Sequencing technologies the next generation

Transcription

1 Sequencing technologies the next generation Ann-Kathrin Wagner 1 July 2015

2 Overview 1. Introduction and repetition 2. Next generation sequencing based on fluorescence dyes 3. Comparison of methods used by different instruments # 2

3 Introduction US$ 1000 Human Genome Project by 2014 Personalized genomics for medical purposes # 3

4 Repetition: DNA Sequence: order of nucleotides within DNA # 4

5 Repetition: DNA Sequence: order of nucleotides within DNA # 5

6 Repetition: DNA Sequence: order of nucleotides within DNA px-Aminoacids_table.png # 6

7 Addition to Sanger sequencing è use of fluorophores instead of radiophosphates # 7

8 NGS fluorescence based 1. Fluorophore attachment 2. Template prep 3. Sequencing 4. Data analysis # 8

9 Fluorescence signal base linked fluorescent nucleotides 3 -O-azidomethyl * 3 -O-allyl * ** * * Terminating functional group ** inhibitory function Metzker, M.; Nature Reviews (2010) # 9

10 Fluorescence signal - phospholinked è no terminating group Metzker, M.; Nature Reviews (2010) # 10

11 Template prep clonally amplified Metzker, M.; Nature Reviews (2010) # 11

12 Template prep single molecule C: hybridization to primer D: attachment to solid support E: polymerases attachment Metzker, M.; Nature Reviews (2010) # 12

13 Sequencing cyclic reversible termination 4-color 1-color at cyclic a time reversible termination Metzker, M.; Nature Reviews (2010) # 13

14 Sequencing sequencing by ligation SoLiD: è decode DNA sequence Metzker, M.; Nature Reviews (2010) # 14

15 Sequencing - SMRT Metzker, M.; Nature Reviews (2010) # 15

16 Zero Mode Waveguides (ZMW) è subwavelength holes in metal film è studies at µm concentration & µs temporal resolution Levene, M.; Science (2003) # 16

17 ZMW è circular polarized light S(z) = I(z)p(z) V eff = πd 2 4 p(z) p(z)+ C ( S(z)dz) 2 S 2 (z)dz eff. observation profile eff. observation volume è limits V eff to first nm Levene, M.; Science (2003) # 17

18 Signal detection & data analysis 532nm 643nm Eid, J.; Science (2009) # 18

19 Signal detection & data analysis ssdna 2-base artificial sequence pattern (CG) è sensitive to ph and concentration change Eid, J.; Science (2009) # 19

20 Signal detection & data analysis Long-read DNA sequencing for 2 bases è suggesting two polymerization rates Eid, J.; Science (2009) # 20

21 Four-color sequencing Signal detection & data analysis interpulse duration: Eid, J.; Science (2009) # 21

22 Summary of commercially available techniques Metzker, M.; Nature Reviews (2010) # 22

23 Summary of commercially available techniques Metzker, M.; Nature Reviews (2010) # 23

24 Summary of commercially available techniques ion torrent en.wikipedia.org/wiki/ion_semiconductor_sequencing Metzker, M.; Nature Reviews (2010) # 24

25 Comparison è PacBio max. read length Illumina max. throughput Ion torrent fastest sequencing van Dijk, E.; Elsevier (2014) # 25

26 US$ 1000 HGP è Illumina`s HiSeq X Ten system National Human Genome Research Institute # 26

27 Summary 3 major improvements of NGS methods: 1. Prep in cell free environment è no cloning 2. Lots of parallel measurements 3. Direct detection è no electrophoresis 2 types of fluorophore attachment ZMW è Reduce of cost and time & increase of throughput # 27

28 Literature Metzker, M.; Sequencing technologies the next generation; Nature Reviews (2010) Levene, M.; Zero-mode waveguides for single-molecule analysis at high concentrations; Science (2003) Eid, J.; Real-time DNA sequencing from single polymerase molecules; Science (2009) van Dijk, E.; Ten years of next-generation sequencing technology; Elsevier (2014) Pushkarev, D.; Single-molecule sequencing of an individual human genome; Nature Biotechnology (2009) Metzker, M.; Emerging technologies in DNA sequencing; Cold Spring Harbor Laboratory Press (2005) wikipedia/dna-sequencing datasheet-hiseq-x-ten from Illumina PacBio_RS_II_Brochure from Pscific Biotechologies Seo, T.; Four-color DNA sequencing by synthesis on a chip using photocleavable fluorescent nucleotides; PNAS (2005) # 28

29 Thank you for your attention! # 29