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Illumina (sequencing by synthesis) method
- 1. Bioinformatics (Biot 604) Lecture slides on Illumina (sequencing by synthesis) method By: Fekadu Korsa Submitted to Dr. Abiy Zegeye June 7, 2020 Addis Ababa, Ethiopia UNIVERSITY 1 ADDIS ABABA Addis Ababa University, School of Graduate Studies, Institute of Biotechnology
- 2. Outline of the Presentation Definition steps of Sequencing by synthesis Analytic procedures performed on the out put UNIVERSITY 2 ADDIS ABABA
- 3. Definition What is Sequencing-by-Synthesis? is the most widely used NGS technology in the world. developed by Illumina. generates > 90% of the sequencing data of the world . generates many millions of highly accurate reads making much faster and cheaper than other sequencing methods. UNIVERSITY 3 ADDIS ABABA
- 4. Steps of Sequencing by synthesis 1. DNA sample preparation . 2.Attach the DNA to surface of flow cell UNIVERSITYADDIS ABABA Cut DNA into smaller fragment by using enzyme digestion Ligate adapter to both ends of DNA Fragment Primer was loaded on the flow cell DNA fragments are washed across the flow cell. Denature DNA strand by heating to 95 oC
- 5. Flow cell UNIVERSITY 5 ADDIS ABABA Surface of flow cell coated with a lawn of oligo pairs DNA that doesn’t attach is washed away. Single DNA binds to primers on the surface of the flow cell
- 6. 3. Bridge Amplification 4. Formation of dsDNA UNIVERSITYADDIS ABABA Single-stranded molecule flips over & forms a bridge by hybridizing to complementary prime Hybridized primer is extended by polymerases Reagents such as Primers, buffer, Nucleotides, DNA Polymerase were added on the flow well
- 7. 5. Denaturation of DNA Bridge UNIVERSITYADDIS ABABA ds DNA bridge is broken down by using heat. Two copies of ssDNA are formed and covalently attached to flow cell
- 8. 6. Completes Amplification UNIVERSITY 8 ADDIS ABABA Cycle of new strand synthesis and Denaturation to make multiple copies of the same sequence (amplification) Fragments Become Double Stranded Denature the Double Strand Molecules
- 9. 7. First base determination 8.Take image of first base UNIVERSITYADDIS ABABA Add sequencing reagents such as Primer, DNA polymerase, Fluorescently labeled nucleotides and Buffer First base is incorporated Remove unincorporated bases Detect Signal & take image of base Deblock and remove the fluorescent signal for new cycle
- 10. 9. Determine second base 10.Take image of second base UNIVERSITYADDIS ABABA Add sequencing reagents Primer,DNA polymerase, Fluorescently labeled nucleotides and Buffer Second base is incorporated Remove unincorporated bases Detect Signal & take image of base Deblock and remove the fluorescent signal for new cycle
- 11. 11.Reading sequence (Out put) UNIVERSITYADDIS ABABA The identity of each base of a cluster is read off from sequential images Data analysis is done by transforming images into base calls and reads
- 12. 12. Align data UNIVERSITYADDIS ABABA After the sequencing is finished they are aligned and analyzed Reads obtained grouped based on their index sequences sequences with similar reads are clustered Forward and reverse reads are paired to form contiguous sequences. The data are aligned and compared to a reference, and sequencing differences are identified.
- 13. Data Analysis UNIVERSITY 13 ADDIS ABABA .Images transformed into base calls and reads DNA sequence is analyzed base-by-base The sequence generated can be aligned to a reference sequence, this looks for matches or changes in the sequenced DNA Illumina sequencing method is a highly accurate method.