Next Generation Sequencing -...
Transcript of Next Generation Sequencing -...
![Page 1: Next Generation Sequencing - uni-mainz.demolgen.biologie.uni-mainz.de/F1-Vorbesprechungen/NGS.pdf · Next Generation Sequencing - Pyrosequenzierung DNA template, Primer, DNA polymerase,](https://reader031.fdocuments.in/reader031/viewer/2022021801/5a7c09117f8b9a563b8c96bb/html5/thumbnails/1.jpg)
Next Generation Sequencing
![Page 2: Next Generation Sequencing - uni-mainz.demolgen.biologie.uni-mainz.de/F1-Vorbesprechungen/NGS.pdf · Next Generation Sequencing - Pyrosequenzierung DNA template, Primer, DNA polymerase,](https://reader031.fdocuments.in/reader031/viewer/2022021801/5a7c09117f8b9a563b8c96bb/html5/thumbnails/2.jpg)
Hochdurchsatz-DNA-Sequenzierung
96 Spuren x 900 Basen = ca. 90 000 Basen in 2,5 Std.
![Page 3: Next Generation Sequencing - uni-mainz.demolgen.biologie.uni-mainz.de/F1-Vorbesprechungen/NGS.pdf · Next Generation Sequencing - Pyrosequenzierung DNA template, Primer, DNA polymerase,](https://reader031.fdocuments.in/reader031/viewer/2022021801/5a7c09117f8b9a563b8c96bb/html5/thumbnails/3.jpg)
Microfabricated capillary array electrophoresis (µCAE)
Seperation length: 15,9 cm
~ 430 bp reads max -> 800 bp
24 min run time, 5 x of Sanger
0,01% error on 80% read length
![Page 4: Next Generation Sequencing - uni-mainz.demolgen.biologie.uni-mainz.de/F1-Vorbesprechungen/NGS.pdf · Next Generation Sequencing - Pyrosequenzierung DNA template, Primer, DNA polymerase,](https://reader031.fdocuments.in/reader031/viewer/2022021801/5a7c09117f8b9a563b8c96bb/html5/thumbnails/4.jpg)
Next Generation Sequencing
(Nature Reviews; 2009)
![Page 5: Next Generation Sequencing - uni-mainz.demolgen.biologie.uni-mainz.de/F1-Vorbesprechungen/NGS.pdf · Next Generation Sequencing - Pyrosequenzierung DNA template, Primer, DNA polymerase,](https://reader031.fdocuments.in/reader031/viewer/2022021801/5a7c09117f8b9a563b8c96bb/html5/thumbnails/5.jpg)
Next Generation Sequencing – Solid/ABI sequencing byligation
ABI - Solid- Plattform
![Page 6: Next Generation Sequencing - uni-mainz.demolgen.biologie.uni-mainz.de/F1-Vorbesprechungen/NGS.pdf · Next Generation Sequencing - Pyrosequenzierung DNA template, Primer, DNA polymerase,](https://reader031.fdocuments.in/reader031/viewer/2022021801/5a7c09117f8b9a563b8c96bb/html5/thumbnails/6.jpg)
Next Generation Sequencing – Solid/ABI sequencing by ligation
![Page 7: Next Generation Sequencing - uni-mainz.demolgen.biologie.uni-mainz.de/F1-Vorbesprechungen/NGS.pdf · Next Generation Sequencing - Pyrosequenzierung DNA template, Primer, DNA polymerase,](https://reader031.fdocuments.in/reader031/viewer/2022021801/5a7c09117f8b9a563b8c96bb/html5/thumbnails/7.jpg)
Next Generation Sequencing – Solid/ABI sequencing by ligation
![Page 8: Next Generation Sequencing - uni-mainz.demolgen.biologie.uni-mainz.de/F1-Vorbesprechungen/NGS.pdf · Next Generation Sequencing - Pyrosequenzierung DNA template, Primer, DNA polymerase,](https://reader031.fdocuments.in/reader031/viewer/2022021801/5a7c09117f8b9a563b8c96bb/html5/thumbnails/8.jpg)
Solid/ABI sequencing by ligation: Decoding
![Page 9: Next Generation Sequencing - uni-mainz.demolgen.biologie.uni-mainz.de/F1-Vorbesprechungen/NGS.pdf · Next Generation Sequencing - Pyrosequenzierung DNA template, Primer, DNA polymerase,](https://reader031.fdocuments.in/reader031/viewer/2022021801/5a7c09117f8b9a563b8c96bb/html5/thumbnails/9.jpg)
Roche - FLX- Plattform
![Page 10: Next Generation Sequencing - uni-mainz.demolgen.biologie.uni-mainz.de/F1-Vorbesprechungen/NGS.pdf · Next Generation Sequencing - Pyrosequenzierung DNA template, Primer, DNA polymerase,](https://reader031.fdocuments.in/reader031/viewer/2022021801/5a7c09117f8b9a563b8c96bb/html5/thumbnails/10.jpg)
Next Generation Sequencing - Pyrosequenzierung
DNA template, Primer, DNA polymerase, ATP sulfurylase, luciferase, apyrase, adenosine 5´ phosphosulfate (APS) and luciferin (no dNTPs)
(DNA)n + dNTP (DNA)n+1 + PPiPolymerase
One dNTPs is added
Nucleotide detection
Incubation Degradation of unincorporated dNTPs and excess ATP
Pyrogram
Addition of dNTPs is performed one at a time
Nucleotide sequence is determined from the signal peak in the pyrogram
The light produced in the luciferase-catalyzed reaction is detected by a CCD camera
![Page 11: Next Generation Sequencing - uni-mainz.demolgen.biologie.uni-mainz.de/F1-Vorbesprechungen/NGS.pdf · Next Generation Sequencing - Pyrosequenzierung DNA template, Primer, DNA polymerase,](https://reader031.fdocuments.in/reader031/viewer/2022021801/5a7c09117f8b9a563b8c96bb/html5/thumbnails/11.jpg)
Next Generation Sequencing – Polonysequencing
- kurze Leseweiten
- Diffusion der Polymerase
- langsame Einbaurate
- Probleme vor allem bei homopolymeren Regionen
![Page 12: Next Generation Sequencing - uni-mainz.demolgen.biologie.uni-mainz.de/F1-Vorbesprechungen/NGS.pdf · Next Generation Sequencing - Pyrosequenzierung DNA template, Primer, DNA polymerase,](https://reader031.fdocuments.in/reader031/viewer/2022021801/5a7c09117f8b9a563b8c96bb/html5/thumbnails/12.jpg)
- relativ kurze Leseweiten (<400 bp)
- Probleme bei repeats > 5 bp
- geringe Fehlerrate
- optimal zur Detektion von SNPs
Next Generation Sequencing – Emulsion PCR
![Page 13: Next Generation Sequencing - uni-mainz.demolgen.biologie.uni-mainz.de/F1-Vorbesprechungen/NGS.pdf · Next Generation Sequencing - Pyrosequenzierung DNA template, Primer, DNA polymerase,](https://reader031.fdocuments.in/reader031/viewer/2022021801/5a7c09117f8b9a563b8c96bb/html5/thumbnails/13.jpg)
Life technologies – Ion Torrent
![Page 14: Next Generation Sequencing - uni-mainz.demolgen.biologie.uni-mainz.de/F1-Vorbesprechungen/NGS.pdf · Next Generation Sequencing - Pyrosequenzierung DNA template, Primer, DNA polymerase,](https://reader031.fdocuments.in/reader031/viewer/2022021801/5a7c09117f8b9a563b8c96bb/html5/thumbnails/14.jpg)
NGS Platforms – Ion Torrent
- sehr schnell- Recht hohe Leseweiten
Beim Einbau eines Nukleotides wird ein H+ frei-gesetzt.
Auf Halbleitertechnologie basierende ionen-Sensoren Messen die pH-Änderung in den Micro-Well-Platten
![Page 15: Next Generation Sequencing - uni-mainz.demolgen.biologie.uni-mainz.de/F1-Vorbesprechungen/NGS.pdf · Next Generation Sequencing - Pyrosequenzierung DNA template, Primer, DNA polymerase,](https://reader031.fdocuments.in/reader031/viewer/2022021801/5a7c09117f8b9a563b8c96bb/html5/thumbnails/15.jpg)
Illumina - HiSeq 2000 - Plattform
![Page 16: Next Generation Sequencing - uni-mainz.demolgen.biologie.uni-mainz.de/F1-Vorbesprechungen/NGS.pdf · Next Generation Sequencing - Pyrosequenzierung DNA template, Primer, DNA polymerase,](https://reader031.fdocuments.in/reader031/viewer/2022021801/5a7c09117f8b9a563b8c96bb/html5/thumbnails/16.jpg)
Next Generation Sequencing – Gene Analyzer Illumina/Solexa
![Page 17: Next Generation Sequencing - uni-mainz.demolgen.biologie.uni-mainz.de/F1-Vorbesprechungen/NGS.pdf · Next Generation Sequencing - Pyrosequenzierung DNA template, Primer, DNA polymerase,](https://reader031.fdocuments.in/reader031/viewer/2022021801/5a7c09117f8b9a563b8c96bb/html5/thumbnails/17.jpg)
Illumina/Solexa - Bridge Amplification
![Page 18: Next Generation Sequencing - uni-mainz.demolgen.biologie.uni-mainz.de/F1-Vorbesprechungen/NGS.pdf · Next Generation Sequencing - Pyrosequenzierung DNA template, Primer, DNA polymerase,](https://reader031.fdocuments.in/reader031/viewer/2022021801/5a7c09117f8b9a563b8c96bb/html5/thumbnails/18.jpg)
Cluster Generation: Bridge Amplification
![Page 19: Next Generation Sequencing - uni-mainz.demolgen.biologie.uni-mainz.de/F1-Vorbesprechungen/NGS.pdf · Next Generation Sequencing - Pyrosequenzierung DNA template, Primer, DNA polymerase,](https://reader031.fdocuments.in/reader031/viewer/2022021801/5a7c09117f8b9a563b8c96bb/html5/thumbnails/19.jpg)
Illumina/Solexa - Bridge Amplification
![Page 20: Next Generation Sequencing - uni-mainz.demolgen.biologie.uni-mainz.de/F1-Vorbesprechungen/NGS.pdf · Next Generation Sequencing - Pyrosequenzierung DNA template, Primer, DNA polymerase,](https://reader031.fdocuments.in/reader031/viewer/2022021801/5a7c09117f8b9a563b8c96bb/html5/thumbnails/20.jpg)
NGS Platforms – Pacific Biosciences
PacBio RSSMRT® Technology (single molecule real time)
![Page 21: Next Generation Sequencing - uni-mainz.demolgen.biologie.uni-mainz.de/F1-Vorbesprechungen/NGS.pdf · Next Generation Sequencing - Pyrosequenzierung DNA template, Primer, DNA polymerase,](https://reader031.fdocuments.in/reader031/viewer/2022021801/5a7c09117f8b9a563b8c96bb/html5/thumbnails/21.jpg)
NGS Platforms – Pacific Biosciences
![Page 22: Next Generation Sequencing - uni-mainz.demolgen.biologie.uni-mainz.de/F1-Vorbesprechungen/NGS.pdf · Next Generation Sequencing - Pyrosequenzierung DNA template, Primer, DNA polymerase,](https://reader031.fdocuments.in/reader031/viewer/2022021801/5a7c09117f8b9a563b8c96bb/html5/thumbnails/22.jpg)
NGS Platforms -Nanopore
![Page 23: Next Generation Sequencing - uni-mainz.demolgen.biologie.uni-mainz.de/F1-Vorbesprechungen/NGS.pdf · Next Generation Sequencing - Pyrosequenzierung DNA template, Primer, DNA polymerase,](https://reader031.fdocuments.in/reader031/viewer/2022021801/5a7c09117f8b9a563b8c96bb/html5/thumbnails/23.jpg)
NGS Platforms
![Page 24: Next Generation Sequencing - uni-mainz.demolgen.biologie.uni-mainz.de/F1-Vorbesprechungen/NGS.pdf · Next Generation Sequencing - Pyrosequenzierung DNA template, Primer, DNA polymerase,](https://reader031.fdocuments.in/reader031/viewer/2022021801/5a7c09117f8b9a563b8c96bb/html5/thumbnails/24.jpg)
Sequenzierung „kurzer“ DNA-Fragmente:
Klonierung der DNA in einen Standard-Vektor, z.B. pUC 18oder Amplifikation der DNA per PCR, wenn bereits Sequenz-
abschnitte bekannt sind
MCS
MCS
Sequenzierung mit Standard-oder bereits bekannten Primern
![Page 25: Next Generation Sequencing - uni-mainz.demolgen.biologie.uni-mainz.de/F1-Vorbesprechungen/NGS.pdf · Next Generation Sequencing - Pyrosequenzierung DNA template, Primer, DNA polymerase,](https://reader031.fdocuments.in/reader031/viewer/2022021801/5a7c09117f8b9a563b8c96bb/html5/thumbnails/25.jpg)
Bei Integrat-Längen bis 1000 - 1500 bp ist es möglich, mit zwei Sequenzierungsreaktionen die vollständige
Basenabfolge zu ermitteln.
Univ. Seq.
Rev. Seq.
Sequenzierung „kurzer“ DNA-Fragmente:
![Page 26: Next Generation Sequencing - uni-mainz.demolgen.biologie.uni-mainz.de/F1-Vorbesprechungen/NGS.pdf · Next Generation Sequencing - Pyrosequenzierung DNA template, Primer, DNA polymerase,](https://reader031.fdocuments.in/reader031/viewer/2022021801/5a7c09117f8b9a563b8c96bb/html5/thumbnails/26.jpg)
![Page 27: Next Generation Sequencing - uni-mainz.demolgen.biologie.uni-mainz.de/F1-Vorbesprechungen/NGS.pdf · Next Generation Sequencing - Pyrosequenzierung DNA template, Primer, DNA polymerase,](https://reader031.fdocuments.in/reader031/viewer/2022021801/5a7c09117f8b9a563b8c96bb/html5/thumbnails/27.jpg)
![Page 28: Next Generation Sequencing - uni-mainz.demolgen.biologie.uni-mainz.de/F1-Vorbesprechungen/NGS.pdf · Next Generation Sequencing - Pyrosequenzierung DNA template, Primer, DNA polymerase,](https://reader031.fdocuments.in/reader031/viewer/2022021801/5a7c09117f8b9a563b8c96bb/html5/thumbnails/28.jpg)
![Page 29: Next Generation Sequencing - uni-mainz.demolgen.biologie.uni-mainz.de/F1-Vorbesprechungen/NGS.pdf · Next Generation Sequencing - Pyrosequenzierung DNA template, Primer, DNA polymerase,](https://reader031.fdocuments.in/reader031/viewer/2022021801/5a7c09117f8b9a563b8c96bb/html5/thumbnails/29.jpg)