Next Generation Sequencing Dylan Young Biomedical Engineering
What is DNA? Molecule composed of Adenine (A) Guanine (G) Cytosine (C) Thymine (T) Paired as either AT or CG Provides genetic instructions for every living organism
What is sequencing? Process of determining the order of nucleotides within DNA Includes any technology that is used to determine the order or A, T, C, and G This information impacts Medical diagnoses Biotechnology Forensic Biology Virology
Long-term Goal of Sequencing To achieve cost and time effective whole-genome sequencing for individual patients
Human Genome Project International project to sequence the entire human genome Remains the world s largest collaborative biological project United States, United Kingdom, France, Germany, Canada, China, Japan Began in 1990 Project sequenced a small group of individuals then assembled them together to form a complete sequence for each chromosome Employed the Sanger Sequencing Method
Sanger Method Invented in 1970 Process Short primer is added to a strand of DNA DNA polymerase is then used to add complementary nucleotides Reaction is stopped by removing an oxygen from the ribonucleotide (termination) Chain terminating nucleotide marked by inserting dye Extension products then separated by Capillary Electrophoresis Electrical field moved negatively charged DNA fragments towards a positive electrode Laser excited the fragments as they move through a window, and a unique wavelength is emitted for each base pair
Human Genome Project Results Project was completed in 2003 3 Billion base pairs were sequenced Total Cost for complete genome sequencing $2.7 Billion
Where to go next Increase sequencing speed Decrease sequencing cost Achieve practical, patient specific whole-genome sequencing By 2005, Sanger sequencing could Sequence 18 kilobases per one sequencing run Cost ~$10million per whole genome sequence
Next Generation Sequencing (NGS) NGS was invented in 2005 Utilized most successfully by Illumina Inc. Repetitively Analyzes short sequences of DNA (100-150 base pairs) in parallel Allows for millions of fragments to be analyzed simultaneously
How it works 1. Strand of DNA is cleaved into segments 2. Cleaved segment is ligated to adaptors and annealed to a slide 3. Slide is flooded with fluorescently labeled nucleotides and DNA polymerase 1. Terminator is added to each nucleotide to ensure only one base is added at a time 4. DNA polymerase then adds corresponding base pair to the section of cleaved DNA and an image is taken of the slide 5. Terminator is then removed to allow the next base to be added 6. This process is repeated for every base pair of the cleaved segment 7. Images are then run through a machine that detects the base pair at each site by color 8. Full sequence is then reconstructed
Efficacy and Price In 2005, NGS could sequence 1 Gigabase of data per run As of 2014, 1.4 Terabases of data per run 45 Human genomes can be sequenced in a single day $1000
Discussion NGS has provided 1000X increase in data/run Can sequence a whole-genome 200,000X faster than the Sanger method Costs 3,000,000X less than the original Human Genome Project Clear superiority over Sanger Method
References 1] Morozova, O. and Marra, M. (2008). Applications of next-generation sequencing technologies in functional genomics. Genomics, 92(5), pp.255-264. [2] van Dijk, E., Auger, H., Jaszczyszyn, Y. and Thermes, C. (2014). Ten years of next-generation sequencing technology. Trends in Genetics, 30(9), pp.418-426. [3] EMBL-EBI Train online. (2017). Illumina sequencing. [online] Available at: https://www.ebi.ac.uk/training/online/course/ebinext-generation-sequencing-practical-course/what-next-generation-dna-sequencing/illumina- [Accessed 26 Sep. 2017]. [4] National Human Genome Research Institute (NHGRI). (2017). DNA Sequencing Costs: Data. [online] Available at: https://www.genome.gov/sequencingcostsdata/ [Accessed 26 Sep. 2017]. [5] National Human Genome Research Institute (NHGRI). (2017). The Cost of Sequencing a Human Genome. [online] Available at: https://www.genome.gov/27565109/the-cost-of-sequencing-a-human-genome/ [Accessed 26 Sep. 2017]. [6] National Human Genome Research Institute (NHGRI). (2017). DNA Sequencing Fact Sheet. [online] Available at: https://www.genome.gov/10001177/dna-sequencing-fact-sheet/ [Accessed 26 Sep. 2017]. [7] Illumina.com. (2017). DNA Sequencing Understanding the genetic code with NGS. [online] Available at: https://www.illumina.com/techniques/sequencing/dna-sequencing.html [Accessed 26 Sep. 2017]. [8] Illumina. (2017). An introduction to Next-Generation Sequencing Technology. [online] Available at: https://www.illumina.com/content/dam/illumina-marketing/documents/products/illumina_sequencing_introduction.pdf [Accessed 25 Sep. 2017]