Next Generation Sequencing (NGS) also known as high-throughput sequencing

Next Generation Sequencing (NGS), also known as high-throughput sequencing, is a revolutionary technology that enables the rapid sequencing of large amounts of DNA or RNA. Unlike traditional Sanger sequencing, which sequences one DNA fragment at a time, NGS can simultaneously sequence millions of fragments in parallel, making it faster, more accurate, and cost-effective for large-scale genomic studies.

1. Next Generation Sequencing (NGS) Techniques

2. Next Generation Sequencing (NGS) Techniques Sanger Sequencing Microarray Technology Next Generation Sequencing (NGS) Applications

3. First-generation DNA sequencing First-generation DNA sequencing methods consist of 2 methods: Maxam-Gilbert (chemical degradation) and Sanger (chain termination) methods which are based on the amplification of template DNA and gel electrophoresis

4. Chain termination DNA sequencing

5. Human Genome Sequencing Project Shotgun sequencing using Sanger sequencing method Genomic DNA Fragmentation Sanger sequencing Assembly

6. Human Genome Sequencing Project Too expensive and time consuming! Completed in 2003, took ~13 years Cost: USD 3 billion

7. Drawbacks of Sanger sequencing method Low-throughput Time consuming and expensive when applied at a large-scale

8. Microarray technology For genotyping, gene expression studies etc. Use of DNA probes – hybridization The sequence has to be known

9. Advantages of NGS over traditional sequence Advantages Fast and cost effective Sensitive towards low complexity sequences (AAAAAAAATT) Less sample requirement Massive production of short sequences.(read sequences) High throughput technique discovery process. Simultaneous analysis of different samples of different genes. Quantitative detection of genomic mutations No requirement of Cloning process. No requirement of any follow up method of sequences. Shortfalls of microarray are overcome with NGS techniques Disadvantage: Multiplexing Its analysis it is computational dependent More chances of errors Application : More frequent in cancer diagnosis Clearer picture of tumor heterogeneity

10. Microarray technology

11. Drawbacks of Microarray technology Only works with known sequences Not capable of identifying new genes or sequencing new genomes Indirect method Reproducibility issue

12. Emergence of Next Generation Sequencing (NGS) Technologies Needed : Direct sequencing method high- throughput, accurate and reproducible cost-effective Target : Human genome sequencing for $1000

13. Benefits of NGS Technologies Ability to sequence thousands of genes or genomic regions simultaneously Ability to directly sequence unknown genomic fragments or genomes Capability to sequence a large number of samples in a short time More power to detect low frequency variants Cost- effective for processing a large number of samples

14. Applications NGS Technologies Genomics Transcriptomics Epigenomics

15. NGS

16. NEXT-GENERATION SEQUENCING Also known as high-throughput sequencing: Read sequences : Raw sequences Roche 454/ Pyrosequencing sequencing Virtual Terminator sequencing SOLiD sequencing Illumina/ Solexa These recent technologies allow us to sequence DNA and RNA much more quickly and cheaply than the previously used Sanger sequencing.

17. Next (second) Generation Sequencing New technologies allowing the massive production of tens of millions of short sequencing fragments. It is also called: “ Massively parallel sequencing”. These techniques could be used to deal with similar problems than microarrays. They raised the promise of personalized medicine

18. High-throughput sequencing workflow High-throughput sequencing workflow. There are three main steps in high-throughput sequencing: Sample Preparation, Immobilization, And Sequencing. Preparation of the sample for high throughput sequencing involves random fragmentation of the genomic DNA and addition of adapter sequences to the ends of the fragments. The prepared sequencing library fragments are then immobilized on a solid support to form detectable sequencing features .

19. High-throughput sequencing workflow Finally, massively parallel cyclic sequencing reactions are performed to interrogate the nucleotide sequence

20. Template amplification strategies.

21. Animation of Emulsion and Bridge Amplification https://www.youtube.com/watch?v=bFNjxKHP8Jc&t=1s https://www.youtube.com/watch?v=fCd6B5HRaZ8&t=2s

22. Emulsion PCR Emulsion PCR is a PCR variation that some NGS technologies use to replicate DNA sequences. It is conducted on a bead surface within tiny water bubbles floating on an oil solution.

23. One Fragment = One Bead = One Read

24. Emulsion PCR

25. Bridge Amplification Sequencing templates are immobilized on a proprietary flow cell surface designed to present the DNA in a manner that facilitates access to enzymes while ensuring high stability of surface bound template and low non-specific binding of fluorescently labeled nucleotides. Solid-phase amplification creates up to 1,000 identical copies of each single template molecule in close proximity (diameter of one micron or less). densities on the order of ten million single-molecule clusters per square centimeter are achieved. Illumina flow cell

26. Bridge Amplification

27. Second Generation Sequencing Techniques Technologies employed in the second-generation sequencing platforms are of two main types based on sequencing chemistry: ‘‘sequencing by synthesis’’ and ‘‘sequencing by ligation’’ Pyrosequencing: sequencing-by-synthesis Reversible termination :sequencing is one of the sequencing-by-synthesis strategies popularized by Illumina/ Solexa

28. Pyrosequencing Pyrosequencing is a DNA sequencing technique that is based on the detection of released pyrophosphate (PPi) during DNA synthesis. In a cascade of enzymatic reactions, visible light is generated that is proportional to the number of incorporated nucleotides The general principle behind different Pyrosequencing reaction systems.

29. Pyrosequencing principle Pyrosequencing technology is based on the sequencing by synthesis principle. After successful incorporation of a nucleotide by a polymerase using a single-stranded PCR (or RT-PCR) fragment as template, the released PPi is converted to light by an enzyme cascade: ATP sulfurylase converts PPi to ATP in the presence of APS. This ATP drives the luciferase -mediated conversion of luciferin to oxyluciferin that generates visible light, which is detected by CCD sensors and seen as a peak in the raw data output (Pyrogram®). Apyrase continuously degrades unincorporated nucleotides and ATP. The height of each peak (light signal) is proportional to the number of nucleotides incorporated. Sequential addition of nucleotides allows quantitative decoding of the sequence to analyze

30. Pyrosequencing There are two different Pyrosequencing strategies that are currently available: Solid phase Pyrosequencing And Liquid Phase Pyrosequencing . Solid phase Pyrosequencing utilizes immobilized DNA in the three-enzyme system described previously. In this system a washing step is performed to remove the excess substrate after each nucleotide addition.

31. Solid-phase Pyrosequencing

32. Liquid-phase Pyrosequencing Primed DNA template and four enzymes involved in liquid-phase Pyrosequencing are placed in a well of a Microtitre plate . The four different nucleotides are added stepwise and incorporation is followed using the enzyme ATP sulfurylase and luciferase. The nucleotides are continuously degraded by nucleotide-degrading enzyme allowing addition of subsequent nucleotide. dXTP indicates one of the four nucleotides.

33. Liquid-phase Pyrosequencing

34. Pyrogram Pyrogram of the raw data obtained from liquid-phase Pyrosequencing. Proportional signals are obtained for one, two, three, and four base incorporations. sequence is indicated above the Pyrogram.

35. Applications of Pyrosequencing Genotyping of Single-Nucleotide Polymorphisms Microbial Typing Resequencing Tag Sequencing Analysis of Difficult Secondary Structures advantages This approach requires no gels, fluorescent dyes, or ddNTPs . https://www.youtube.com/watch?v=nFfgWGFe0aA

36. 454 Pyrosequencing This technology is a sequencing-by-synthesis method that involves a combination of emulsion PCR and Pyrosequencing. https://www.youtube.com/watch?v=bFNjxKHP8Jc

37. 454 Pyrosequencing