1. Prototyping Presentation The best marketing tool for your business.

2. CONTENT 1.Introduction 2.What is NGS 3.STEPS OF NGS PREPARATION 4.SEQUENCING AND THEIR PRINCIPLE 5.DATA ANALYSIS 6.APPLICATIONS 7.LIMITATIONS 8.CONCLUSION 9.REFERENCES

3. INTRODUCTION DNA sequencing is figuring out the order of DNA nucleotides, or bases ATGC, in our genome that make up an organism’s DNA. The sequencing of the human genome was completed in 2003, after 13 years of international collaboration and investment of USD 3 billion. The human genome project used Sanger sequencing, the principal method of DNA sequencing since its invention in the 1970s. Today, the demand for sequencing is growing exponentially, with a large amounts of genomic DNA needing to be analysed quickly, cheaply, and accurately. Thanks to new sequencing technologies known collectively as Next Generation Sequencing, it is now possible to sequence and entire genome in a matter of hours.

4. ERA OF SEQUENCING

5. Next-generation sequencing also known as high throughout full parallel sequencing, is the all encompassing term used to describe a number of different modern sequencing technologies. These include Illumina sequencing, Roche 454 sequencing, ion torrent; proton/PGM sequencing and solid sequencing.With the advent of the technologies sequencing DNA and RNA has become much more facile and affordable in comparison to the previously used to Shankar sequencing for these reasons NGS has been the game changer in the field of modern genomics and molecular biology. A common starting point for template preparation of NGS platforms is random fragmentation of target DNA and addition of platform specific adapter sequences to flanking ends. Protocol typically use sonication to share input DNA, coupled with several rounds of enzymatic modification to produce a sequence are ready product. What is NGS ? Over the past 20 years, gene expression profiling, a revolutionary technique, has been widely used for genomic identification, genetic testing, drug discovery, and disease diagnosis, among other things.The field of genomics and proteomics research has undergone neoteric fluctuations as a result of next-generation sequencing (NGS), a paradigm-shifting technology that provides higher accuracy, larger throughput and more applications than the microarray platform.

6. The use of massively parallel sequencing has increasingly been the object of study in recent years. The NGS technologies are implemented for several applications, including whole genome sequencing, de novo assembly sequencing, resequencing, and transcriptome sequencing at the DNA or RNA level. For instance, de novo assembly sequencing assembles the genome of a particular organism without a reference genome sequence ( which may lead to a better understanding at the genomic level and may assist in predicting genes, protein coding regions, and pathways. In addition, resequencing the organism with a known genome can help in understanding the relationship between genotype and phenotype and identify the differences among reference sequences (6,7). In addition, NGS technologies have been widely used to analyze small RNAs,including identification of differentially expressed microRNAs (miRNAs), prediction of novel miRNAs, and annotation of other small non-coding RNAs. Currently, there are several companies implementing different NGS technologies, such as Illumina (http://www.Illumina.com), Roche (http://www.454.com), ABI Life Technologies (http://www.lifetechnologies.com), Helicos Biosciences (http://www.helicosbio.com), Pacific Bioscience (http://www.pacificbiosciences.com), and Oxford Nanopore (http://www.nanoporetech.com).

7. Steps of NGS Sequencing The principle behind Next Generation Sequencing (NGS) is similar to that of Sanger sequencing, which relies on capillary electrophoresis. The genomic strand is fragmented, and the bases in each fragment are identified by emitted signals when the fragments are ligated against a template strand. The NGS method uses array-based sequencing which combines the techniques developed in Sanger sequencing to process millions of reactions in parallel, resulting in very high speed and throughput at a reduced cost. The genome sequencing projects that took many years with Sanger methods can now be completed in hours with NGS, although with shorter read lengths (the number of bases that are sequenced at a time) and less accuracy. 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.

8. ●Amplification: 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. 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 Bridge PCR: this process leads to clonal clusters of localised identical strands Emulsion PCRBridge PCR

9. Sequencing and their principle : Sequencing : DNA is sequenced using one of several different approaches Several competing methods of Next Generation Sequencing have been developed by different companies. NGS methods are well – established and share many features in common. The preparation of target DNA for DNA sequencing is almost same in all of these methods the only thing differs is the exact sequence process.  454 Pyrosequencing  Ion torrent semiconductor sequencing  Sequencing by ligation {SOLiD}  Illumina / Reversible terminator sequencing

10. Pyrosequencing :-  Pyrosequencing technology is a recently established non electrophoretic, sequencing-by-synthesis technique which uses an enzymatic system based on luciferase (an enzyme which emits light in the presence of ATP ) to monitor DNA production.  Pyros (Greek for “fire”, because light is produced)  Pyrosequencing provides the analysis of single nucleotide polymorphisms (SNPs) as well as whole-genome sequencing.  Read lengths are around 200-300 bases.  400,000 reads of parallel sequencing  100 mb of output per run  Run time 7.5 hours.  Principle : (DNA)n +dNTP DNA polymerase (DNA)n+1 + PPi coupled rkn PPi + APS ATP Sulfurylase ATP ATP + Luciferin + O2 Luciferase Oxyluciferin + Light + AMP + PPi +CO2 ATP + dNTP Apyrase ADP + dNDP + 2Pi degradation rkn ADP + dNDP Apyrase AMP + dNMP + 2Pi

11. MECHANISM {inside the well}:-

12. ION TORRENT SEQUENCING :- Ion Torrent sequencing technology combines semiconductor sequencing technology with biochemistry, enabling the direct translation of chemical information into digital sequence data. PRINCIPLE :-  DNA chain is fixed in the semiconductor chip micro holes, and followed by the incorporation of ACGT.  With the incorporation of each base, hydrogen ions are released, which can be detected at the bottom of each hole through the detection of H+.  If there are two identical bases on the DNA strand, the output voltage is doubled, and the chip records two identical bases.  Hydrogen ions are detected on ion semiconductor or sequencing chips.  The transistors and circuits are then pattern-transferred and subsequently etched onto the wafers using photolithography. Compared with other sequencing technologies, this sequencing system is :- simple, faster, affordable, cost-effective and more flexible.

13. GENOME ANALYZER {SOLEXA} :-  The Genome analyzer was developed by Solexa (now part of illumina).  It can be used for conventional DNA sequencing as well as for transcriptome analysis. PRICIPLE :-  This method is based on a two-step mechanism. It combines single molecule amplification technology and novel reversible terminator-based sequencing.  DNA, randomly fragmented by shear stress,is ligated with adapters at both sides of their chain. Then the DNA is attached to the internal surface of a flow cell.  This flow cell is derivatized with oligonucleotides forming a dense layer of primers, which are complementary to the adapters.  The DNA fragments hybridized with the primers in a bridging way initialise solid-phase bridge amplification immediately and the fragments become double-stranded.  With further steps including denaturation, renaturation and synthesis, a high-density of equal DNA fragments is generated in a small area. Approximately one million double-stranded DNA copies are produced per cluster, which is representing one single fragment. This guarantees the required signal intensity for detection during sequencing  Real sequencing reaction :- All four dNTPs carrying a base-unique fluorescent dye are added and incorporated by a DNA polymerase gradually. Following each base incorporation step, an image is made by laser excitation for each cluster.  The identity of the first base is recordable. Then the elimination of the chemically blocked 3’ -OH group and the dye follows.  Within every new cycle, the DNA chain is elongated and more images are recorded.  A base-calling algorithm assigns the sequences and evaluates the analysis quality. With read length of 25–35 bases the reading frame is tenfold smaller than with common pyrosequencing.  Solexa read length is limited by interference between DNA polymerase and the used fluorochromes, which results in a reduced enzyme activity. Most strands get terminated early.  Fluorochromes used by Solexa and also by SOLiD generate stronger signals than the luciferase in pyrosequencing allowing a much higher density of reactions.

14. SOLiD Sequencing :- o Sequencing by Oligonucleotide Ligation and Detection. o Developed by Applied Biosystems {Life technologies }. It is based on polony sequencing (Polymerase +colony). o The sequencer adopts the technology of two –base sequencing based on ligation sequencing. o Each sequencing involves 5 rounds of cyclic steps and each round involve multiple ligation steps between 16 possible Di-base probes having in total of 4 fluorescent dye {among 16 Di-base probes} attached to them. o Then the sequence is determined and cross checked using a Di- base color coding system and sequence alignment. This can also be done by computer program  ADVANTAGES: higher accuracy and precision  DISADVANTAGES : shorter read length {85 bp} and high cost.

16. Target audience

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