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Basic Steps of the NGS Method

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This presentation covers the basics on what Next Generational Sequencing is and some terminology needed in order to understand the process.

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Basic Steps of the NGS Method

  1. 1. Next Generation Sequencing Basic Steps of NGS Method
  2. 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. 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. 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. 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. 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. 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. 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. 9. Next Generation Sequencing Sanger Dideoxy

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