Next generation sequencing

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1. SUBMITTED T O S.PALANIANANTH Ist MSC Microbiology Dr . S.Sivasankara Narayani SUBMITTED BY Presented to Next generation sequencing

2. INTRODUCTION: Next Generation Sequencing ( NGS ) is a powerful platform that has enabled the sequencing of thousands to millions of DNA molecules simultaneously. 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. The high demand for low-cost sequencing has driven the development of high-throughput sequencing which produce thousands or millions of sequences at once. They are intended to lower the cost of DNA sequencing beyond what is possible with standard dye-terminator methods. Thus, 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 revolutionized the study of genomics and molecular biology.

3. Classification: Classified to different generations, NGS has led to overcome the limitations of conventional DNA sequencing methods and has found usage in a wide range of molecular biology applications. The generations it is classified into include: First Generation Sanger Sequencing Second Generation Sequencing Pyrosequencing Sequencing by Reversible Terminator Chemistry Sequencing by Ligation   Third Generation Sequencing Single Molecule Fluorescent Sequencing Single Molecule Real Time Sequencing Semiconductor Sequencing Nanopore Sequencing Fourth Generation Sequencing Aims conducting genomic analysis directly in the cell.

4. Sanger Sequencing: Sanger Sequencing utilizes a high fidelity DNA-dependent polymerase to generate a complimentary copy to a single stranded DNA template In each reaction a single primer, complementary to the template, initiates a DNA synthesis reaction from its 3’ end. Deoxynucleotides or nucleotides , which are the monomers of DNA, are added one after the other in a template-dependent manner forming phospho-diester bonds between the 3’ hydroxyl of the growing end of the primer and the 5’ tri-phosphate group of the incoming nucleotide Each reaction also contains a mixture of four di- deoxynucleotides , one for each DNA base (i.e. A, G, T, and C). These di- deoxynucleotides resemble the DNA monomers enough to allow incorporation into the growing strand, however, they differ from natural deoxynucleotides in two ways: 1) they lack a 3’ hydroxyl group which is required for further DNA extension resulting in chain termination once incorporated in the DNA molecule, and 2) each di- deoxynucleotide has a unique fluorescent dye attached to it allowing for automatic detection of the DNA sequence

5. Sanger Sequencing: Many copies of different-length DNA fragments are generated in each reaction , terminated at all of the nucleotide positions of the template molecule by one of the di- deoxynucleotides The reaction mixtures are loaded on the sequencing machine, either manually onto slab gels or automatically with capillaries, and are electrophoresed to separate the DNA molecules by size. The DNA sequence is read through the fluorescent emission of the di- deoxynucleotide as it flows through the gel ( Modern day Sanger Sequencing instruments use capillary based automated electrophoresis, which typically analyzes 8–96 sequencing reactions simultaneously.

6. Next generation methods of DNA sequencing have three general steps: Library preparation : libraries are created using random fragmentation of DNA, followed by ligation with custom linkers Amplification: the library is amplified using clonal amplification methods and PCR Sequencing: DNA is sequenced using one of several different approaches

7. LIBRARY PREPARATION: Firstly, DNA is fragmented either enzymatically or by sonication (excitation using ultrasound) to create smaller strands . Adaptors (short, double-stranded pieces of synthetic DNA) are then ligated to these fragments with the help of DNA ligase, an enzyme that joins DNA strands. The adaptors enable the sequence to become bound to a complementary counterpart. Adaptors are synthesised so that one end is 'sticky ' whilst the other is 'blunt' (non-cohesive) with the view to joining the blunt end to the blunt ended DNA. This could lead to the potential problem of base pairing between molecules and therefore dimer formation. To prevent this, the chemical structure of DNA is utilised , since ligation takes place between the 3′-OH and 5′-P ends . By removing the phosphate from the sticky end of the adaptor and therefore creating a 5′-OH end instead, the DNA ligase is unable to form a bridge between the two termini

8. LIBRARY PREPARATION In order for sequencing to be successful, the library fragments need to be spatially clustered in PCR colonies or 'polonies' as they are conventionally known, which consist of many copies of a particular library fragment. Since these polonies are attached in a planar fashion, the features of the array can be manipulated enzymatically in parallel. This method of library construction is much faster than the previous labour intensive procedure of colony picking and E. coli cloning used to isolate and amplify DNA for Sanger sequencing, however , this is at the expense of read length of the fragments.

9. AMPLIFICATION: Library amplification is required so that the received signal from the sequencer is strong enough to be detected accurately . With enzymatic amplification, phenomena such as 'biasing' and 'duplication' can occur leading to preferential amplification of certain library fragments. Instead, there are several types of amplification process which use PCR to create large numbers of DNA clusters.

10. Emulsion PCR: Emulsion oil, beads, PCR mix and the library DNA are mixed to form an emulsion which leads to the formation of micro wells In order for the sequencing process to be successful, each micro well should contain one bead with one strand of DNA (approximately 15% of micro wells are of this composition). The PCR then denatures the library fragment leading two separate strands , one of which (the reverse strand) anneals to the bead. The annealed DNA is amplified by polymerase starting from the bead towards the primer site. The original reverse strand then denatures and is released from the bead only to re-anneal to the bead to give two separate strands.

11. Emulsion PCR: These are both amplified to give two DNA strands attached to the bead. The process is then repeated over 30-60 cycles leading to clusters of DNA. This technique has been criticised for its time consuming nature, since it requires many steps (forming and breaking the emulsion, PCR amplification, enrichment etc ) despite its extensive use in many of the NGS platforms. It is also relatively inefficient since only around two thirds of the emulsion micro reactors will actually contain one bead. Therefore an extra step is required to separate empty systems leading to more potential inaccuracies

12. Bridge PCR: The surface of the flow cell is densely coated with primers that are complementary to the primers attached to the DNA library fragments (The DNA is then attached to the surface of the cell at random where it is exposed to reagents for polymerase based extension. On addition of nucleotides and enzymes, the free ends of the single strands of DNA attach themselves to the surface of the cell via complementary primers, creating bridged structures. Enzymes then interact with the bridges to make them double stranded, so that when the denaturation occurs, two single stranded DNA fragments are attached to the surface in close proximity. Repetition of this process leads to clonal clusters of localised identical strands. In order to optimise cluster density, concentrations of reagents must be monitored very closely to avoid overcrowding.

13. Bridge PCR: SEQUENCING: Sequencing: DNA is sequenced using one of several different approaches

14. Pyrosequencing:   principle: Pyrosequencing is non-electrophoretic, bioluminescence method that measures the release of inorganic pyrophosphate by proportionally converting it into visible light using a series of enzymatic reaction. 454 sequencing was the first commercially available advanced sequencing technique. It was introduced in 2005 by the 454 Corporation. Step-1 DNA is first denatured into single strands ,joined to adapters at either end of the fragmented DNA and attached to microscopic beads Step-2 The DNA on the beads is amplified by an emulsion PCR. PCR amplified allowing up to 1 million identical fragments around one bead 

15. Pyrosequencing:   Step-3 Each bead is then placed in a well of a Pico Titer tube, which is put into a flow cell where it is incubated with DNA polymerase, ATP sulfurylase , luciferase, and apyrase long with the substrates luciferin and adenosine 5’- phosphosulfate (ASP). Step-4 The first of four deoxyribonucleotide triphosphates ( dNTP ) is added to the reaction. DNA polymerase catalyzes the incorporation of the deoxyribon Nucleotide triphosphate into the DNA strand, if it is complementary to the base in the template strand. Each incorporation event is accompanied by release of pyrophosphate( PPi ) in a quantity equimolar to the amount of incorporated nucleotide

16. Pyrosequencing:   Step-5 ATP sulfuryla sequantitatively converts PPito ATP in the presence of adenosine5 phosphosulfate (APS). This ATP drives the luciferase mediated conversion of luciferin to oxyluciferin that generates visible light in amounts that are proportion alto the amount of ATP. The light produced in the luciferase- catalyzed reaction is detected by a charge coupled device (CCD) camera and seen as a peak in a PyrogramT”4 . The height of each peak (light signal) is proportion alto the number of nucleotides incorporated  Step-6 Apyrase, a nucleotide degrading enzyme, continuously degrades ATP and unincorporated dNTPs . This switches off the light and regenerates the reaction solution. The next dNTP is then added.

17. Pyrosequencing:   Step-7 Addition of dNTPs is performed one at a time. It should be noted that deoxyadenosine alfa-thio triphosphate ( dATPalfaS ) is used as a substitute for the natural deoxyadenosine triphosphate ( dATP ) since it is efficiently used by the DNA polymerase, but not recognized by the luciferase. As the process continues, the complementary DNA strand is built up and the nucleotide sequence is determined from the signal peaks in the Pyrogram .

18. Ion torrent semiconductor sequencing: Ion torrent sequencing uses a "sequencing by synthesis" approach, in which a new DNA strand, complementary to the target strand, is synthesized one base at a time. A semiconductor chip detects the hydrogen ions produced during DNA polymerization Following polony formation using emulsion PCR, the DNA library fragment is flooded sequentially with each nucleoside triphosphate ( dNTP ), as in pyrosequencing. The dNTP is then incorporated into the new strand if complementary to the nucleotide on the target strand. Each time a nucleotide is successfully added, a hydrogen ion is released, and it detected by the sequencer's pH sensor. As in the pyrosequencing method, if more than one of the same nucleotide is added, the change in pH/signal intensity is correspondingly larger. Ion torrent sequencing is the first commercial technique not to use fluorescence and camera scanning; it is therefore faster and cheaper than many of the other methods. Unfortunately, it can be difficult to enumerate the number of identical bases added consecutively. For example, it may be difficult to differentiate the pH change for a homorepeat of length 9 to one of length 10, making it difficult to decode repetitive sequences.

19. Illumina/ Solexa sequencing Principle : Illumina sequencing was initially developed by Solexa Inc. and introduced in 2006; shortly after its introduction, Solexa was purchased by Illumina Inc. This technique uses a flow cell surface that allows for the detection of over 1 million individual reads. This flow surface has eight channels on it, so eight separate sequencing experiments can be run per cell. The process uses the four nucleotides labeled with four different fluorescent mole clues and involves the   following steps. Step 1: Prepare Genomic DNA Sample A strand cannot be sequenced if it is tolarge or if it is double stranded. Therefore the DNA is fragmented i nto between 300 and 800 bps long through–  Sonication  Nebulization  Enzyme digestion Adapters are needed to be ligated no the ends of the fragments in order to get the sequence to anneal to where the DNA sequence can be determined by the sequencing machine. Denature dsDNA into ssDNA by heating to 95°

20. Illumina/ Solexa sequencing Step-2- Attach DNA to Surface In bridge amplification, the adapter constructs added to the DNA sequence of interest have flow cell binding sites, P5 and P7, which allow the P5 and P7 regions of the single-stranded library fragments to anneal to their complimentary oligo's on the flow cell surface. This means if one of the DNA fragments P5 anneals to the flow cell, it will anneal by attaching to a P7 oligosthatis attached to the flow cell, and vise versa. Several samples can be loaded the 8 lane flow cell for simultaneous analysis  Step 3: Bridge Amplification In this step the sequence makes a kind of bridge shape when it is being copied. Unlabeled nucleotides and polymerase enzyme are added to initiate the solid phase bridge amplification Flow cell

21. Illumina/ Solexa sequencing Step-4-Fragments Become Double Stranded The reagents needed to sequence are added such as primers to start the sequencing, nucleotides to form the new sequence, polymerase to actually sequence the nucleotides together, and buffer to pH at an optimal level for the enzymatic reaction. The flow cell oligo's act as primers and a strand complimentary to the library fragment is synthesized. step 5 Denature the Double-Stranded Molecules The original strand is washed a way, leaving behind fragment copies that are covalently bonded to the flow cell surface in a mixture of orientations

22. Illumina/ Solexa sequencing step-6-Complete Amplification The steps through of the addition of the sequencing reagents, creation of double stranded DNA, and the denaturation are repeated multiple times to create thousands of identical copies of the same sequence in each cluster. In other word search cluster is made of up thousands of the identical sequence and each cluster is a different fragment of the original larger sequence of interest. Step-7-Determine First Base The P5 region is cleaved, resulting in clusters containing only fragments which are attached by the P7 region. This ensures that all copies are sequenced in the same direction. The sequencing primer anneals to the P5 end of the fragment, and begins the sequencing by synthesis process. The reagents needed to sequence are added such as primers to start the sequencing, fluorescently labeled nucleotides to form and detect the base added to each cluster ,polymerase to actually add the nucleotides to the forming strand, and buffer to keep the pH at an optimal level for the enzymatic reaction

23. Illumina/ Solexa sequencing Step-8-Image First Base The unincorporated bases, which are the bases that were not attached by a polymerase to one of newly forming sequences in the clusters, are washed away. Then the machine can clearly detect the fluorescent signals that are left and record the base that was added to each of the clusters. After that the block on the newly added base’s 3 prime part of the sugar is removed so that another base will be able to added to it by polymerase. The fluorescent is also removed so tha tonly the new base’s fluorescent is detected in the new cycle.

24. Illumina/ Solexa sequencing step-9-Determine Second Base • Add sequencing reagents • Primers • Polymerase • Fluorescently labeled nucleotides • Buffer • Second base incorporated Step-10-Image Second Chemistry Cycle Again the image is detected by first washing a way the un incorporated bases. Then the flow cell is prepared for the next cycle by removing the fluorescent and blocking the newly added base so that polymerase can add another base to it. Step-11-Sequencing Over Multiple Chemistry Cycles The sequencing cycles are repeated to determine the sequence of bases in a fragment ,one base at a time.

25. Illumina/ Solexa sequencing Step-12-Align Data • After the sequencing is finished they are aligned • Each was once one larger sequence that had been fragmented • Needs To be realigned to find the original sequence of the larger sequence . Bioinformatics tools are used to do this using a reference sequence. A reference sequences digital nucleic acid sequence data base, assembled by scientists as are presentative example of a species 'set of genes. SNPs are also call by the alignment tools as well. SNP stands for Single Nucleotide Polymorphism . Using this variations in the new sequence can be found population in which a Single Nucleotide — A, T, C or G — in the genome differs between members of a biological species or paired chromosomes

26. Advantages of NGS - 1. Construction of a sequencing library for clonal amplification to generate sequencing features. 2. No invivo cloning, transformation, colony picking 3. Array-based sequencing 4. Higher degree of parallelism than capillary-based sequencing