Human genome Project and genomics

• Aims of the Human Genome Project

  • Determine the sequences of the 3 billion chemical base pairs that make up human DNA
  • Identify all the approximately 20,000-25,000 genes in human DNA
  • Improve tools for data analysis
  • Store this information in databases and make available to researchers
  • Transfer related technologies to the private sector
  • Address the ethical, legal, and social issues (ELSI) that may arise from the project
• Hierarchical shotgun approach

  Map-based, clone-by-clone, BAC-by-BAC- vector that can incorporate fragments 150-350 kb. Useful for sequencing genomes that contain repetitive sequences.
• BAC (Bacterial Artificial Chromosome)


  A man-made piece of DNA that can replicate inside a bacterial cell and can incorporate large DNA fragments
• How to construct a BACs library

  1. Break genome into smaller fragments

  2. Construction of ordered clone maps

  3. Sequencing of ordered clones
• Whole-genome shotgun sequencing


  Cut the DNA of an entire chromosome into overlapping fragments short enough for sequencing, clone the fragments, sequence each fragment, and order the sequences into one overall sequence with computer software
• The human genome was first broken down into very large DNA fragments (each approximately 100,000 nucleotide pairs) and cloned into BACs. The order of the BACs along a chromosome was determined by comparing the pattern of restriction enzyme cleavage sites in a given BAC clone with that of the whole genome.
• Approximately 30,000 BAC clones were sequenced to complete the human genome.
• Next generation sequencing

  Allows much faster and easier genome reading, uses include drug treatment to see which one won't affect the host

  Instead of using bacterial cells to generate libraries, made using PCR amplification of billions of DNA fragments each attached to a solid support
• Illumina Sequencing

  Genomic DNA is fragmented

  2. Each fragment is placed in a droplet with a bead

  3. Using PCR, 10^6 copies of each fragment are made, each attached to the bead by 5' end

  4. A solution of each of the four nucleotides is added to all wells and then washed off. The entire process is then repeated.
• Nanopore sequencing


  Motor protein records each signal as DNA passes through nanopore
• The cost of genome sequencing has decreased from $100 million in 2001 to about a thousand dollars by the end of 2014.
• Advantages of next-generation / 3rd generation sequencing


  • Long reads with no amplification

  Direct detection of epigenetic modifications on native DNA

  Direct sequencing through regions of the genome inaccessible or difficult to analyse by short read platforms

  Uniform coverage of the genome as they are not sensitive to GC content as short-read platforms
• What can you do with a genome sequence?

  • Store the sequence – databases (NCBI Genbank)

  Identify all genes (and therefore "all" proteins)

  Compare with other species – comparative and evolutionary genomics
• How to identify protein-coding genes and understand their function

  Scan the stored sequences for those that represent transcriptional and translational start and stop signals, RNA-splicing sites or promoter sequences

  Search for open reading frames: ATG...TAG/TGA/TAA

  Search for similarity with known genes – BLAST programs

  Predict transcriptional start sites and exons using consensus sequences

  Compare the similarities between species
• BLASTN

  Nucleotide query vs. nucleotide database
• BLASTP


  Protein query vs. protein database
• The 1000 Genomes Project, which was set up in 2008, aimed to study the small fraction of genetic material that varies among people to help explain individual differences in susceptibility to disease, response to drugs or reaction to environmental factors.
• The 1000 Genomes Project has sequenced 2,504 people across five continental regions.
• Practical benefits of genome projects

  • Understanding individual differences in susceptibility to disease, response to drugs or reaction to environmental factors

  Personalising medicine to use more effective treatment

  Tracking influenza virus infections to help design the annual flu vaccine

  Engineering new microbes for applications like detecting toxic metals

  Stopping dangerous side effects and influencing how medicines work for an individual
• Which technique did the international human genome project use

  Hierarchial shotgun approach
• Describe Celera genomics

  Publicly funded

  responsibile for 30% of the sequencing
• How to enact hierarchical sequencing

  1. locate genes on chromosome
  2. Arrange overlapping large clones in order
  3. DNA sequencing of individual clones
• Second generation sequencing


  if a nucleotide is joined to growning strand PPi is released causing a flash of light unique to that base

  If the nucleotide (dntP) is not complementary to the the next template base its is not joined to strand so NO flash

  The pattern of flashes reveals the sequence
• Describe the cancer genome atlas


  Sequencing genome of thousands of cancer samples and types

  To look to see for genes that trigger cancer
• Describe the use of DNA sequencing in agriculture

  Use to select desirable traits in plants or animals
• describe the use of DNA sequencing in enhanced forensics

  DNA analysis for human identification e.g finding a missing person
• Describe the use of DNA sequencing with Microbes and microbiomes

  Track annual flu shot

  Used to detect poisonous metals
• Describe the use of DNA sequencing in Pharmacogenomics

  stops dangerous side effects

  influences how medicine works to increase it to be better for the individual