Your SlideShare is downloading. ×
EVE161 Lecture 2
Upcoming SlideShare
Loading in...5
×

Thanks for flagging this SlideShare!

Oops! An error has occurred.

×

Saving this for later?

Get the SlideShare app to save on your phone or tablet. Read anywhere, anytime - even offline.

Text the download link to your phone

Standard text messaging rates apply

EVE161 Lecture 2

1,319
views

Published on

EVE 161

EVE 161

Published in: Education, Technology

0 Comments
0 Likes
Statistics
Notes
  • Be the first to comment

  • Be the first to like this

No Downloads
Views
Total Views
1,319
On Slideshare
0
From Embeds
0
Number of Embeds
4
Actions
Shares
0
Downloads
43
Comments
0
Likes
0
Embeds 0
No embeds

Report content
Flagged as inappropriate Flag as inappropriate
Flag as inappropriate

Select your reason for flagging this presentation as inappropriate.

Cancel
No notes for slide

Transcript

  • 1. EVE 161:
 Microbial Phylogenomics ! Lecture #2: Evolution of Sequencing ! UC Davis, Winter 2014 Instructor: Jonathan Eisen Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !1
  • 2. Where we are going and where we have been • Previous lecture: ! 1. Introduction • Current Lecture: ! 2. Sequencing Evolution • Next Lecture: ! 3. Era I: The Tree of Life Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !2
  • 3. Main Paper Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !3
  • 4. Annu. Rev. Genom. Human Genet. 2008.9: by Universidad Nacional Autonom ther k links to ontent online, his volume articles ve search Next-Generation DNA Sequencing Methods Elaine R. Mardis Departments of Genetics and Molecular Microbiology and Genome Sequencing Center, Washington University School of Medicine, St. Louis MO 63108; email: emardis@wustl.edu Annu. Rev. Genomics Hum. Genet. 2008. 9:387–402 First published online as a Review in Advance on June 24, 2008 The for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Annual Review of Genomics and Human Genetics Slides Slides for UC at EVE161 Course Taught by Jonathan Eisen is online Davisgenom.annualreviews.orgWinter 2014 !4
  • 5. Many other papers of interest • http://www.microbialinformaticsj.com/ content/2/1/3/ • http://www.hindawi.com/journals/bmri/ 2012/251364/abs/ • http:// m.cancerpreventionresearch.aacrjourna ls.org/content/5/7/887.full Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !5
  • 6. Sequencing Technology Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !6
  • 7. Key Issues • • • • • • Cost / bp Read length Paired end Ease of feeding Error profiles Barcoding potential Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !7
  • 8. Timeline Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !8
  • 9. Timeline 1977 2010 Sanger sequencing method by F. Sanger (PNAS ,1977, 74: 560-564) 1983 1953 2000 1990 1980 Approaching to NGS PCR by K. Mullis (Cold Spring Harb Symp Quant Biol. 1986;51 Pt 1:263-73) Discovery of DNA structure Human Genome Project (Cold Spring Harb. Symp. Quant. Biol. 1953;18:123-31) (Nature , 2001, 409: 860–92; Science, 2001, 291: 1304–1351) 1993 Development of pyrosequencing (Anal. Biochem., 1993, 208: 171-175; Science ,1998, 281: 363-365) Single molecule emulsion PCR 1998 Founded Solexa 1998 Founded 454 Life Science 2000 454 GS20 sequencer (First NGS sequencer) 2005 Solexa Genome Analyzer (First short-read NGS sequencer) Illumina acquires Solexa (Illumina enters the NGS business) 2006 2006 ABI SOLiD (Short-read sequencer based upon ligation) Roche acquires 454 Life Sciences From Slideshare presentation of Cosentino Cristian http://www.slideshare.net/cosentia/highthroughput-equencing (Roche enters the NGS business) 2007 2007 GS FLX sequencer (NGS with 400-500 bp read lenght) NGS Human Genome sequencing (First Human Genome sequencing based upon NGS technology) 2008 2008 Hi-Seq2000 (200Gbp per Flow Cell) Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 2010 !9
  • 10. Timeline 1977 2010 Sanger sequencing method by F. Sanger (PNAS ,1977, 74: 560-564) 1983 1953 2000 1990 1980 Approaching to NGS PCR by K. Mullis (Cold Spring Harb Symp Quant Biol. 1986;51 Pt 1:263-73) Discovery of DNA structure (Cold Spring Harb. Symp. Quant. Biol. 1953;18:123-31) Human Genome Project (Nature , 2001, 409: 860–92; Science, 2001, 291: 1304–1351) 1993 Development of pyrosequencing (Anal. Biochem., 1993, 208: 171-175; Science ,1998, 281: 363-365) Single molecule emulsion PCR 1998 Founded Solexa 1998 Founded 454 Life Science 2000 454 GS20 sequencer (First NGS sequencer) 2005 Solexa Genome Analyzer (First short-read NGS sequencer) Illumina acquires Solexa (Illumina enters the NGS business) 2006 2006 ABI SOLiD (Short-read sequencer based upon ligation) Roche acquires 454 Life Sciences (Roche enters the NGS business) 2007 2007 GS FLX sequencer (NGS with 400-500 bp read lenght) NGS Human Genome sequencing (First Human Genome sequencing based upon NGS technology) From Slideshare presentation of Cosentino Cristian http://www.slideshare.net/cosentia/highthroughput-equencing 2008 2008 Hi-Seq2000 (200Gbp per Flow Cell) 2010 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Miseq Roche Jr Ion Torrent PacBio Oxford !10
  • 11. Generation I: Manual Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !11
  • 12. Maxam-Gilbert Sequencing Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !12
  • 13. Maxam-Gilbert Sequencing http://www.pnas.org/content/74/2/560.full.pdf Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !13
  • 14. Maxam-Gilbert Sequencing Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !14
  • 15. Maxam-Gilbert Sequencing Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !15
  • 16. Sanger Sequencing http://www.ncbi.nlm.nih.gov/pmc/articles/PMC431765/ Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !16
  • 17. PhiX174 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !17
  • 18. Sanger Sequencing Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !18
  • 19. Sanger Sequencing Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !19
  • 20. Nobel Prize 1980 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !20
  • 21. Generation II: Automation Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !21
  • 22. Sanger method with labeled dNTPs Automation I The Sanger mehtods is based on the idea that inhibitors can terminate elongation of DNA at specific points Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !22
  • 23. Sanger method with labeled dNTPs Automation I The Sanger mehtods is based on the idea that inhibitors can terminate elongation of DNA at specific points Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !23
  • 24. Automation 2 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !24
  • 25. Automated Sanger Highlights • • • • • • • 1991: ESTs by Venter 1995: Haemophilus influenzae genome 1996: Yeast, archaea genomes 1999: Drosophila genome 2000: Arabidopsis genome 2000: Human genome 2004: Shotgun metagenomics Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !25
  • 26. Generation III: Clusters not clones Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !26
  • 27. Generation III: Clusters not clones Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !27
  • 28. Next Gen Sequencingplatforms Outline Next-generation sequencing From Slideshare presentation of Cosentino Cristian http://www.slideshare.net/cosentia/ high-throughput-equencing Isolation and purification of target DNA Sample preparation Amplification Emulsion PCR Sequencing by synthesis with  3’-blocked reversible terminators Pyrosequencing Imaging Cluster generation on solid-phase Sequencing Library validation Sequencing by ligation Four colour imaging Sequencing by synthesis with  3’-unblocked reversible terminators Single colour imaging Data analysis Illumina GAII Roche 454 ABi SOLiD Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Helicos HeliScope !28
  • 29. 454 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !29
  • 30. 454 Roche 454 Sanger method ABi SOLiD Illumina GAII Pyrosequencing From Slideshare presentation of Cosentino Cristian http://www.slideshare.net/ cosentia/high-throughputequencing HeliScope Nanopore Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !30
  • 31. Roche 454 Roche 454 Sanger method ABi SOLiD Illumina GAII Pyrosequencing From Slideshare presentation of Cosentino Cristian http://www.slideshare.net/ cosentia/high-throughputequencing HeliScope Nanopore Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !31
  • 32. 454 -> Roche • 1st “Next-generation” sequencing system to become commercially available, in 2004 • Uses pyrosequencing • Polymerase incorporates nucleotide • Pyrophosphate released • Eventually light from luciferase released • Three main steps in 454 method • Library prep • Emulsion PCR • Sequencing Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !32
  • 33. 454 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !33
  • 34. 454 Step1: Library Prep a DNA library preparation 4.5 hours Ligation B •Genome fragmented by nebulization A •No cloning; no colony picking Selection (isolate AB fragments only) A B A B •sstDNA library created with adaptors •A/B fragments selected using avidin-biotin purification gDNA gDNA b fragmented by nebulization or Emulsion PCR sonication 8 hours sstDNA library Fragments are endrepaired and ligated to adaptors containing universal priming sites Fragments are denatured and AB ssDNA are selected by avidin/biotin purification (ssDNA library) From Mardis 2008. Annual Rev. Genetics 9: 387. Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !34
  • 35. only) A B with adaptors •A/B fragments selected using avidin-biotin purification 454 Step 2: Emulsion PCR A B gDNA sstDNA library b Emulsion PCR 8 hours Anneal sstDNA to an excess of DNA capture beads sstDNA library c Emulsify beads and PCR reagents in water-in-oil microreactors Clonal amplification occurs inside microreactors Break microreactors and enrich for DNA-positive beads Bead-amplified sstDNA library From Mardis 2008. Annual Rev. Genetics 9: 387. Sequencing 7.5 hours Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !35
  • 36. Anneal sstDNA to an excess of DNA capture beads Emulsify beads and PCR reagents in water-in-oil microreactors Clonal amplification occurs inside microreactors Break microreactors and enrich for DNA-positive beads 454 Step 3: Sequencing sstDNA library Bead-amplified sstDNA library c Sequencing 7.5 hours •Well diameter: average of 44 µm •400,000 reads obtained in parallel •A single cloned amplified sstDNA bead is deposited per well Amplified sstDNA library beads 390 Mardis Quality filtered bases From Mardis 2008. Annual Rev. Genetics 9: 387. Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !36
  • 37. 454 Step 3: Sequencing Pyrosequencing Roche 454 Sanger method Annu. Rev. Genomics Hum. Genet., 2008, 9: 387-402 Nature Reviews genetics, 2010, 11: 31-46 - 44 µm ABi SOLiD Illumina GAII HeliScope Nanopore From Slideshare presentation of Cosentino Cristian http://www.slideshare.net/ cosentia/high-throughputequencing Pyrosequecning Reads are recorded as flowgrams Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !37
  • 38. 454 Key Issues • Number of repeated nucleotides estimated by amount of light ... many errors • Reasonable number of failures in EMPCR and other steps • Systems • • • • • GS20 FLX FLX Titanium FLX Titanium XL Junior Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !38
  • 39. Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !39
  • 40. X DISCONTINUED Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !40
  • 41. Solexa Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !41
  • 42. Sequecning by synthesis with reversible terminator Solexa From Slideshare presentation of Cosentino Cristian http:// www.slideshare.net/ cosentia/highthroughput-equencing Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !42
  • 43. Sequecning by synthesis with reversible terminator Illumina From Slideshare presentation of Cosentino Cristian http:// www.slideshare.net/ cosentia/highthroughput-equencing Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !43
  • 44. Illumina • • • • Sequencing by synthesis 1st system released in 2006 by Solexa Acquired by Illumina in 2007 Systems • • • • • GA GAII HiSeqs HiScan MiSeq Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !44
  • 45. Instrumentation GAII Accessories ction le tion Bioanalyzer 2100 Cluster station ers ation ng by esis sis ne h hput Paired-end module Genome Analyzer IIx Linux server Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !45
  • 46. Illumina Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !46
  • 47. Illumina a b Illumina’s library-preparation work flow DNA fragments OH OH Blunting by fill-in and exonuclease P P P P A A Phosphorylation P7 P5 Grafted flow ce P Addition of A-overhang Ligation to adapters P First denatura c 3' 5' Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 O Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Fluor !47
  • 48. mina GAII oduction Flow cell Illumina Flow Cell ample paration usters lification encing by nthesis nalysis peline High oughput From Slideshare presentation of Cosentino Cristian http:// www.slideshare.net/ cosentia/highthroughput-equencing !48 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014
  • 49. 13 May 2013 16:4 Illumina b library-preparation work flow DNA fragments OH OH Blunting by fill-in and exonuclease P A A Phosphorylation First cycle annealing First cycle extension Second cycle denaturation Fluor HN O N O PPP 3' n = 35 total Cleavage site Block Second cycle denaturation Incorporate Detect Deblock Cleave fluor O HN 5' DNA O N O O 3' 5' Denaturation P O Excitation Initial extension P 5' Emission Template hybridization Addition of A-overhang Ligation to adapters 3' P7 P5 Grafted flow cell First denaturation P Second cycle annealing Second cycle extension Cluster amplification P5 linearization Block with ddNTPs x OH free 3' end Denaturation and sequencing primer hybridization Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 brary-construction process. (b) Illumina cluster generation by bridge amplification. (c) Sequencing by synthesis with !49
  • 50. continues for a specific number of cycles, as determined by user-defined instrument settings, which permits discrete read lengths of 25–35 read and a quality checking pipeline evaluates the Illumina data from each run, removing poor-quality sequences. Illumina Step 1: Attach DNA a Adapter DNA fragment DNA Dense lawn of primers Adapter Adapters Prepare genomic DNA sample Attach DNA to surface Randomly fragment genomic DNA and ligate adapters to both ends of the fragments. Bind single-stranded fragments randomly to the inside surface of the flow cell channels. From Mardis 2008. Annual Rev. Genetics 9: 387. The Illumina sequencing-by-synthesis approach. Cluster strands created by bridge amplification are primed and all four fluorescently labeled, 3′-OH blocked nucleotides are added to the flow cell with DNA polymerase. The Nucleotides cluster strands are extended by one nucleotide. Following the incorporation step, the unused nucleotides and DNA polymerase molecules are washed away, a scan buffer is added to the flow cell, and the optics system scans each lane of the flow cell by imaging units called tiles. Once imaging is completed, chemicals that effect cleavage of the fluorescent labels and the 3′-OH blocking groups are added to the flow cell, which prepares the cluster strands for another round of fluorescent nucleotide incorporation Attached Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !50 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014
  • 51. Prepare genomic DNA sample Attach DNA to surface Randomly fragment genomic DNA and ligate adapters to both ends of the fragments. Bind single-stranded fragments randomly to the inside surface of the flow cell channels. Illumina Step 2: Bridge PCR Nucleotides Attached Bridge amplification Add unlabeled nucleotides and enzyme to initiate solidphase bridge amplification. Denature the double stranded molecules From Mardis 2008. Annual Rev. Genetics 9: 387. The Illumina sequencing-by-synthesis approach. Cluster strands created by bridge amplification are primed and Figure 2 ′ all four Illumina sequencing-by-synthesis approach. Cluster strands created added toamplification are with DNA all four fluorescently The fluorescently labeled, 3 -OH blocked nucleotides are by bridge the flow cell primed and polymerase. The cluster strands are extended by are added to the flowFollowing the incorporationcluster strands are extended by one and labeled, 3′ -OH blocked nucleotides one nucleotide. cell with DNA polymerase. The step, the unused nucleotides nucleotide. Following the incorporation step, the unused scan buffer DNA polymerase flow cell, and the optics system DNA polymerase molecules are washed away, a nucleotides andis added to the molecules are washed away, a scan buffer is added to the flow cell, and the cell system scans units called flow Once imaging is completed, chemicals that effect scans each lane of the flow opticsby imaging each lane of thetiles.cell by imaging units called tiles. Once imaging is completed, chemicals that effect cleavage of the fluorescent labels and the 3′ -OH blocking groups are added to the flow cell, which prepares the ′ cleavagestrands for another round of fluorescent nucleotideblocking groups are added to the flow cell, which prepares the cluster of the fluorescent labels and the 3 -OH incorporation. cluster strands for another round of fluorescent nucleotide incorporation Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !51 392 Mardis Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014
  • 52. Clusters Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !52
  • 53. sity of California - Davis on 01/09/14. For personal use only. P Ligation to adapters Sequencing by Synthesis First denaturation 3' c 5' HN O N O PPP Emission A C T G Excitation C A Second cycle denaturation Sec an Cluster amplification line Fluor O 3' Fi an Cleavage site Block Incorporate Detect Deblock Cleave fluor T O G A HN 5' DNA O N O O T G C 3' 5' x OH free 3' end Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Figure 3 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !53
  • 54. lengths for SOLiD are user defined between 25–35 bp, and each sequencing run yields between 2–4 Gb of DNA sequence data. Once from a capillary sequencer and (b) each nextgeneration read type has a unique error model different from that already established for Illumina Step 3: Sequencing b First chemistry cycle: determine first base To initiate the first sequencing cycle, add all four labeled reversible terminators, primers, and DNA polymerase enzyme to the flow cell. Image of first chemistry cycle Before initiating the next chemistry cycle After laser excitation, capture the image of emitted fluorescence from each cluster on the flow cell. Record the identity of the first base for each cluster. The blocked 3' terminus and the fluorophore from each incorporated base are removed. Laser From Mardis 2008. Annual Rev. Genetics 9: 387. GCTGA... Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for read over multiple chemistry by Jonathan Eisen Winter 2014 Sequence UC Davis EVE161 Course Taughtcycles !54
  • 55. umina GAII Illumina SBS technologySBS troduction Sample reparation Clusters mplification quencing by synthesis Analysis pipeline High hroughput From Slideshare presentation of Cosentino Cristian http:// www.slideshare.net/ cosentia/highthroughput-equencing Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !55
  • 56. Illumina Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !56
  • 57. ABI Solid Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !57
  • 58. ABI Solid Sequecning by ligation ABi SOLiD Sanger method Roche 454 Illumina GAII HeliScope Nanopore From Slideshare presentation of Cosentino Cristian http:// www.slideshare.net/ cosentia/highthroughput-equencing Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !58
  • 59. G09-20 ARI 25 July 2008 14:57 ABI Solid Details The ligase-mediated sequencing approach of the Applied Biosystems SOLiD sequencer. In a SOLiD™ substrate Di base probes Template manner similar to Roche/454 emulsion PCR 2nd base 3 'TTnnnzzz5 ' 1 μm amplification, DNA fragments for SOLiD A C G T bead 5' Template sequence 3' P1 adapter A 3 'TGnnnzzz5 ' sequencing are amplified on the surfaces of 1C G μm magnetic beads to provide sufficient signal 3 'TCnnnzzz5 ' T during the sequencing reactions, and are then 3 'TAnnnzzz5 ' deposited onto a flow cell slide. LigaseGlass slide Cleavage site mediated sequencing begins by annealing a 5. Repeat steps 1–4 to extend sequence 1. Prime and ligate P OH primer to the shared adapter sequences on Ligation cycle 1 2 3 4 5 6 7 ... (n cycles) + Ligase Primer round 1 each amplified fragment, and then DNA ligase GC TT CT GT TT CA AT TA CG GT GA CA AA AA 3' is provided along with specific fluorescentUniversal seq primer (n) AT 3' 1 μm 3' labeled 8mers, whose 4th and 5th bases are bead P1 adapter Template sequence TA 6. Primer reset encoded by the attached fluorescent group. 2. Image Excite Fluorescence Each ligation step is followed by fluorescence Universal seq primer (n–1) detection, after which a regeneration step 1. Melt off extended 3' 2. Primer reset sequence removes bases from the ligated 8mer 3' 1 μm TA 3' bead (including the fluorescent group) and 3. Cap unextended strands concomitantly prepares the extended primer 7. Repeat steps 1–5 with new primer Phosphatase for another round of ligation. (b) Principles of PO two-base encoding. Because each fluorescent Primer round 2 1 base shift 3' –1 group on a ligated 8mer identifies a two-base Universal seq primer (n–1) A A C A C G T C A A T A C C 4. Cleave off fluor 3' combination, the resulting sequence reads can 1 μm 3' Cleavage agent bead T GT G C A G T T A T GG be screened for base-calling errors versus true HO AT polymorphisms versus single base deletions P 3' TA by aligning the individual reads to a known Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !59 8. Repeat Reset with , n–2, n–3, n–4 primers From Mardis 2008. Annual Rev. Genetics 9: 387. high-quality reference sequence. a 1st base 4 Read position Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
  • 60. Comparison in 2008 Roche (454) Illumina SOLiD Chemistry Pyrosequencing Polymerase-based Ligation-based Amplification Emulsion PCR Bridge Amp Emulsion PCR Paired ends/sep Yes/3kb Yes/200 bp Yes/3 kb Mb/run 100 Mb 1300 Mb 3000 Mb Time/run 7h 4 days 5 days Read length 250 bp 32-40 bp 35 bp Cost per run (total) Cost per Mb $8439 $8950 $17447 $84.39 $5.97 $5.81 From “Introduction to Next Generation Sequencing” by Stefan Bekiranov prometheus.cshl.org/twiki/pub/Main/CdAtA08/ Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 CSHL_nextgen.ppt Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !60
  • 61. Comparison in 2012 Roche (454) Illumina SOLiD Chemistry Pyrosequencing Polymerase-based Ligation-based Amplification Emulsion PCR Bridge Amp Emulsion PCR Paired ends/sep Yes/3kb Yes/200 bp Yes/3 kb Mb/run 100 Mb 1300 Mb 3000 Mb Time/run 7h 4 days 5 days Read length 250 bp 32-40 bp 35 bp Cost per run (total) Cost per Mb $8439 $8950 $17447 $84.39 $5.97 $5.81 From “Introduction to Next Generation Sequencing” by Stefan Bekiranov prometheus.cshl.org/twiki/pub/Main/CdAtA08/ Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 CSHL_nextgen.ppt Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !61
  • 62. Bells and Whistles • • • • Multiplexing Paired end Mate pair ???? Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !62
  • 63. Multiplexing Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !63
  • 64. Small amounts of DNA Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !64
  • 65. Illumina GAII Capture Methods High throughput sample preparation Introduction Sample preparation RainDance Microdroplet PCR Roche Nimblegen Salid-phase capture with customdesigned oligonucleotide microarray Clusters amplification Sequencing by synthesis Analysis pipeline High throughput Reported 84% of capture efficiency From Slideshare presentation of Cosentino Cristian http://www.slideshare.net/cosentia/high-throughput-equencing Nature Methods, 2010, 7: 111-118 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Reported 65-90% of capture efficiency Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !65
  • 66. Illumina GAII Introduction Sample preparation Capture preparation Methods High throughput sample Agilent SureSelect Solution-phase capture with streptavidin-coated magnetic beads Clusters amplification Sequencing by synthesis Analysis pipeline High throughput Reported 60-80% of capture efficiency From Slideshare presentation of Cosentino Cristian http://www.slideshare.net/cosentia/ high-throughput-equencing Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !66
  • 67. Moleculo DNA Large fragments Isolate and amplify Sublibrary w/ unique barcodes CACC GGAA TCTC ACGT AAGG GATC AAAA Sequence w/ Illumina Assemble seqs w/ same codes Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !67
  • 68. Generation 3.5 • Even faster Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !68
  • 69. Ion Torrent PGM Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !69
  • 70. Applied Biosystems Ion Torrent PGM Workflow similar to that for Roche/454 systems. ! Not surprising, since invented by people from 454. Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !70
  • 71. Ion Torrent PGM Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !71
  • 72. Generation IV?? • Single molecule sequencing Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !72
  • 73. Pacific Biosciences Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !73
  • 74. Pacific Biosciences Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !74
  • 75. Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !75
  • 76. Analytical Chemistry Pacific Biosciences REVIEW Figure 2. Schematic of PacBio’s real-time single molecule sequencing. (A) The side view of a single ZMW nanostructure containing a single DNA polymerase (Φ29) bound to the bottom glass surface. The ZMW and the confocal imaging system allow fluorescence detection only at the bottom surface of each ZMW. (B) Representation of fluorescently labeled nucleotide substrate incorporation on to a sequencing template. The corresponding a Figure 2. Schematic of PacBio’s real-time single molecule sequencing. (A) The side view of temporal fluorescence detection with respect to each of the five incorporation steps is shown below. Reprinted with permission from ref 39. Copyright single ZMW nanostructure containing a single DNA polymerase (Φ29) bound to the bottom 2009 American Association for the Advancement of Science. glass surface. The ZMW and the confocal imaging system allow fluorescence detection only Φ29 polymerase. Each amplified product of a circularized Each hybridization and ligation cycle is followed by fluorescent at theisbottom surface of each ZMW. (B) Representation of fluorescently subsequently regeneration labeled nucleotide fragment called a DNA nanoball (DNB). DNBs are selectively imaging of the DNB spotted chip and attached to a hexamethyldisilizane (HMDS) coated silicon chip template. The corresponding temporal is repeated of the DNBs with a formamide solution. This cycle substrate incorporation on to a sequencing that is photolithographically patterned with aminosilane active until the library of probes and fluorescence detection with respect to each of the fiveentire combinatorialthesteps is shown anchors is below. sites. Figure 3A illustrates the DNB array design. examined. incorporation use of unchained reads and This formula of The use of the DNBs coupled with the highly patterned array regeneration of the sequencing fragment reduces reagent conoffers several advantages. The production of DNBs et al. Analytical Chemistry 83: 4327. 2011. errors that can sumption and eliminates potential accumulation From Niedringhaus increases signal intensity by simply increasing the number of hybridization arise in other sequencing technologies that require close to sites available for probing. Also, the for UC Davis EVE161 Course Taught by Jonathan Eisen Winter reaction.19,52,53 size of the DNB is on the completion of each sequencing 2014 Slides !76 Complete Genomics Winter 2014 same length scale as the active site orfor UC Davis EVE161 Course Taught by Jonathan Eisen showcased their DNB array and cPAL “sticky” spot patterned on Slides
  • 77. Complete Genomics Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !77
  • 78. Complete Genomics REVIEW Figure 3. Schematic of Complete Genomics’ DNB array generation and cPAL technology. (A) Design of sequencing fragments, subsequent DNB synthesis, and dimensions of the patterned nanoarray used to localize DNBs illustrate the DNB array formation. (B) Illustration of sequencing with a set of common probes corresponding to 5 bases from the distinct adapter site. Both standard and extended anchor schemes are shown. omplete Genomics’ DNB array generation and cPAL technology. (A) Design of sequencing fragments, subsequent DNB f the patterned nanoarray used to localize DNBs illustrate the DNB array formation. (B) Illustration of sequencing with a set onding to 5 bases from the distinct adapter site. Both standard and extended anchor schemes are shown. Reprinted with pyright XXXX American Association for the Advancement of Science. From Niedringhaus et al. Analytical Chemistry 83: 4327. 2011. gure 3. Schematic of Complete Genomics’ DNB array generation and cPAL technology. (A) Design of sequencing fragments, subsequent8 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !7 DN ased the number of false positive gene sitosterolemia phenotype localize DNBs after comparison of nthesis, and dimensions of the patterned nanoarray used towere determinedTaught by the DNB array formation. (B) Illustration of sequencing with a Slides for UC Davis EVE161 Course illustrate Jonathan Eisen Winter 2014 ely reduced the number gene candidates the patient’s genome to a collection of reference genomes.
  • 79. Oxford Nanopore Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !79
  • 80. Oxford Nanopore From Oxford Nanopores Web Site This diagram shows a protein nanopore set in an electrically resistant membrane bilayer. An ionic current is passed through the nanopore by setting a voltage across this membrane.
 
 If an analyte passes through the pore or near its aperture, this event creates a characteristic disruption in current. By measuring that current it is possible to identify the molecule in question. For example, this system can be used to distinguish the four standard DNA bases and G, A, T and C, and also modified bases. It can be used to identify target proteins, small molecules, or to gain rich molecular information for example to distinguish the enantiomers of ibuprofen or molecular binding dynamics. Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !80 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014
  • 81. ytical Chemistry • Oxford Nanopore REV Figure6. BiologicalnanoporeschemeemployedbyOxfordNanopore. (A)SchematicofRHLproteinnanoporemutantdepictingthepositionsofthe cyclodextrin (at residue 135) and glutamines (at residue 139). (B) A detailed view of the β barrel of the mutant nanopore shows the locations of the arginines (at residue 113) and the cysteines. (C) Exonuclease sequencing: A processive enzyme is attached to the top of the nanopore to cleave single nucleotides from the target DNA strand and pass them through the nanopore. (D) A residual current-vs-time signal trace from an RHL protein nanopore that shows a clear discrimination between single bases (dGMP, dTMP, dAMP, and dCMP). (E) Strand sequencing: ssDNA is threaded through a protein nanopore and individual bases are identified, as the strand remains intact. Panels A, B, and D reprinted with permission from ref 91. Copyright 2009 Nature Publishing Group. Panels C and E reprinted with permission from Oxford Nanopore Technologies (Zoe McDougall).# Figure6. BiologicalnanoporeschemeemployedbyOxfordNanopore.(A)SchematicofRHLproteinnanoporemutantdepictingthepositionsofthe cyclodextrin (at residue 135) and glutamines (at residue 139). (B) A detailed view of the β barrel of the mutant nanopore shows the locations of the arginines (at residue 113) and the cysteines. (C) Exonuclease sequencing: A processive enzyme is e 6. attached to the top of the nanopore to cleave single nucleotidesOxfordtarget DNA strand and pass them through the nanopore. (D) A residual current-vs-time signal trace fromtheRHL protein Biological nanopore scheme employed by from the Nanopore. (A) Schematic of RHL protein nanopore mutant depicting an positions nanopore that shows a clear discrimination between single bases (dGMP, dTMP, dAMP, and dCMP). (E) Strand sequencing: ssDNA is threaded through a protein nanopore and individual bases ar dextrin (at residue 135) and glutaminesand D reprinted with permission fromdetailed view of Natureβ barrel Group. Panels C and Enanopore shows the locati (at residue 139). (B) A ref 91. Copyright 2009 the Publishing of the mutant reprinted with permission from Oxford identified, as the strand remains intact. Panels A, B, ginines (at residue 113) and the cysteines. (C) Exonuclease sequencing: A processive enzyme is attached to the top of the nanopore to cleave Nanopore Technologies (Zoe McDougall). From Niedringhaus et al. Analytical Chemistry 83: 4327. RHL protein nan otides from the target DNA strand and pass them through the nanopore. (D) A residual current-vs-time signal trace from an2011. Slides single Davis EVE161 Course Taught and dCMP). (E) Winter 2014 1 hows a clear discrimination between for UC bases (dGMP, dTMP, dAMP, by Jonathan EisenStrand sequencing: ssDNA is threaded!8thro n nanopore and individual bases are identified, as the strand Course Taught by Jonathanand D reprinted with permission from ref 91. Cop Slides for UC Davis EVE161 remains intact. Panels A, B, Eisen Winter 2014
  • 82. Analytical Chemistry Nanopores REVIEW igure 5. Nanopore DNA sequencing using electronicmeasurements and optical readout as detection methods. (A) In electronic nanoporenanopo Nanopore DNA sequencing using electronic measurements and optical readout as detection methods.(A)In electronic schemes ignal is obtained through ionic current,73 tunneling current,78 and voltage difference79 measurements. Each method must produce a characteristic signa schemes, signal is obtained through ionic current,73 tunneling current, and voltage difference measurements. Each method o differentiate the four DNA bases. Reprinted with permission from ref 83. Copyright 2008 Annual Reviews. (B) In the optical readout nanopore design ach nucleotide is converted to a preset oligonucleotide sequence the four DNAwith labeled markersoptical detected during translocation of the DNA must produce a characteristic signal to differentiate and hybridized bases. (B) In the that are readout nanopore design, each ragment through is converted to a preset oligonucleotide sequence and hybridized with labeled markers that are detected during nucleotide the nanopore. Reprinted from ref 82. Copyright 2010 American Chemical Society. translocation of the DNA fragment through the nanopore. would result, in theory, inFrom Niedringhaus etthrough detectably altered current flow al. Analyticalbilayer, using ionic current blockage method. The author lipid Chemistry 83: 4327. 2011. he pore. Theoretically, nanopores could also be designed to Taught by Jonathansingle nucleotides could be discriminated as lon predicted that Eisen Winter 2014 Slides for UC Davis EVE161 Course measure tunneling current across the pore as bases, each with a as: (1) each nucleotide produces a unique signal signature; (2 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 istinct tunneling potential, could be read. The nanopore apthe nanopore possesses proper aperture geometry to accommo
  • 83. Oxford Nanopore From Oxford Nanopores Web Site Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !83
  • 84. Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !84