Week 6 - P2 - L2 - Detecting Genetic Variation and Variant Calling

A genetic variant is defined as any variation in nucleotide identity at any position within a genome that deviates from a reference sequence.
The reference genome serves as the standardized "wild type" or normal sequence against which an individual or group's genome is compared.
To identify a genetic variant, there must be a baseline reference to determine what constitutes a deviation.
The human genome contains a massive number of genetic variants in any single individual.
The presence of a genetic variant does not automatically imply a biological consequence; the majority of mutations acquired by individuals are neutral.
A primary challenge in genomics is determining which specific variants contribute to or directly cause genetic diseases.

Inherited Mutations: These are passed from parents to offspring and are present in every cell of the individual's body.
Acquired Mutations: These are developed throughout a person's lifetime and are not present in every cell. They can result from environmental factors or exposure to mutagens.
De Novo Mutations: This specific class of mutations is not found in the somatic cells of the parents. They typically arise from a mutation in the sperm, the egg, or very early during the gestation period when there are very few cells.
Mosaicism: This occurs when a de novo mutation happens in one of the few cells early in gestation. All cells derived from the mutated cell will carry the mutation, while cells derived from the non-mutated cells will not. This can result in certain organs or tissues carrying a mutation while others do not, although it is more common for de novo mutations to be present in all cells of the body.

Whole Genome Sequencing (WGS): This approach sequences the entire genome, providing data on variations across all regions, including:
- Introns
- Exons
- Regulatory regions
- Intergenic regions
Exome Sequencing (WES): This focuses exclusively on the exome regions, which represent the coding sequences of the genome. While it neglects most introns and regulatory regions, it does include the $5'$ and $3'$ untranslated regions ( $UTRs$ ) as they are part of the exons.
The choice between WGS and WES depends on the specific goals of the research and where the clinician or researcher expects to find genomic variations.

Fragmentation: The first step involves breaking the whole genome into smaller DNA fragments.
Exon Separation: Because WES targets only exons, specific $cDNA$ probes are used to bind to exon regions within the fragmented genome via complementary base pairing.
Capture: Once the $cDNA$ probes have bound to the target sequences, all non-binding DNA (non-exonic material) is removed.
Captured Population: The remaining material is a population of DNA sequences that match the exons.
Amplification and Sequencing: Each captured DNA fragment is amplified and sequenced to produce a signal. This signal is read to determine the DNA sequence for each fragment, known as a DNA read.
Alignment: The resulting DNA reads are computationally aligned against the known exon sequences of the organism to complete the sequence of the individual's exome.

Variant calling is the systematic process of identifying where variants exist in an individual's genome. It follows a multi-step workflow:

Step 1: DNA Extraction: DNA is collected and isolated from the individual of interest.
Step 2: DNA Sequencing: Performing either WGS or WES based on the type of genetic variations being investigated.
Step 3: Read Alignment: The DNA reads produced during sequencing are aligned against a reference genome.
Step 4: Genotyping: Identifying the specific genotype at every single nucleotide position (e.g., position $1$ , $2$ , $3$ , etc.) in the genome or exome.
- The genotype at any position will be one of the four nucleotides: adenine ( $A$ ), thymine ( $T$ ), cytosine ( $C$ ), or guanine ( $G$ ).
- An individual can be homozygous (carrying two identical alleles) or heterozygous (carrying different alleles) at any specific nucleotide position depending on what was inherited from the mother and father.
Step 5: Variant List Generation: Comparing the identified genotypes to the reference genome to create a list of every position where the individual's sequence differs from the reference. This list can contain thousands or even more variants and specifies if they are homozygous or heterozygous.
Step 6: Interpretation and Annotation: The final step involves prioritizing and assessing the variant list to determine which variations are likely to be pathogenic (disease-causing) versus benign.

Steps $3$ , $4$ , $5$ , and $6$ of the variant calling process require significant computational power and specialized bioinformatics programs.
Read Alignment Confidence: The confidence in a specific nucleotide identity at a certain position is determined by "read depth."
- Example: If $12$ separate reads indicates a $T$ at a specific position, there is high confidence in that call.
- Example: If only $6$ reads cover a position, there is lower confidence compared to a higher read depth.
Visualizing Zygosity:
- Homozygous Variant: In a specific position, the reference might show a guanine ( $G$ ), but every single read from the target individual shows an adenine ( $A$ ).
- Heterozygous Variant: In a specific position, the reference might show a guanine ( $G$ ), but the individual's reads show an adenine ( $A$ ) in half of the reads and a guanine ( $G$ ) in the other half, indicating inheritance of different nucleotides from each parent.

Annotation Tools: Online tools help identify and visualize the most important variants and allow users to manually search for specific genes or regions of interest.
Pathogenicity Assessments: Annotation does not always definitively label a mutation as pathogenic or benign unless it matches a known mutation. Instead, it provides suggestive information.
Manual Verification: Users often must perform additional research or experimentation to establish if a specific variant of interest is actually responsible for a disease state.