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Deciphering DNA sequences is essential for virtually all branches of biological research. With the
advent of capillary electrophoresis (CE)-based Sanger sequencing, scientists gained the ability to
elucidate genetic information from any given biological system. This technology has become widely
adopted in laboratories around the world, yet has always been hampered by inherent limitations in
throughput, scalability, speed, and resolution that often preclude scientists from obtaining the essential
information they need for their course of study. To overcome these barriers, an entirely new technology
was required—Next-Generation Sequencing (NGS), a fundamentally different approach to sequencing
that triggered numerous ground-breaking discoveries and ignited a revolution in genomic science.

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  1. 1. What is sequencing?? • Deciphering the code hidden in biological sequences like DNA, polypeptides etc. • Method and technologies that enables us to determine the order of nucleotides and amino acids in DNA and Polypeptide respectively.
  2. 2. Traditional methods of Sequencing and its limitations • Maxam-Gilbert Method  Use of radioactive labels.  Sanger Method  It utilize the fluorescent dye for labeling.  separation of extended fragments of DNA with the addition of di-deoxynucleotides (lack a 3’-OH group) Thus, chain termination. Limitation Slow High cost per run.
  3. 3. Automated Sanger method 1. Bacterial cloning or PCR template purification 2. labelling of DNA fragments using the chain termination method with energy transfer 3. dye-labelled di-de oxynucleotides and a DNA polymerase 4. capillary electrophoresis 5. fluorescence detection that provides four-colour plots to reveal the DNA sequence.
  4. 4. NEXT GENERATION SEQUENCING •Also known as ▫High throughput sequencing or ▫ultra-deep sequencing or ▫massively parallel sequencing.
  5. 5. What is next generation sequencing ?? • Automated Sanger method (1st generation) • Technologies developed after that are known as next generation sequencing. • NGS enables the sequencing of biological codes at a very rapid pace with low cost per operation. • This is the primary advantage over conventional methods. • For example Billions of short reads can be sequenced in one operation.
  6. 6. Major Platforms for NGS •454 ( By Roche) •SOLiD (By Applied Biosystems) •Solexa (By Illumina)
  7. 7. • Above mentioned platform varies in strategies, application and type of data generated. • However, all technologies are common in ▫ That they generate sequences on an unprecedented scale ▫ DNA cloning is not required ▫ and very low operation cost.
  8. 8. What NGS Consists of Next generation technologies for sequencing is combination of strategies for • template preparation • sequencing and imaging • genome alignment • assembly methods
  9. 9. Template preparation As even most sensitive imaging technique is not able to detect single molecule, amplification of templates is inevitable. • Clonally amplified templates ▫ By emulsion PCR (emPCR) e.g. 454 and SOLiD ▫ Solid phase amplification e.g. illumina • Single-molecule templates
  10. 10. Template preparation: Traditional vs. NGS • Immobilization of templates fragments over bead /glass plate allows billions of the sequencing reaction run simultaneously.
  11. 11. sequencing and imaging • Sequencing ▫ cyclic reversible termination(CRT) e.g. illumina/solexa ▫ single-nucleotide addition (SNA) e.g. 454/roche ▫ real-time sequencing: R&D going on (pacific Bioscience) ▫ Sequencing by ligation (SBL) e.g. SOLiD • Imaging ▫ measuring bioluminescent signals ▫ four-colour imaging of single molecular events e.g. illumina/solexa.
  12. 12. Genome alignment and assembly After NGS reads have been generated, they are aligned to either • a known reference sequence or • assembled de novo
  13. 13. 454 (Pyrosequencing) • DNA is fragmented, joined to adapters at either end of the fragmented DNA • amplified in an emulsion PCR (includes agarose bead with complimentary adaptors to fragmented DNA) • PCR amplified allowing up to 1 million identical fragments around one bead and finally dropped into a PicoTitreTube (PTT)
  14. 14. PCR amplification Pico Titre Tube • Adapter containing the universal priming site are ligated to target ends • Same primer can be used for amplification
  15. 15. • In Pico titre tube reaction of fluorescence occurs with the addition of nucleotides Nucleotide addition
  16. 16. Nucleotide addition Output
  17. 17. SOLiD (support oligonucleotide ligation detection) • Sequencing by Oligo/Ligation and Detection. • Steps ▫ Library Preparation  two types of libraries sequencing-fragment or mate-paired are prepared. ▫ Emulsion PCR/Bead Enrichment  amplification of template fragments is done in same manner as 454. ▫ Bead Deposition Deposit 3’ modified beads onto a glass slide.
  18. 18. Sequencing by Ligation • Primers hybridize to the P1 adapter sequence on the templated beads • The method uses two-base- encoded probes(4 probes), which has the primary advantage of improved accuracy. • Multiple cycles of ligation, detection and cleavage are performed. • Extension product is removed and the template is reset with a primer complementary to the n-1 position for a second round of ligation cycles.
  19. 19. Illumina • Breaking up DNA • Adding adaptors, but in this case attach not to a bead but to a slide • Fold-back PCR is then used to amplify the fragmented DNA into a cluster
  20. 20. Sequential addition of nucleotides are added using a polymerase
  21. 21. NGS and Bioinformatics • Alignment of sequence reads to a reference BLAST doesn’t blast here Short read aligners side-lines BLAST Software • Bowtie • MAQ • BWA
  22. 22. • Above strategy works if reference genome exist. • de novo assembly from paired or unpaired reads • base-calling and/or polymorphism detection • structural variant detection • genome browsing.
  23. 23. Application of NGS • Variants discovery in targeted region or whole genome by re-sequencing • Reassembling genome of lower organism by de novo method. • Cost-effective sequencing of complex samples at remarkable scale and speed. • Sequencing entire transcriptome. • In Meta genomics : Sequencing genome of entire biological communities • Replacing ChIP-on-chip with ChIP-seq in case of multicellular eukaryotes. • Personalized genome for personalized medicine
  24. 24. • Further Readings 1. Branton, D. et al. The potential and challenges of nanopore sequencing. Nature Biotech. 26 1146–1153 (2008). 2. Wang, Z., Gerstein, M. & Snyder, M. RNA-Seq: a revolutionary tool for transcriptomics. Nature Rev. Genet. 10, 57–63 (2009). 3. Petrosino, J. F., Highlander, S., Luna, R. A., Gibbs, R. A. & Versalovic, J. Metagenomic pyrosequencing and microbial identification. Clin. Chem. 55, 856–866 (2009). 4. Park, P. J. ChIP–seq: advantages and challenges of a maturing technology. Nature Rev. Genet. 10, 669–680 (2009).