End-to-End NGS Analysis & Clinical Variant Interpretation Pipeline
From sequencer FASTQ files through alignment, deduplication, BQSR, variant calling (germline & somatic), functional annotation, clinical repositories (gnomAD/ClinVar/COSMIC/OMIM), to an exhaustive deconstruction of all 28 ACMG/AMP evidence criteria, Bayesian combining matrices, and automated clinical report generation.
Curriculum Modules
Click on any module below for deep-dive production code, architectural diagrams, bash pipelines, and diagnostic criteria.
1: The Missing Bridge: Connecting Bioinformatics to Clinical Decision Making
Why raw VCF coordinates are clinically silent, the distinction between research discovery and diagnostic pathology, and the 4-phase transformation from raw data to patient care.
2: End-to-End Pipeline Architecture & Workflow Flowchart
Complete unbroken workflow diagram from sequencer FASTQ files through alignment, deduplication, BQSR, variant calling, annotation, and clinical diagnostic reporting.
3: Step 1 — Quality Control (QC) & Preprocessing
FASTQ four-line structure, Phred logarithmic quality score calculations, FastQC metric evaluation, and automated adapter/low-quality base trimming with FastP and Trimmomatic.
4: Step 2 — Read Alignment & Reference Genome Mapping
Human reference builds (GRCh38 vs hs37d5/hg19), Burrows-Wheeler Transform index construction, BWA-MEM read group injection, and STAR 2-pass spliced alignment.
5: Step 3 — Post-Alignment Processing: Deduplication & GATK BQSR
Coordinate sorting, optical vs PCR duplicate marking with GATK MarkDuplicates, and Base Quality Score Recalibration (BQSR) modeling systematic machine error.
6: Step 4 — Variant Calling: Germline vs. Somatic Architecture
Path A: Germline calling with GATK HaplotypeCaller (GVCF), CNNScoreVariants, and DeepVariant vs Path B: Somatic calling with MuTect2, Strelka2, and cross-sample contamination filtering.
7: Step 5 — Functional Annotation Engines & In Silico Predictors
Converting chromosomal coordinates into biological consequences with Ensembl VEP, ANNOVAR, and SnpEff, plus missense impact benchmarking with CADD, REVEL, and AlphaMissense.
8: Step 6 — Clinical & Population Repositories: gnomAD, ClinVar, COSMIC & OMIM
Population allele frequency filtering with gnomAD (BA1/BS1 cutoffs), ClinVar submission evaluation & 4-star hierarchy, somatic hotspots in COSMIC, and OMIM phenotype linkage.
9: Step 7 — The ACMG/AMP 2015 Masterclass: All 28 Criteria Deconstructed
Exhaustive criteria analysis: PVS1 decision tree, PS1-4 strong evidence, PM1-6 moderate evidence, PP1-5 supporting evidence, stand-alone BA1, BS1-4, and BP1-7 benign criteria.
10: ACMG Combining Rules, Bayesian Framework & Clinical Report Generation
Combining rules matrix for 5-tier classification, Tavtigian Bayesian odds-of-pathogenicity formulation, resolving conflicting VUS evidence, and automated clinical PDF reporting.