Lecture 10a Polymorphism Discovery and Genotyping

Lecture 10a - Polymorphism Discovery and Genotyping - BIO4BI3 Bioinformatics

• Lecture covered polymorphism discovery and genotyping methods within the context of bioinformatics.
• The workflow encompasses DNA sequencing, quality control, assembly, genome annotation, expression analysis, marker-trait associations, population analysis, genotyping, and polymorphism discovery.

Learning Objectives

• Understand polymorphisms and their four broad types.
• Describe genotyping and the different methods used.
• Identify some genotyping software tools.
• Recognize challenges associated with genotyping.
• Explain genome imputation and its various approaches.

Types of Polymorphisms

• A polymorphism is a difference in an individual's genome relative to a reference genome.
• Genomes evolve through mutation, duplication, and deletion.
• Four broad categories of polymorphisms:
  • SNPs (single nucleotide polymorphisms): Changes in single bases within a genome.
  • Indels (insertions/deletions): Insertion or deletion of 1 or more bases compared to a reference.
  • CNVs (copy number variations): Differences in the number of copies of a genetic element.
  • Structural variants: Changes larger than 1kb, including insertions, deletions, inversions, translocations, and rearrangements.

What is Genotyping

• Genotyping is determining the alleles at specific positions in an individual's genome relative to a reference.
• Genotyping positions are often predetermined and separate from polymorphism discovery.
• Several methods exist for genotyping:
  • Genome sequencing-based methods (low cost per data point, whole genome sequencing).
  • Microarray-based genotyping (fixed panels of SNPs).
  • PCR-derived methods (Tag-Man, KASP, HRM - rapid, reliable, lower throughput).

DNA Sequencing-Based Genotyping

• Advances in sequencing technology lead to more sequencing-based genotyping approaches.
• Approaches include whole-genome sequencing (low coverage), targeted amplification, and reduced representation.
• Exome sequencing and GBS are examples of reduced representation methods.

Genotyping-by-Sequencing

• A detailed diagram of the genotyping-by-sequencing process was provided.
• Steps include: Constructing restricted representation libraries (RRLs) using restriction enzymes, ligating custom barcoded adaptors, pooling samples, performing PCR amplification, and using barcodes to assign sequences and produce DNA sequence data for each sample.

Polymorphism Discovery vs. Genotyping

• Genotyping approaches (microarrays, PCR) separate polymorphism discovery and genotyping.
• Sequencing-based approaches (GBS, exome) perform both simultaneously.
• Genotyping known positions provides highly consistent results, but often involves technological investment.
• Sequencing-based approaches are typically cost-effective per individual but may not consistently produce genotypes at the same positions.

Tools for Discovery and Genotyping

• Modern polymorphism discovery utilizes whole-genome sequencing and long-read technologies.
• Sequencing-based genotyping relies on mapping sequencing reads to a reference genome. Key software tools like Samtools, GATK, and Deep Variant assist in calling SNPs/variants.
• GATK pipeline details the steps involved in processing DNA.

Deep Variant

• A deep neural network approach used for SNP/variant discovery and genotyping.
• Claims greater accuracy than other approaches (e.g., GATK) by analyzing image representations of sequencing data.

Discovery Challenges

• Sequencing errors impact read mapping, reducing reliability of results.
• Alignment errors and complex genomes create challenges in accuracy.
• Genotyping in polyploidy genomes is complicated; requires more genome coverage for sufficient statistical certainty.
• Genome duplications introduce challenges in accurate genotype determination.

Why We Genotype

• Understand genetic relationships among individuals.
• Understand genetic diversity in a population.
• Undertake genealogical research.
• Predict phenotype from genotype through disease classification, marker-assisted backcrossing, or genomic selection.
• Perform genome-wide association studies to identify phenotype/genotype relationships.

Phased Genotyping

• Genomes usually contain one maternal and one paternal copy of each chromosome.
• Phased genotyping aims to determine which copy came from which parent for specific genomic locations.
• Phased assembly is increasingly applied toward heterozygous individuals and enables more accurate imputation.

Imputing Genotypes

• All genotyping methods have data gaps.
• Genome imputation fills in missing data based on a reference panel of known genotypes, improving data completeness.
• Imputation algorithms use statistical relationships, statistical allele frequencies, and parental haplotype maximization.
• Common imputation software includes PLINK, BEAGLE, MACH, and GLIMPSE.

This quiz covers essential concepts in polymorphism discovery and genotyping methods in bioinformatics. It explores DNA sequencing, quality control, genome annotation, and the challenges associated with genotyping. Test your understanding of polymorphisms and the tools used in this field.