DNA sequencing methods

DNA sequencing methods and next generation sequensing

1. DNA sequencing methods 1 Presented by: Sepideh Saroughi

2. DNA sequencing is the process of determining the nucleic acid sequence – the order of nucleotides in DNA Medical diagnosis Biotechnology F orensic biology V irology 1970s : two-dimensional chromatography, fluorescence-based sequencing methods 2

3. Sequencing of full genomes Basic methods Large-scale sequencing and de novo sequencing High-throughput methods Long-read sequencing methods Short-read sequencing methods Methods in development 3

4. Sequencing of full genomes Whole genome sequencing Whole genome sequencing (WGS), also known as full genome sequencing Saliva , Epithelial cells, Bone marrow, H air, S eeds , Plant leaves High-throughput sequencing (HTS) methods Personalized Medicine 4

5. Basic methods 1. Maxam-Gilbert sequencing This method is based on nucleobase- specific partial chemical modification of DNA and subsequent cleavage of the DNA backbone at sites adjacent to the modified nucleotides 5

6. 2. Chain-termination methods S ingle-stranded DNA molecules Polyacrylamide gel S hort oligonucleotide + template = primer D ideoxynucleotides ( ddNTPs: ddATP , ddCTP, ddGTP, and dNTP) Not all DNA polymerases can be used for sequencing 6

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8. The most significant innovations in Sanger sequencing have been: The development of fluorescent dyes The use of thermal-cycle sequencing Software developments to interpret and analyze the sequences Licor , Amersham , MilliGen , Perkin Elmer and Dupont , all of them except the AB machines 8

9. Large-scale sequencing and denovo sequencing Very long DNA pieces Shotgun sequencing Analysis of DNA sequences longer than 1000 base pairs 9

10. High-throughput methods Long-read sequencing methods 1. Single molecule real time (SMRT) sequencing 2. Nanopore DNA sequencing Short-read sequencing methods 1 . Massively parallel signature sequencing (MPSS ) 2. Polony sequencing 3 . 454 pyrosequencing 4 . Illumina (Solexa) sequencing 5 . SOLiD sequencing 6. Ion Torrent semiconductor sequencing 7 . DNA nanoball sequencing 8. Helicos single molecule fluorescent sequencing 10

11. Long-read sequencing methods 1. Single molecule real time (SMRT) sequencing Real-time sequencing also enables read lengths to be longer Read lengths of up to 20 000 bp 11

12. 2. Nanopore DNA sequencing Using nanopore sequencing, a single molecule of DNA or RNA can be sequenced without the need for PCR amplification or chemical labeling of the sample MinION Mkl Problem: 1. reduce the high error rates of base 2. optimize the speed of DNA 12

13. Short-read sequencing methods 1. Massively parallel signature sequencing (MPSS ) MPSS is a method for determining expression levels of mRNA by counting the number of individual mRNA molecules produced by each gene. 13

14. 2. Polony sequencing M ultiplex sequencing technique U sed to “read” millions of immobilized DNA sequences in parallel P aired-end tags library DNA template is of 135 bp in length with two 17–18 bp paired genomic tags separated The current read length of this technique is 26 bases per amplicon and 13 bases per tag, leaving a gap of 4–5 bases in each tag . The protocol of Polony sequencing can be broken into three main parts, which are the paired end-tag library construction , template amplification and DNA sequencing . 14

15. Polony sequencing 15

16. 3. 454 pyrosequencing H igh throughput , Second generation A single strand of DNA with a length of 400-500bp The 4 required enzymes are: DNA Polymerase , ATP Sulfurylase , Luciferase , Apyrase 16

17. 4. Illumina (Solexa) sequencing Reversible I dentification of single nucleotides Illumina sequencing technology works in three basic steps: amplify, sequence, and analyze. P urified DNA 17

18. 5. SOLiD sequencing N ext-generation DNA sequencing 99.94% 18

19. 6. Ion Torrent semiconductor sequencing H ydrogen ions S equencing by synthesis 19

20. A major advantage of the system is that no camera, light source or scanner is needed 20

21. 7. DNA nanoball sequencing H igh throughput sequencing Genomic sequence R olling circle replication Fluorescence 21

22. 8. Helicos single molecule fluorescent sequencing T he first commercial NGS S ingle molecule fluorescent sequencing M13 bacteriophage S uccessfully sequenced the human genome Clinical evaluation & Sequenced RNA 2 STEP 1. Preparing the DNA ( Fragmenting the DNA; Tailing; Blocking) 2. DNA sequencing ( Sample loading; Filling and locking ; Sequencing ) 22

23. Methods in development Tunnelling currents DNA sequencing Sequencing by hybridization Microfluidic Sanger sequencing Microscopy-based techniques RNAP sequencing In vitro virus high-throughput sequencing 23

24. 1. Tunnelling currents DNA sequencing Tunneling current measurements in liquid have been considered a promising way to identify the sequence of base molecules in RNA and DNA A pair of metal electrodes 24

25. 2. Sequencing by hybridization Small changes relative to a known DNA sequence DNA chips , microarrays , synthetic oligonucleotides Affymetrix NABsys 3. Microfluidic Sanger sequencing 25

26. 4. Microscopy-based techniques Microscopic evaluation of specimens S ymptomatic patient 26

27. 5. RNAP sequencing RNA polymerase (RNAP ) Polystyrene bead 27

28. 6. In vitro virus high-throughput sequencing Protein interactions C ombination of 454 pyrosequencing and an in vitro virus mRNA display method 7. Sequencing with mass spectrometry MALDI-TOF MS Size Easily detect differences between RNA fragments Single-nucleotide polymorphisms in human Up to 100 base pairs 28

29. Next-generation sequencing Library (Breakage of DNA, Immobilization, Amplification) Up of thousands or millions of DNA fragments S ingle experiment 29

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31. Methods There is no electrophoresis or any other fragment separation step. Reversible terminator sequencing Pyrosequencing Illumina se q uencing 31

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33. Thanks for your attention 33