Exome seuencing (steps, method, and applications)
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This document summarizes exome sequencing methodology. It describes that exome sequencing sequences all expressed genes in a genome, which contains around 180,000 exons totaling 30 million base pairs in humans. There are two main steps: 1) selecting the exonic regions of DNA using either array-based or in-solution capture techniques, and 2) sequencing the captured exonic DNA using various platforms such as Sanger, 454, Illumina, or SOLiD sequencing. Exome sequencing has applications in rare variant mapping, discovering causes of Mendelian disorders, and clinical diagnostics.
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Exome seuencing (steps, method, and applications)
- 1. Presented By: Hamza Khan Registration # FA16-R02-019 1
- 2. Introduction Exome sequencing, also known as whole exome sequencing Sequencing all of the expressed genes in a genome Humans have about 180,000 exons, 30 million base pairs 2
- 3. Methodology 2 steps: 1. Select only the subset of DNA that encodes proteins (exons) 2. Sequencing of the exonic DNA using DNA sequencing technology 3
- 4. 1. Selection of Exons (Target Enrichment) 2 strategies Array-based capture In-solution capture 4
- 6. In-Solution Capture Use pool of custom oligonucleotides (probes) Synthesized and hybridized in solution Probes are labeled with beads After hybridization beads can be pulled down and washed Fragments can be sequenced after beads removal 6
- 7. 7
- 8. 2. Sequencing Several sequencing platforms are available: 1. Sanger Sequencing 2. Next Generation Sequencing: 454 Pyrosequencing (454 Life Sciences) Illumina (Solexa) sequencing SOLiD Sequencing (Applied Bio-systems) 8
- 9. Sanger Sequencing Chain termination method Use of labeled ddNTPs Lack 3’-OH group required for phosphodiester bond formation 9
- 10. 10
- 12. DNA fragmentation and in vitro adaptor ligation 11 Library preparation 12
- 13. DNA fragmentation and in vitro adaptor ligation emulsion PCR 1 2 1 2 Library preparation Clonal amplification 13
- 14. DNA fragmentation and in vitro adaptor ligation emulsion PCR bridge PCR 1 2 1 2 Library preparation Clonal amplification 14
- 15. Introduction to NGS http://ueb.ir.vhebron.net/NGS DNA fragmentation and in vitro adaptor ligation emulsion PCR bridge PCR Pyrosequencing 1 2 3 1 2 3 Cyclic array sequencing Library preparation Clonal amplification 454 sequencing 15
- 16. Introduction to NGS http://ueb.ir.vhebron.net/NGS DNA fragmentation and in vitro adaptor ligation bridge PCR Pyrosequencing Sequencing-by-ligation 1 emulsion PCR 2 3 1 2 3 454 sequencing SOLiD platform Cyclic array sequencing Library preparation Clonal amplification 16
- 17. Introduction to NGS http://ueb.ir.vhebron.net/NGS DNA fragmentation and in vitro adaptor ligation bridge PCR Pyrosequencing Sequencing-by-ligation Sequencing-by-synthesis 1 emulsion PCR 2 3 1 2 3 454 sequencing SOLiD platform Solexa technology Cyclic array sequencing Library preparation Clonal amplification 17
- 21. Applications Rare variant mapping in complex disorders Discovery of Mendelian disorders Clinical Diagnostics 21
- 22. 22
Editor's Notes
- A Mendelian trait is one that is controlled by a single locus in an inheritance pattern. In such cases, a mutation in a single gene can cause a disease that is inherited according to Mendel's laws. Examples include sickle-cell anemia, Tay-Sachs disease, cystic fibrosis and xeroderma pigmentosa.