Next-Generation Sequencing Methods and Gene Filtering

Next-Generation Sequencing Methods

• Whole Exome Sequencing (WES): Focuses on protein-coding regions of the genome, efficient for identifying protein-altering mutations.
• Whole Genome Sequencing (WGS): Analyzes the entire genome; more comprehensive but often more resource-intensive than WES.
• Both WES and WGS are high-throughput methods used for rapid disease-gene identification and personalized medicine.

Candidate Gene Filtering using WES

• Filtering Process:
  • Removes common genetic variants, focusing on rare mutations.
  • Prioritizes protein-altering variants over non-coding ones.
• Validation:
  • Reduces a large number of variants to a manageable few candidate genes.
  • Confirms findings with family segregation analysis and Sanger sequencing.

Functional Evidence for Causality

• Further Tests: Checks gene expression, detects protein in patient samples, investigates how the mutation impacts protein function or tissue development.
• Model Organisms: Investigates pathways/mechanisms using in vitro/in vivo models. Validation through knockdown/overexpression studies to observe resulting phenotypic effects.

Cell Culture (In Vitro)

• Overview: Growing animal cells in controlled conditions outside the body.
• Benefits: Cost-effective, rapid, repeatable, and reduces reliance on live animals.
• Types:
  • Primary cells: Limited growth potential.
  • Immortalized cell lines: Continuous growth.
  • Tissue-specific cell lines: Widely available.

Gene Knockdown Techniques

• RNA Interference (RNAi): Used to reduce expression of specific genes.

  • shRNA: Delivered via DNA plasmid and transcribed in cells.
  • siRNA: Chemically synthesized and directly introduced into cells, bypassing transcription.

• shRNA Function: Simulates microRNA gene silencing function, the process involves the Dicer enzyme and RISC complex to degrade mRNA.

Induced Pluripotent Stem Cells (iPSCs)

• Creation: Skin fibroblasts are reprogrammed into pluripotent stem cells.
• Use: Useful for studying development and disease, but lacks the complexity of a complete organism.

Animal Models in Research

• Mice: Genetically similar to humans, short lifespan, easy to handle.
  • Precise gene editing is possible (e.g., using Cre-Lox system).
• Zebrafish: Small, rapid reproduction and development outside the mother.
  • Gene knockdown is done using morpholinos.

Gene Editing: CRISPR-Cas9

• Method: Powerful gene editing technique utilizing guide RNA (gRNA) to target specific DNA sequences.
• Mechanism: Cas9 enzyme creates double-strand DNA breaks, repaired via:
  • NHEJ: Can lead to indels (insertions/deletions).
  • HDR: Allows precise edits using a repair template.

Genetic Analysis Approaches

• Forward Genetics: Starts with a phenotype and determines the underlying genetic cause.
• Reverse Genetics: Begins with a gene mutation and studies its associated phenotypic effects.