Next Generation Sequencing (NGS)

1. Next Generation Sequencing (NGS) Pankoj Kumar Das

2. Background • DNA • 1869: DNA, Friedrich Miescher • 1953: Double helix model, Watson & Crick • Sequencing • 1995: Foundation for sequencing, Frederick Sanger • 1970: location-specific primer extension strategy; Ray Wu • 1977: DNA sequencing with chain-terminating inhibitors; Sanger • 1977 & 1980: New sequencing method, Maxam & Gilbert

3. Genome sequenced • Bacteriophage ΦX174, 1997 • Haemophilus influenzae, 1995 • Saccharomyces cerevisiae, 1996 • Caenorhabditis elegans, 1998 • Drosophila melanogaster, 1999 • Human genome, 2000 (2003) http://www.genomenewsnetwork.org/resources/timeline Human Genome, 2011 https://en.wikipedia.org/wiki/Human_genome

4. Earlier Sequencing Methods • Basic methods • Maxam-Gilbert sequencing • Chemical modification (radioactivity) • SangerSequencing • Chain termination (ddNTPs) • Advanced methods • Shotgun sequencing • 1000 base pairs target DNA are broken into random fragments and reassembled • 2001, draft of the human genome

5. NGS: What? • DNA sequencing methods developed in the mid to late 1990s are called the "next-generation" or "second-generation" sequencing (NGS) methods, in order to distinguish them from the aforementioned earlier methods, like Sanger Sequencing. • Entire genome to be sequenced at once • Fragmenting the genome into small pieces • Randomly sampling for a fragment • Multiple fragments are sequenced at once • Massively parallel sequencing • Low cost NGS: Why? • Opened the door to more room for error. • Homopolymers • Background noise • Short read • Complex algorithm

6. High-throughput methods • Massively parallel signature sequencing (MPSS) • Polony sequencing • 454 pyrosequencing • Illumina (Solexa) sequencing • Combinatorial probe anchor synthesis (cPAS) • SOLiD sequencing

7. 454 sequencing • Background • 454 Life Sciences: founder Jonathan Rothberg • Roche in 2007 and shut down by Roche in 2013 (production ceased mid-2016) • Project "Jim": 2007 • Process • DNA library preparation and emPCR • Adaptor addition • Emulsion mixing & PCR • Sample loading (Pico Titer Plate) • Pyrosequencing • Addition of dNTP & release of PPi • PPi binds with Ammonium per sulfate • Creates ATP -> attached with luciferin • Oxidized into oxyluciferin • Generate light • Data analysis

8. Illumina sequencing Four Basic Steps Sample preparation Cluster formation Sequencing Data analysis

9. High-throughput sequencing methods

10. Third generation sequencing • Single molecule real time (SMRT) sequencing • Nanopore DNA sequencing • Advantages • Longer reads • Epigenetics • Portability • Speed

11. Nanopore Sequencing Principles Ohm’s Law (I=V/R) Types Biological Alpha hemolysin MspA (Mycobacterium smegmatis porin A) Solid Tunneling current Fluorescence Challenges Rapid rate (1 to 5μs per base) cause background noise Fail to get single base resolution Exonuclease approach Porin

12. Methods in development • Tunnelling currents DNA sequencing • Sequencing with mass spectrometry • Microfluidic Sanger sequencing • Microscopy-based techniques • RNAP sequencing • In vitro virus high-throughput sequencing

13. References • Watson, JD and Crick, FHC Molecular Structure of Nucleic Acids; Nature, April 25, 1953 • "Introducing 'dark DNA' – the phenomenon that could change how we think about evolution". • Behjati, S., & Tarpey, P. S. (2013). What is next generation sequencing?. Archives of Disease in Childhood-Education and Practice, 98(6), 236-238. • Min Jou W, Haegeman G, Ysebaert M, Fiers W (May 1972). "Nucleotide sequence of the gene coding for the bacteriophage MS2 coat protein". Nature. 237 (5350): 82–8. Bibcode:1972Natur.237...82J. • Fiers W, Contreras R, Duerinck F, Haegeman G, Iserentant D, Merregaert J, Min Jou W, Molemans F, Raeymaekers A, Van den Berghe A, Volckaert G, Ysebaert M (April 1976). "Complete nucleotide sequence of bacteriophage MS2 RNA: primary and secondary structure of the replicase gene". Nature. 260 (5551): 500–7. • Sanger F, Air GM, Barrell BG, Brown NL, Coulson AR, Fiddes CA, Hutchison CA, Slocombe PM, Smith M (February 1977). "Nucleotide sequence of bacteriophage phi X174 DNA". Nature. 265 (5596): 687–95. • https://www.genome.gov/11006943/human-genome-project-completion-frequently-asked-questions/ • Fleischmann RD, Adams MD, White O, Clayton RA, Kirkness EF, Kerlavage AR, Bult CJ, Tomb JF, Dougherty BA, Merrick JM (July 1995). "Whole-genome random sequencing and assembly of Haemophilus influenzae Rd". Science. 269 (5223): 496–512. • Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, et al. (February 2001). "Initial sequencing and analysis of the human genome". Nature. 409 (6822): 860–921. • Astier, Y; Braha O; Bayley H (2006-02-08). "Toward single molecule DNA sequencing: direct identification of ribonucleoside and deoxyribonucleoside 5'-monophosphates by using an engineered protein nanopore equipped with a molecular adapter". J Am Chem Soc. 128 (5): 1705–10. • Stoddart D; Heron A; Mikhailova E; Maglia G; Bayley H (2009). "Single-nucleotide discrimination in immobilized DNA oglionucleoties with a biological nanopore". Proc. Natl. Acad. Sci. USA. 106 (19): 7702–7707. • https://en.wikipedia.org/wiki/DNA_sequencing#/media/File:Historic_cost_of_sequencing_a_human_genome.svg • Wolters, J. F., Chiu, K., & Fiumera, H. L. (2015). Population structure of mitochondrial genomes in Saccharomyces cerevisiae. BMC genomics, 16(1), 451.

14. Questions?