NGS Linkage and GWAS Analysis

This repository contains the analyses performed as part of a Next-Generation Sequencing (NGS) methodologies project, focusing on the genetic investigation of two diseases with distinct genetic architectures:

• Clouston disease (monogenic, autosomal dominant)
• Rheumatoid arthritis (complex, multifactorial)

The project combines genetic linkage analysis and association studies using standard statistical genetics tools.

tools

• R (paramlink, qqman)
• PLINK
• MERLIN
• FBAT

📁 repository structure

simplified structure for project submission, the report in docs/

.
├── code/
│   └── main.ipynb
├── assets/
├── report/
│   └── NGS_Methodologies_Report.pdf
└── README.md

Project Overview

Clouston Disease (Monogenic Disorder)

For Clouston disease, family-based analyses were performed to identify the disease-associated locus.

1. Genetic Linkage Analysis (LOD score)

• Parametric linkage analysis using the LOD score method
• Analysis conducted on chromosome 13 using microsatellite markers
• Evaluation of different recombination fractions (θ values)

Example family structure used in the analysis:

Figure 1: Example pedigree used for linkage analysis. Affected individuals are shown in black.

LOD score curve for a representative marker:

Figure 2: LOD score as a function of recombination fraction (θ). The maximum LOD score is observed at θ ≈ 0.

2. Familial Association Analysis (TDT)

Following linkage analysis, candidate variants within the GJB6 gene were tested using the Transmission Disequilibrium Test (TDT).

Figure 3: TDT results for SNPs within the GJB6 gene. SNP rs76179836 shows significant association.

Linkage disequilibrium verification using Ensembl:

Figure 4: Linkage disequilibrium (r²) between rs76179836 and surrounding variants, indicating the SNP is likely causal.

Rheumatoid Arthritis (Complex Disease)

For Rheumatoid Arthritis (RA), both linkage and genome-wide association analyses were performed.

1. Non-Parametric Linkage Analysis (sib-pairs)

• Analysis conducted using MERLIN
• Identification of suggestive and significant linkage regions

LOD score plot for chromosome X:

Figure 5: Non-parametric linkage (NPL) analysis for chromosome X. The red line indicates the significance threshold.

LOD score plot for chromosome 6:

Figure 6: Suggestive linkage region on chromosome 6 around 50–53 cM.

2. Genome-Wide Association Study (GWAS)

• GWAS performed using PLINK
• Allelic and genotypic association tests
• Bonferroni correction applied for multiple testing

Manhattan plots of GWAS results:

Figure 7: Manhattan plots for allelic (left) and genotypic (right) association tests. No variants surpass the Bonferroni threshold.