Next-Generation Sequencing Methods and Gene Filtering

Study Notes

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.

Description

Explore the advanced techniques involved in Next-Generation Sequencing, focusing on Whole Exome Sequencing (WES) and Whole Genome Sequencing (WGS). This quiz covers candidate gene filtering processes and methods used to validate genetic variants, including functional evidence for causality. Ideal for students and professionals interested in genetics and personalized medicine.