Next generation sequencing

an overview

1. BHARATHIDASAN INSTITUTE OF TECHNOLOGY ANNA UNIVERSITY TIRUCHIRAPPALLI-24 DEPARTMENT OF BIOTECHNOLOGY CLASS SEMINAR SUBJECT CODE/ TITLE: BT6603/GENETIC ENGINEERING & GENOMICS NEXT GENERATION SEQUENCING Presented by Faculty in charge Name : P. Swathipriya Dr . P.S. Sudhakar Gandhi Reg No . : 810014214042 Asst. Professor

2. BHARATHIDASAN INSTITUTE OF TECHNOLOGY ANNA UNIVERSITY TIRUCHIRAPPALLI-24 BHARATHIDASAN INSTITUTE OF TECHNOLOGY ANNA UNIVERSITY TIRUCHIRAPPALLI-24 DEPARTMENT OF BIOTECHNOLOGY CLASS SEMINAR SUBJECT CODE/ TITLE: BT6603/GENETIC ENGINEERING & GENOMICS NEXT GENERATION SEQUENCING Presented by Faculty in charge Name : P. Swathipriya Dr. P.S. Sudhakar Gandhi Reg No. : 810014214042 Asst. Professor

3. WHAT IS SEQUENCING? Genome sequencing  is a method used to figure out the order of DNA nucleotides, or bases in a genome- the order of A, C, G, and T that make up an organism's DNA. IMPORTANCE OF DNA SEQUENCING Better understanding of gene expression. Gene expression has significance in protein creation etc. It is capable to detect various diseases and genetic illnesses. Personalised medicine and disease discovery is possible. Forensics

4. HISTORY OF SEQUENCING 1869 - Discovery of DNA 1909 - Chemical characterisation 1953 - Structure of DNA solved 1977 - Sanger seq. invented -First genome sequenced - (5 kb) 1986 - First automated sequencing machine 1990 - Human Genome Project started 1992 - First “sequencing factory” at TIGR 1995 - First bacterial genome – H. influenzae (1.8 Mb) 1998 - First animal genome – C. elegans (97 Mb) 2003 - Completion of HGP (3 Gb ) – 13 years, $2.7 bn 2005 - First “next-generation” sequencing instrument 2013 - >10,000 genome sequences in NCBI database

5. INVENTION OF DNA SEQUENCERS 1977 First genome (ФX174) Sequencing by synthesis (Sanger) Sequencing by degradation ( Maxam Gilbert) First generation sequencers Second generation sequencers NGS Third generation sequencers Fourth generation sequencers

6. Next-generation sequencing (NGS), also known as high-throughput sequencing, is the catch-all term used to describe a number of different modern sequencing technologies including: Illumina ( Solexa ) sequencing Roche 454 sequencing SOLiD sequencing Ion torrent: Proton / PGM sequencing NEXT GENERATION SEQUENCERS

7. These recent technologies allow us to sequence DNA and RNA much more quickly and cheaply than the previously used Sanger sequencing , and as such have revolutionised the study of genomics and molecular biology. NGS has brought high speed not only to genome sequencing and personal medicine it has also changed the way we do genome research NEXT GENERATION SEQUENCERS

8. OVERVIEW OF NEXT GENERATION SEQUENCING PROTOCOL

9. The sequencing process 1. LIBRARY PREPARATION 2. CLONAL AMPLIFICATION 3. CYCLIC ARRAY SEQUENCING DNA fragmentation and invitro adaptor ligation sequencing 1 2 Emulsion PCR Bridge PCR 3 Pyrosequencing 454 sequencing SOLID platform Solexa technology Sequencing-by-ligation sequencing-by-synthesis

10. IILUMINA / SOLEXA SEQUENCING In NGS, vast numbers of short reads are sequenced in a single stroke. To do this, firstly the input sample must be cleaved into short sections. The length of these sections will depend on the particular sequencing machinery used.

11. . In Illumina sequencing, 100-150bp reads are used. Somewhat longer fragments are  ligated to generic adaptors and annealed to a slide using the adaptors. PCR is carried out to amplify each read, creating a spot with many copies of the same read. They are then separated into single strands to be sequenced. The slide is flooded with nucleotides and DNA polymerase. These nucleotides are fluorescently labelled , with the colour corresponding to the base. They also have a terminator, so that only one base is added at a time . IILUMINA / SOLEXA SEQUENCING

12. IILUMINA / SOLEXA SEQUENCING

13. IILUMINA / SOLEXA SEQUENCING FLOW CELL

14. IILUMINA / SOLEXA SEQUENCING

15. IILUMINA / SOLEXA SEQUENCING

16. Illumina sequencing IILUMINA / SOLEXA SEQUENCING

17. BRIDGE PCR AMPLIFICATION

18. BRIDGE PCR AMPLIFICATION

19. BRIDGE PCR AMPLIFICATION

20. Illumina sequencing IILUMINA / SOLEXA SEQUENCING

21. Illumina sequencing An image is taken of the slide. In each read location, there will be a fluorescent signal indicating the base that has been added IILUMINA / SOLEXA SEQUENCING

22. Illumina sequencing The slide is then prepared for the next cycle. The terminators are removed, allowing the next base to be added, and the fluorescent signal is removed, preventing the signal from contaminating the next image. IILUMINA / SOLEXA SEQUENCING

23. Illumina sequencing The process is repeated, adding one nucleotide at a time and imaging in between computers are then used to detect the base at each site in each image and these are used to construct a sequence. IILUMINA / SOLEXA SEQUENCING

24. All of the sequence reads will be the same length, as the read length depends on the number of cycles carried out. IILUMINA / SOLEXA SEQUENCING

25. IILUMINA / SOLEXA SEQUENCING

26. 454 SEQUENCING

27. PYROSEQUENCING A method of DNA sequencing based on the “sequencing by synthesis" principle. It differs from Sanger sequencing, relying on the detection of pyrophosphate release (hence the name) on nucleotide incorporation, rather than chain termination with dideoxynucleotides . ssDNA template is hybridized to a sequencing primer Incubated with the enzymes DNA polymerase, ATP sulfurylase , luciferase and apyrase , and with the substrates adenosine 5´ phosphosulfate (APS) and luciferin .

28. PYROSEQUENCING

29. PYROSEQUENCING CHEMISTRY

30. PYROSEQUENCING

31. PYRO SEQUENCING The addition of one of the four deoxynucleotide triphosphates ( dNTPs )(in the case of dATP we add dATPαS which is not a substrate for a luciferase ) initiates the second step. DNA polymerase incorporates the correct, complementary dNTPs onto the template. This incorporation releases pyrophosphate ( PPi ) stoichiometrically . PYROSEQUENCING

32. PYRO SEQUENCING ATP sulfurylase quantitatively converts PPi to ATP in the presence of adenosine 5´ phosphosulfate . This ATP acts as fuel to the luciferase -mediated conversion of luciferin to oxyluciferin that generates visible light in amounts that are proportional to the amount of ATP. PYROSEQUENCING

33. The light produced in the luciferase -catalyzed reaction is detected by a camera and analyzed in a program. Unincorporated nucleotides and ATP are degraded by the apyrase , and the reaction can restart with another nucleotide PYROSEQUENCING

34. ABI SOLID Sequencing

35. NGS APPLICATION

36. NGS ADVANTAGES

37. Array- basedsequencing Sanger sequencing Next-generation sequencing Advantages of NGS- Construction of a sequencing library for clonal amplification to generate sequencing features. No invivo cloning, transformation, colony picking Array-based sequencing Higher degree of parallelism than capillary-based sequencing

38. Reason Observation And Experience -the holy trinity of science Thank you SWATHI PRABHAKAR -ROBERT GREEN INGERSOLL