🧬Genomics Unit 2 – DNA Sequencing Technologies and Genome Assembly

DNA Basics and Structure

• DNA (deoxyribonucleic acid) is the hereditary material in humans and almost all other organisms
• Consists of two strands that coil around each other to form a double helix structure
  • Each strand is made up of a sugar-phosphate backbone with nitrogenous bases attached
  • Four types of nitrogenous bases: adenine (A), thymine (T), guanine (G), and cytosine (C)
• Bases on opposite strands pair specifically: A with T and C with G, held together by hydrogen bonds
• The sequence of these bases along the backbone encodes genetic information for the development, functioning, growth and reproduction of all known organisms
• DNA segments that carry this genetic information are called genes
• During cell division, DNA is replicated semi-conservatively, meaning each strand serves as a template for the production of the complementary strand

Sequencing Methods Overview

• DNA sequencing determines the precise order of nucleotides within a DNA molecule
• Enables us to decipher genetic information and understand the blueprint of life
• Sanger sequencing, developed by Frederick Sanger in 1977, was the first widely adopted method
  • Uses dideoxynucleotides (ddNTPs) to terminate DNA synthesis at specific bases
  • Produces DNA fragments of varying lengths, which are then separated by size using gel electrophoresis
• Maxam-Gilbert sequencing, another early method, uses chemical cleavage of DNA at specific bases followed by electrophoresis
• Pyrosequencing detects the release of pyrophosphate (PPi) during DNA synthesis, which is converted into light by a series of enzymatic reactions
• Sequencing by ligation (SBL) uses DNA ligase to identify the nucleotide present at a given position in a DNA sequence
• Single-molecule real-time (SMRT) sequencing captures the addition of fluorescently labeled nucleotides during DNA synthesis in real-time

Next-Generation Sequencing Technologies

• High-throughput sequencing methods that parallelize the sequencing process, producing thousands or millions of sequences concurrently
• Illumina (Solexa) sequencing uses reversible dye-terminators to detect single nucleotides as they are incorporated into growing DNA strands
  • Amplifies DNA fragments on a glass slide and uses reversible dye terminators to perform sequencing by synthesis
  • Most widely used NGS platform due to its high accuracy and low per-base cost
• Roche 454 sequencing uses pyrosequencing technology on a micro-scale, detecting light emitted during nucleotide incorporation
• Ion Torrent sequencing detects hydrogen ions released during DNA polymerization using semiconductor technology
• SOLiD (Sequencing by Oligonucleotide Ligation and Detection) uses ligation-based sequencing and a two-base encoding system for improved accuracy
• PacBio SMRT sequencing captures the addition of fluorescently labeled nucleotides during DNA synthesis in real-time, enabling long read lengths
• Oxford Nanopore sequencing detects changes in electrical current as DNA molecules pass through a protein nanopore, allowing for ultra-long read lengths

Genome Assembly Strategies

• Process of aligning and merging DNA sequence fragments to reconstruct the original genome
• Two main approaches: de novo assembly and reference-guided assembly
  • De novo assembly reconstructs the genome from scratch without using a reference genome
  • Reference-guided assembly aligns sequence reads to a pre-existing reference genome of a closely related organism
• Overlap-Layout-Consensus (OLC) algorithm finds overlaps between reads, constructs a graph, and determines the most likely genome sequence
  • Works well for long reads (e.g., Sanger, PacBio) but computationally intensive for short reads
• De Bruijn Graph (DBG) approach breaks reads into shorter k-mers, constructs a graph from these k-mers, and finds a path through the graph to assemble the genome
  • Suitable for high-coverage, short-read data (e.g., Illumina)
• Greedy algorithm selects the highest-scoring overlap at each step and merges the corresponding reads until no more overlaps are found
• Hybrid assembly combines the advantages of both short and long reads by using long reads to resolve complex regions and short reads for accuracy

Bioinformatics Tools for Assembly

• Software programs designed to handle the vast amounts of data generated by NGS technologies and assist in genome assembly
• Quality control tools (e.g., FastQC, PRINSEQ) assess the quality of raw sequence data and perform necessary filtering and trimming steps
• Short-read assemblers:
  • Velvet uses a de Bruijn graph approach and handles both single-end and paired-end reads
  • SOAPdenovo is a de Bruijn graph-based assembler designed for large genomes and supports multiple k-mer sizes
  • ABySS (Assembly By Short Sequences) is a distributed de Bruijn graph assembler that can handle large genomes using minimal computing resources
• Long-read assemblers:
  • Canu is a fork of the Celera Assembler designed for high-noise single-molecule sequencing (e.g., PacBio, Oxford Nanopore)
  • Falcon is a Hierarchical Genome Assembly Process (HGAP) that uses DAG-Chainer to construct contigs from long reads
• Hybrid assemblers (e.g., SPAdes, MaSuRCA) leverage the strengths of both short and long reads to produce high-quality assemblies
• Genome scaffolding tools (e.g., SSPACE, BESST) order and orient contigs into larger scaffolds using paired-end or mate-pair information
• Assembly quality assessment tools (e.g., QUAST, REAPR) evaluate the quality and completeness of genome assemblies using various metrics and benchmarks

Challenges and Limitations

• Repetitive sequences, such as transposable elements and tandem repeats, can lead to ambiguities and gaps in the assembly
  • Short reads may not span the entire length of a repeat, making it difficult to resolve their exact location and copy number
• Sequencing errors, particularly in long-read technologies, can introduce false variations and complicate the assembly process
• Heterozygosity in diploid organisms can result in the assembly of separate contigs for each allele, increasing assembly complexity
• Computational resources and storage requirements can be limiting factors, especially for large, complex genomes
• Incomplete or fragmented assemblies may miss important genomic regions, leading to an incomplete understanding of the organism's biology
• Low-complexity regions, such as homopolymers and GC-rich areas, can be challenging to sequence accurately and assemble correctly
• Contamination from other organisms (e.g., bacteria, viruses) can introduce foreign sequences into the assembly, requiring careful filtering and validation
• Validation and benchmarking of assembly quality can be difficult, particularly for non-model organisms lacking a reference genome

Applications in Research and Medicine

• Comparative genomics: studying the similarities and differences between genomes of different species to understand evolutionary relationships and identify conserved functional elements
• Variant detection: identifying single nucleotide polymorphisms (SNPs), insertions, deletions, and structural variations associated with diseases or traits of interest
• Transcriptomics: sequencing and quantifying RNA transcripts to study gene expression patterns and identify novel transcripts or alternative splicing events
• Metagenomics: sequencing DNA from environmental samples to study microbial communities and their interactions without the need for cultivation
• Personalized medicine: using an individual's genome sequence to tailor medical treatments, predict disease risk, and develop targeted therapies
  • Pharmacogenomics: studying how genetic variations influence drug response and toxicity to optimize medication dosing and minimize adverse effects
• Agrigenomics: applying genomic technologies to improve crop yields, resistance to pests and diseases, and nutritional quality
• Forensics: using DNA sequencing to identify individuals, determine kinship, or link suspects to crime scenes based on genetic evidence
• Ancient DNA analysis: sequencing DNA from historical or archaeological samples to study past populations, migrations, and evolutionary changes

Future Trends in Sequencing and Assembly

• Increasing read lengths and accuracy of long-read sequencing technologies (e.g., PacBio HiFi, Oxford Nanopore) will improve the contiguity and completeness of genome assemblies
• Integration of multiple sequencing technologies (e.g., short reads, long reads, linked reads) will become more common to leverage the strengths of each approach
• Advances in computational methods, such as machine learning and artificial intelligence, will help to automate and optimize the assembly process
• Cloud computing and distributed systems will enable the assembly of large, complex genomes using scalable resources and collaborative platforms
• Portable, real-time sequencing devices (e.g., Oxford Nanopore MinION) will facilitate in-field sequencing and rapid outbreak response
• Single-cell sequencing will provide insights into cellular heterogeneity and enable the assembly of genomes from rare or unculturable organisms
• Improved algorithms for haplotype phasing and diploid genome assembly will help to resolve allelic variations and provide a more complete view of an organism's genetic makeup
• Standardization of assembly quality metrics and benchmarking datasets will facilitate the comparison and reproducibility of genome assembly studies