Basic Steps of the NGS Method
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Next generation sequencing (NGS) involves chemical assays other than traditional Sanger sequencing to sequence DNA. The basic steps of NGS are: 1) library preparation where DNA is fragmented and adapters are ligated, 2) clonal amplification where fragments are amplified, and 3) sequencing reactions using techniques like pyrosequencing or reversible dye terminators to determine the sequence. The sequenced fragments are then analyzed bioinformatically to map reads and assemble sequences.
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Basic Steps of the NGS Method
- 1. Next Generation Sequencing Basic Steps of NGS Method
- 2. Terminology • Next Generation Sequencing (NGS) – DNA sequencing methods that involve chemical assays other than the traditional Sanger deoxy- chain-termination method (1st Gen Seq) • NGS AKAs – Deep Sequencing – Massively Parallel Sequencing – Second and Third Generation Sequencing • 2nd : Undergoes amplification of the template molecules • 3rd : Single molecule sequencing
- 3. Generic Overview of NGS Library Preparation Clonal Amplification Sequencing Voelkerding KV (2010) Next Generation Sequencing for Clinical Diagnostics-Principles and Application to Targeted Resequencing for Hypertrophic Cardiomyopathy. Journal of Molecular Diagnostics 5(12): 539-551
- 4. 1. Library Preparation • Input DNA Fragmented – Shearing by • Sonication • Nebulization • Enzyme digestion • Fragments have terminal overhangs – Blunt-end repair and phosphorylation • Adapter ligation – Platform-specific adapter are ligated to the fragments • Final Library – Short DNA fragments with platform-specific adaptors Voelkerding KV (2010) Next Generation Sequencing for Clinical Diagnostics-Principles and Application to Targeted Resequencing for Hypertrophic Cardiomyopathy. Journal of Molecular Diagnostics 5(12): 539-551
- 5. 2. Clonal Amplification • Amplify the fragments – Emulsion PCR – oil-in-water based • One Bead = One Fragment = One Sequence Bead – Bridge Amplification – solid surface, flow-cell based • One Cluster = One Fragment = One Sequence Bead Voelkerding KV (2010) Next Generation Sequencing for Clinical Diagnostics-Principles and Application to Targeted Resequencing for Hypertrophic Cardiomyopathy. Journal of Molecular Diagnostics 5(12): 539-551
- 6. 3. Sequencing • Pyrosequencing • Sequence incorporation of nucleotides luminescence • Sequencing by Ligation • Introduction of oligonucleotide probes fluorescence • Reversible dye terminators • Incorporation of reversible dye terminators fluorescence Voelkerding KV (2010) Next Generation Sequencing for Clinical Diagnostics-Principles and Application to Targeted Resequencing for Hypertrophic Cardiomyopathy. Journal of Molecular Diagnostics 5(12): 539-551
- 7. Figure from Wang et. al, RNA-Seq: a revolutionary tool for transcriptomics, Nat. Rev. Genetics 10, 57-63, 2009). Sample preparation Data analysis: Mapping reads Visualization (Gbrowser) De novo assembly Quantification Next generation sequencing (NGS) 4. Analysis
- 8. Work Cited • Ambry Genetics. Making Sense of NextGent Sequencing. Kelly Gonzalez, MS, CGC, and Senior Manager of Clinical Genomics. Available at http://www.ambrygen.com/sites/default/files/pdfs/NERRG_4-10- 12_Making_Sense_of_NetGen_Sequencing_KG(3).pdf. Access verified May 21, 2014. • Omixon. Allen Van Deynze, 2010; Solanaceae Coordinated Agricultural Project. Next Generation Sequencing. Available at http://www.omixon.com/the-basics- of-next-generation-sequencing/. Access verified May 21, 2014. • The Illumina HiSeq 2000 Sequencing Technology. (Feb 08, 2011). dkfz. German Cancer Research Center. http://www.dkfz.de/gpcf/hiseq_technology.html Access verified May 21, 2014.
- 9. Next Generation Sequencing Sanger Dideoxy
Notas del editor
- In this video we will be going over the basic step taken in most Next Generation Sequencing methods.
- First we will go over what Next Generation Sequencing is and some other common names that it is referred to as. Next Generation Sequencing is DNA sequencing methods that involve chemical assays other than the traditional Sanger deoxy-chain-termination method which is also referred to as first generation sequencing. The Sanger method is a very long and work extensive method of sequencing and the next generation sequencing is much faster and is able to sequence more than one strand of DNA at a time. Other names for next generation sequencing is deep sequencing or massively parallel sequencing. It can also be split up into 2nd and 3rd generation sequencing by whether or not the DNA being sequence needs to be amplified or not. In 2nd generation sequencing the DNA undergoes amplification, or the production of thousand of copies of the template molecule, by either PCR or emulsion amplification. 3rd Generation sequencing is also referred to as single molecule sequencing because it does not need to be amplified and sequences the single DNA molecule.
- The basic steps are Library Preparation, Clonal Amplification if it is 2nd Generation Sequencing, and then the Sequencing itself. After the sequencing is finished the data must then be process and analyzed as well.
- In sequencing the library is a collection of DNA fragments that is stored and propagated in a population of micro-organisms through the process of molecular cloning. In order to prepare the library it must first be fragmented into smaller pieces of about 300 bps, that can then be sequenced. The process of cutting up a large strand of DNA, sequencing it, and then putting it back together is referred to as shotgun sequencing. There are various ways to fragment DNA. One common method used is sonication which shears DNA by exposing it to periods of high sound energy. Another common method is nebulization which shears DNA by forcing it through a small hole in a nebulizer unit. This results in the formation of a mist that is collected. The fragment size is determined by the pressure of the gas used to push the DNA through the nebulizer. After fragmentation the ends of the obtained DNA-fragments are repaired and an A-overhang is added at the 3'-end of each strand. Afterwards, adaptors which are necessary for amplification and sequencing are ligated to both ends of the DNA-fragments. These fragments are then size selected and purified.
- Next is clonal amplification which can be accomplished in two different ways. One is by PCR; in emulsion PCR they use an oil in water based method where one emulsion bead is attached to a single DNA fragment and then that fragment is amplified. Each bead has it’s own fragment. The other method is bridge amplification which uses the solid surface, flow-cell based method; this creates clusters in which each cluster is made up of many of the same DNA fragment sequence. Each of these amplification methods are discussed in greater detail in other available videos.
- There are various methods of sequencing,
- Mapping is the computational process of identifying the specific region of a reference genome form which an individual sequence DNA template originated.
- This is the work cited for this video.
- In the next set of videos we will be going over the various sequencing methods and how each work. The first will be the First Generational Sequencing method of Sanger Dideoxy and the rest that will follow will the various Next Generational Sequencing methods.