Übersicht Sequenziermethoden

Transkript

1 DNA-Sequenzierung

2 Übersicht Sequenziermethoden Sanger-Sequenzierung Pyrosequencing (Roche/454) Illumina/Solexa Pacific Biosciences (PacBio)

3 Sanger-Sequenzierung Didesoxymethode, Kettenabruch- Synthese 1975/77 Sanger & Coulson Zugabe der Basen zum Teil als Didesoxynukleosidtriphosphaten (ddatp, ddctp, ddgtp, ddttp), Abbruch der Synthese Entstehung von DNA-Fragmenten in unterschiedlicher Länge Auftrennung durch Gelelektrophorese Anfang 1990er: ddntps mit Fluoreszenzfarbstoffen markiert Zugabe aller in einer Reaktion Auftrennung durch Kapillarelektrophorese Campbell et al., Biology (2008)

4 Sanger-Sequenzierung Klassische Methode: 4 getrennte Reaktionen pro ddntp Auftrennung in 4 getrennten Spuren auf einem Agarosegel Mardis 2013, Annual Review of Analytical Chemistry

5 Sanger-Sequenzierung

6 Sequenz-Chromatogramm

7 1. Introduction, Basic Laboratory Techniques Beispiel für ein Sequenzierprojekt 2. Extraction of Plasmid DNA and Drosophila Genomic DNA 3. DNA Restriction and Gel Electrophoresis 4. Protein Extraction and Quantification 5. Measurement of Enzymatic Activity 6. Bacterial Growth and Transformation 7. Polymerase Chain Reaction 8. RNA Isolation, Reverse Transcription PCR 9. Cloning of DNA Fragments in Plasmid Vectors 10. Identification and Confirmation of Cloned DNA 11. DNA Sequencing

8 1. Introduction, Basic Laboratory Techniques 2. Extraction of Plasmid DNA and Drosophila Genomic DNA 3. DNA Restriction and Gel Electrophoresis 4. Protein Extraction and Quantification 5. Measurement of Enzymatic Activity 6. Bacterial Growth and Transformation 7. Polymerase Chain Reaction 8. RNA Isolation, Reverse Transcription PCR Beispiel für ein Sequenzierprojekt Aufreinigung des PCR-Produkts DNA Extraktion PCR: Amplifikation des Genes 9. Cloning of DNA Fragments in Plasmid Vectors 10. Identification and Confirmation of Cloned DNA 11. DNA Sequencing Sequenzierung des aufgereinigten PCR-Produkts

9 Aufreinigung von PCR-Produkten Enzymmischung ExoSAP Exonuclease I: Abbau dntps alkalische Phosphatase: Deaktivierung Primer

10 Aufreinigung von PCR-Produkten Enzymmischung ExoSAP Gelextraktion Exonuclease I: Abbau dntps alkalische Phosphatase: Deaktivierung Primer 1. gel electrophoresis, excise PCR product 2. weigh gel slice 3. add 3 volumes of buffer QG to 1 volume of gel (100 mg gel ~ 100µl) C for 10 min to solve gel slice, vortex every 2-3 minutes 5. add 1 volume of isopropanol, mix by inverting 6. add sample (max. 800 µl) to MinElute column, 1 min 13,000 rpm, discard flowthrough 7. repeat step 6, if sample volume is > 800 µl 8. add 500 µl Buffer QG, 1 min 13,000 rpm, discard flow-through 9. add 750 µl Buffer PE, let MinElute column stand for 5 min, 1 min 13,000 rpm, discard flow-through min 13,000 rpm, discard flow-through 11. place MinElute column into 1.5 ml microcentrifuge tube 12. add 10 µl of 70 C H 2 O (or Buffer EB), let Min Elute column stand for 5 min, 1 min 13,000 rpm to elute PCR product

11 Aufreinigung von PCR-Produkten Enzymmischung ExoSAP Gelextraktion Exonuclease I: Abbau dntps alkalische Phosphatase: Deaktivierung Primer 1. gel electrophoresis, excise PCR product 2. weigh gel slice 3. add 3 volumes of buffer QG to 1 volume of gel (100 mg gel ~ 100µl) C for 10 min to solve gel slice, vortex every 2-3 minutes 5. add 1 volume of isopropanol, mix by inverting 6. add sample (max. 800 µl) to MinElute column, 1 min 13,000 rpm, discard flowthrough 7. repeat step 6, if sample volume is > 800 µl 8. add 500 µl Buffer QG, 1 min 13,000 rpm, discard flow-through 9. add 750 µl Buffer PE, let MinElute column stand for 5 min, 1 min 13,000 rpm, discard flow-through min 13,000 rpm, discard flow-through 11. place MinElute column into 1.5 ml microcentrifuge tube 12. add 10 µl of 70 C H 2 O (or Buffer EB), let Min Elute column stand for 5 min, 1 min 13,000 rpm to elute PCR product

12 Aufreinigung von PCR-Produkten Enzymmischung ExoSAP Gelextraktion Exonuclease I: Abbau dntps alkalische Phosphatase: Deaktivierung Primer 1. gel electrophoresis, excise PCR product 2. weigh gel slice 3. add 3 volumes of buffer QG to 1 volume of gel (100 mg gel ~ 100µl) C for 10 min to solve gel slice, vortex every 2-3 minutes 5. add 1 volume of isopropanol, mix by inverting 6. add sample (max. 800 µl) to MinElute column, 1 min 13,000 rpm, discard flowthrough 7. repeat step 6, if sample volume is > 800 µl 8. add 500 µl Buffer QG, 1 min 13,000 rpm, discard flow-through 9. add 750 µl Buffer PE, let MinElute column stand for 5 min, 1 min 13,000 rpm, discard flow-through min 13,000 rpm, discard flow-through 11. place MinElute column into 1.5 ml microcentrifuge tube 12. add 10 µl of 70 C H 2 O (or Buffer EB), let Min Elute column stand for 5 min, 1 min 13,000 rpm to elute PCR product

13 Aufreinigung von PCR-Produkten Enzymmischung ExoSAP Gelextraktion Exonuclease I: Abbau dntps alkalische Phosphatase: Deaktivierung Primer 1. gel electrophoresis, excise PCR product 2. weigh gel slice 3. add 3 volumes of buffer QG to 1 volume of gel (100 mg gel ~ 100µl) C for 10 min to solve gel slice, vortex every 2-3 minutes 5. add 1 volume of isopropanol, mix by inverting 6. add sample (max. 800 µl) to MinElute column, 1 min 13,000 rpm, discard flowthrough 7. repeat step 6, if sample volume is > 800 µl 8. add 500 µl Buffer QG, 1 min 13,000 rpm, discard flow-through 9. add 750 µl Buffer PE, let MinElute column stand for 5 min, 1 min 13,000 rpm, discard flow-through min 13,000 rpm, discard flow-through 11. place MinElute column into 1.5 ml microcentrifuge tube 12. add 10 µl of 70 C H 2 O (or Buffer EB), let Min Elute column stand for 5 min, 1 min 13,000 rpm to elute PCR product

14 1. Introduction, Basic Laboratory Techniques 2. Extraction of Plasmid DNA and Drosophila Genomic DNA 3. DNA Restriction and Gel Electrophoresis 4. Protein Extraction and Quantification 5. Measurement of Enzymatic Activity Beispiel für ein Sequenzierprojekt DNA Extraktion PCR: Amplifikation des Genes 6. Bacterial Growth and Transformation 7. Polymerase Chain Reaction 8. RNA Isolation, Reverse Transcription PCR 9. Cloning of DNA Fragments in Plasmid Vectors 10. Identification and Confirmation of Cloned DNA 11. DNA Sequencing Aufreinigung des PCR-Produkts Sequenzierung des aufgereinigten PCR-Produkts Extraktion des Plasmids Sequenzierung des Inserts (= PCR-Produkt) Klonierung des PCR-Produkts Re-Amplifikation des Inserts (PCR) Aufreinigung des PCR-Produkts Sequenzierung des aufgereinigten PCR-Produkts

15 Sequenzierung des Inserts aus einem Vektor

16 Übersicht Sequenziermethoden Sanger-Sequenzierung Pyrosequencing (Roche/454) Illumina/Solexa Pacific Biosciences (PacBio)

17 Evolution des Datenertrags Mardis 2011, Nature

18 Pyrosequencing (Roche/454) Margulies et al. 2005, Nature Mardis 2007, Trends in Genetics Mardis 2008, Annual Review of Genomics and Human Genetics

19 Mardis 2008, Annual Review of Genomics and Human Genetics Illumina/Solexa Sequenzierung mit Brückensynthese

20 Pacific Biosiences (PacBio) Metzker 2010, Nature Reviews Genetics

21 Methodische Unterschiede Roche/454 Illumina Hi-Seq 2000 Pacific Biosciences RS Library amplification method empcr on bead surface Enzymatic amplification on glass surface NA (single molecule detection) Sequencing method Polymerase-mediated incorporation of unlabelled nucleotides Polymerase- mediated incorporation of end-blocked fluorescent nucleotides Polymerase-mediated incorporation of terminal phosphate labelled fluorescent nucleotides Detection method Light emitted from secondary reactions initiated by release of PPi Fluorescent emission from incorporated dye-labelled nucleotides Real time detection of fluorescent dye in polymerase active site during incorporation Post incorporation method Error model NA (unlabelled nucleotides are added in base-specific fashion, followed by detection) Substitution errors rare, insertion/deletion errors at homopolymers Chemical cleavage of fluorescent dye and 3 blocking group NA (fluorescent dyes are removed as part of PPi release on nucleotide incorporation) End of read substitution errors Random insertion/deletion errors Single pass error rate 1% 0.1% 13% Sanger ABI3730 error rate: 0.001% Mardis 2011, Nature Rhoads and Au 2015, Genomics, Proteomics & Bioinformatics

22 Datenertrag 700 nucleotides 1 million reads 700 Mb 23 hours (Illumina) 250 nucleotides 6 billion reads Gb 11 days > nucleotides reads > 500 Mb 2 hours MacLean et al., Nat Rev Microbiol (2009)

23 Preisentwicklung 2015: Preis pro Mb (Illumina): 0,03$ Delsney et al. 2010, Plant Science Rhoads and Au 2015, Genomics, Proteomics & Bioinformatics