
• Definition and historical development of molecular diagnostics
• The role of molecular diagnostics in clinical laboratories
• Molecular methods in genetic, oncological, infectious disease, and pharmacogenomic diagnostics
• Overview of pre-analytical, analytical, and post-analytical processes
• Concepts of quality, accuracy, and clinical utility in molecular diagnostics
|
|
Week - 2 |
Clinical Sample Types and Pre-Analytical Processes
Content:
• Blood, tissue, FFPE, swab, cerebrospinal fluid, plasma, serum, and urine samples
• Sample collection, transportation, and storage conditions
• Sample stability
• Pre-analytical errors
• Sources of contamination and prevention strategies
• Sample acceptance and rejection criteria in clinical laboratories
|
|
Week - 3 |
Nucleic Acid Isolation
Content:
• Isolation of DNA, RNA, cell-free DNA, and viral nucleic acids
• Manual and automated isolation systems
• Spin-column, magnetic bead-based, and organic extraction methods
• DNA/RNA isolation from FFPE samples
• RNase control in RNA isolation
• Factors affecting isolation yield and quality
|
|
Week - 4 |
Nucleic Acid Quality Control and Sample Preparation
Content:
• Measurement of DNA/RNA concentration
• Spectrophotometric analysis: A260/280 and A260/230 ratios
• Fluorometric quantification methods
• Quality assessment by gel electrophoresis
• RIN, DIN, and fragment analysis
• PCR inhibitors
• Use of internal controls and extraction controls
|
|
Week - 5 |
Conventional PCR and PCR-Based Diagnostic Approaches
Content:
• Basic principles of PCR
• Primer design
• PCR components and reaction optimization
• Conventional PCR
• Nested PCR
• Multiplex PCR
• Allele-specific PCR
• PCR controls
• False-positive and false-negative results
|
|
Week - 6 |
RT-PCR, qPCR, and RT-qPCR
Content:
• Principles of reverse transcription
• Fundamentals of qPCR technology
• SYBR Green-based and probe-based qPCR
• Ct/Cq values and amplification curves
• Melting curve analysis
• Absolute and relative quantification
• ΔCt and ΔΔCt methods
• Applications of RT-qPCR in viral load measurement, gene expression analysis, and pathogen detection
|
|
Week - 7 |
Digital PCR and Advanced Quantification Approaches
Content:
• Principles of digital PCR
• Droplet digital PCR and chip-based digital PCR systems
• Absolute quantification
• Detection of low-frequency variants
• Analysis of copy number variations
• Minimal residual disease monitoring
• Comparison of qPCR and digital PCR
• Clinical advantages and limitations
|
|
Week - 8 |
Sanger Sequencing and Chromatogram Analysis
Content:
• Basic principles of Sanger sequencing
• Chain-termination method
• Capillary electrophoresis
• Chromatogram interpretation
• Identification of heterozygous and homozygous variants
• Evaluation of mosaic variants
• Clinical applications of Sanger sequencing
• Variant confirmation by Sanger sequencing
|
|
Week - 9 |
Introduction to Next-Generation Sequencing Technologies
Content:
• Basic principles of NGS technologies
• Amplicon-based sequencing
• Hybrid capture-based sequencing
• Targeted gene panels
• Clinical exome sequencing
• Whole-exome sequencing
• Whole-genome sequencing
• Short-read and long-read sequencing approaches
• Advantages and limitations of NGS technologies
|
|
Week - 10 |
NGS Library Preparation, Quality Control, and Sequencing Metrics
Content:
• Steps of NGS library preparation
• Fragmentation
• Adapter ligation
• Indexing and barcoding
• Target enrichment
• Pooling strategies
• Library quality control
• Q-score, read length, depth, coverage, and uniformity
• Interpretation of low coverage and technical limitations
|
|
Week - 11 |
NGS Bioinformatics Workflow and Variant Analysis
Content:
• FASTQ, BAM/CRAM, and VCF file formats
• Quality control
• Alignment to the reference genome
• Variant calling
• Variant filtering
• Variant annotation
• Analysis of SNVs, indels, CNVs, structural variants, and gene fusions
• Clinical variant prioritization
• Databases: ClinVar, OMIM, gnomAD, dbSNP, COSMIC, CIViC, and ClinGen
|
|
Week - 12 |
Clinical Interpretation of Genetic Variants
Content:
• Distinction between germline and somatic variants
• ACMG/AMP variant classification system
• Pathogenic, likely pathogenic, VUS, likely benign, and benign classifications
• Population frequency
• Functional studies
• Segregation analysis
• In silico prediction tools
• HGVS nomenclature
• Importance of clinical context in variant interpretation
|
|
Week - 13 |
Validation and Quality Management in Molecular Diagnostic Tests
Content:
• Analytical validation and clinical validation
• Difference between test verification and test validation
• Sensitivity, specificity, accuracy, and precision
• Limit of detection
• Linear range
• Repeatability and reproducibility
• Positive and negative predictive values
• Use of reference materials
• Internal quality control and external quality assessment
• Pipeline validation in NGS tests
• Quality management in molecular diagnostic laboratories
|
|
Week - 14 |
Molecular Reporting, Ethical Principles, and Current Applications
Content:
• Main components of a molecular diagnostic report
• Distinction between technical findings and clinical interpretation
• Reporting of test limitations
• Reporting of variants of uncertain significance
• Secondary and incidental findings
• Informed consent and genetic counseling
• Data security and ethical principles
• Applications in oncology, infectious diseases, pharmacogenomics, and liquid biopsy
• Metagenomic NGS, long-read sequencing, and artificial intelligence-supported molecular diagnostic approaches
• General course evaluation and student presentations
|