Organization of the Human Genome

• Lecture about the organisation of the human genome

Universal Features of Cells

• An estimated 10 million to 100 million living species inhabit Earth
• Humans are eukaryotes comprised of cells from all three life domains
• The human body consists of about 37 trillion human cells
• Approxaimetly 100 trillion bacteria, mainly in the gut, exists in the human body
• Archaea, including methanogens, contribute to less than 1% of intestinal microflora and are responsible for flatulence

Genes and Heredity

• Genes represent the complete sequence of nucleic acids, either DNA or RNA
• Genes contain information crucial for the production of a certain product, like functional RNA or protein
• Genes are the functional units of heredity
• Structural genes form specific DNA sequences, which encode proteins and are the building blocks of life central to biological processes

Genetics vs. Genomics

• Genetics focuses on the study of individual genes inherited from parents/ancestors
• Genetics explores how genes work and what each one is made of, including DNA sequence
• Genetics looks into changes within single genes
• Genomics includes the study of the entirety of human DNA
• Genomics looks in depth on how the DNA functions with other relations
• Genomics looks at changes anywhere in the genome

DNA Sequencing

• DNA sequencing determines the accurate order of nucleotides in DNA.
• Sanger method was introduced in 1975
• Maxam-Gilbert method introduced in 1977
• Automatic sequencing was introduced in 1995
• Next-generation sequencing introduced in 2005

Historical Events in Sequencing Technologies

• 1869: Friedrich Miescher isolates DNA
• 1953: James Watson and Francis Crick's double helix structure of DNA
• 1965: Frederick Sanger etc. published papers for DNA sequencing
• 1977: Walter Gilbert and Allan Maxam developed chemical sequencing
• 1986: ABI develops automated DNA sequencing
• 1995: Sequencing S. cerevesiae genome
• 1996: Development of Pyrosequencers
• 1998: Solexa develops sequencing by synthesis
• 2005: 454 launches high-throughput sequencers

Sanger Sequencing (1977)

• Sanger sequencing determines the nucleotide sequence of DNA
• Dideoxy method, also known as the “chain termination method"
• Stops at random synthesis
• Cannot bind another deoxyribonucleotide
• Create sections of different lengths
• Separation by electrophoresis
• Includes ddATP, ddTTP, ddCTP, ddGTP, each labeled with a unique dye color

Capillary Electrophoresis

• Follows Sanger sequencing to separate DNA molecules
• Peaks should be evenly distributed across the chromatogram, with variation in peak height less than 3-fold, each containing only a single color

Next Generation Sequencing (NGS)

• NGS is an alteration of Sanger sequencing
• This method uses micro- and nanotechnologies that enable massive parallel sequencing
• NGS minimises sample amount and all reaction components
• Parallel sequencing of many DNA sequences at one time

NGS Workflow Steps

• DNA extraction
• Library preparation (fragmentation, adapter ligation, sequencing library creation)
• Sequencing
• Analysis

Key Features of Select NGS Devices

• Pyrosequencing Roche/454 uses clonal amplification and luminescence output
• Sequencing by Ligation Life Tech/SOLID uses clonal amplification and fluorescence output
• H+ Ion Generation Life Tech/Ion Torrent uses clonal amplification and pH Change output
• Reversible Dye Terminators Illumina uses cluster amplification and fluorescence output

Pyrosequencing (2005)

• This method is grounded on the release of pyrophosphate (PPi) through enzymatic DNA synthesis
• No need for electrophoresis or fragment separation
• Sequencing is done to achieve real-time DNA synthesis

Illumina (2007)

• This method involves fragmenting DNA, adding adaptors, attaching to a flowcell, binding to a primer, and conducting PCR extension to form clusters

Third Generation Sequencing (2010)

• Cheaper, faster, and more sophisticated than previous methods
• Uses other principles to decode much longer stretches of DNA
• Single-Molecule Sequencing in Real Time (SMRT) and Oxford Nanopore Technology (ONT)

Oxford Nanopore Technology (ONT)

• Involves very fast and compact sequencing
• Can sequence DNA outside of conventional laboratory settings
• Makes use of MinIONs in different environments for sequencing

Applications of Third-Generation Sequencing

• Genomics for detecting structural variants and identifying tandem repeats
• Transcriptomics to find gene expression
• Epigenomics to discover different nucleotides
• Metagenomics helps determine gene alaysis

Applications of NGS

• Can rapidly sequence whole genomes and target regions
• Utilise RNA sequencing to find novel RNA variants
• Analyses genome-wide DNA methylation and DNA-protein interactions
• Helps to sequence cancer samples to look for rare variations
• Can be used to track the human microbiome

Gene Sequencing Evolution

• First-generation includes Sanger sequencing and it determines the correct order of dNTP
• Second-generation sequencing includes 454 and has short-read sequencing
• Third-generation sequencing includes PacBio and is tens of kb.

Comparison of Sequencing Generations

• First-generation sequencing has DNA purification and cloning
• First-generatuon sequencing uses synthesis and has low output data
• Second-generation sequencing uses bridge amplication with synthesis
• Second-generation sequencing has high output data
• Third-generation sequencing does not use amplifications but direct adaptor ligation
• Single molecule sequencing is a type of sequencing with ~90-95% accuracy and high output data

Human Genome Project (HGP)

• Involved an international research collaboration across many countries
• The HGP's main goal was to define the full sequence of the genetic material
• Defining sequence needs non-coading sequences and regulator elements

History of the HGP

• This was an important scientific project from 1990-2003
• Coordinated by the Human Genome Organization (HUGO) under Victor A. McKusick.
• Started in the United States and was overseen by the National Institute of Health (NIH)
• Read 3.2 billion DNA bases for about $3 billion

Timeline for the HGP

• NIH NIGMS began funding of genome porjects in 1987
• Started human mappiing project under Francis Collins in 1990
• Craig Venter founded TIGR in 1994
• First chromosome was sequecned in 1999
• Finished fully in 2003

Project Objectives

• Created a genetic map, identified genes, determined the sequence of DNA, and created information storage
• It also considered ethical, legal, and social consequences.

Whose Genome Was Sequenced?

• The genome created was a patcvhwork of many people
• Around 30% of the samples came from European ancestry

HUGO Project

• HUGO porject was almost completed around March 2022
• 8% of the genome has not been read and is the Telomere-to-Telomere (T2T)
• HUGO porject was completed around August 2023 with the Y chromosome being sequenced

Numbers of Genomes in the NCBI database

• Scientists can now sequence DNA bases per second
• There is about 213, 850 viruses
• 2 460, 000 bacterial
• 27, 320 archaebacteria
• 47,510 eukaryotic

Results of the HGP

• A aim to understand the function of all human genes
• Found differences between individuals
• Understood location of genes through sequencing
• Human genome cinsists of around 30,000 genes

HGP Discovery

• Found that humans do not have 80-100 thousand genes
• Only around 20-25 thousand genes
• 3.2 billion bp of genomoc DNA
• Ethical problems

HGP Findings

• It was found that humans evolved and different populations were born
• DNA base sequences was able to serve as a data base
• Allowed for gene comparison

Medical Benefits from HGP

• Allowed scientists to chractertize tumors, use Diaganostics, and study functions
• Allowed there to be genetic consultation

Tech Benefits from HGP

• Allowed for gene therapy and databases

Summary of HGP

• Mapping out the human genome means being able to learn if a person is likely to get a disease
• With a list of all the alleles, doctors can give better treatment
• Concerns about who owns the data and if health will decline

National Genomic Projects

• Focused four major categories: normal variance, medical
• Designed to improve personalized medicine

Encyclopedia of DNA Elements (ENCODE) (2003)

• Identifies all functional elements in mice and humans' DNA
• Contains 32 scientific teams
• Is a public research consortium
• This is aimed to explain how the genome works

Human Microbiome Project (2007)

• Collection of the microbes that are on people
• The microbiome is associated with 2008-2016
• This study's purpuse were to measure differences in genetic variation
• Looked at 2,500 to find human genetic
• They wanted to affect disease

EBP Earth Biology Project (2018)

• They want to do the genome sequencing for people
• The project includes mapping and biology
• They want to give social benefits with the 4.7 biilon dollar budget.

All of Us Research Program (2022)

• Used over 100,00 whole genome sequencesto better revolutionze

The goals of the human pangenome are:

• Creating a reference and marking diversity for the scientific community of medicine
• Sequencinf and aiming a for set with around 300 people

Personal Genome Project (2025)

• They use 300 euros to sequence 1000 genomes