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Evolution of DNA Sequencing by Jonathan Eisen

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Evolution of DNA Sequencing by Jonathan Eisen (for UC Davis Workshop in Applied Phylogenetics at Bodega Bay Marine Lab)

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Evolution of DNA Sequencing by Jonathan Eisen

  1. 1. Evolution of Sequencing Workshop in Applied Phylogenetics March 8, 2015 Bodega Bay Marine Lab Jonathan A. Eisen UC Davis Genome Center 1
  2. 2. Notes • Some slides have been kept in here for historical information - do not necessarily use them all in presentations • For most of the methods presented, the source material can be genomic DNA, PCR amplified DNA, RNA converted into cDNA, etc. • I have attempted to include the source of all materials - apologies when this is not done or done incorrectly 15
  3. 3. Review Papers 16 Mardis ER. Next-generation sequencing platforms. Annu Rev Anal Chem 2013;6:287-303. 
 doi: 10.1146/annurev-anchem-062012-092628. 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 Annual Review of Genomics and Human Genetics is online at genom.annualreviews.org This article’s doi: 10.1146/annurev.genom.9.081307.164359 Copyright c⃝ 2008 by Annual Reviews. All rights reserved 1527-8204/08/0922-0387$20.00 Key Words massively parallel sequencing, sequencing-by-synthesis, resequencing Abstract Recent scientific discoveries that resulted from the application of next- generation DNA sequencing technologies highlight the striking impact of these massively parallel platforms on genetics. These new meth- ods have expanded previously focused readouts from a variety of DNA preparation protocols to a genome-wide scale and have fine-tuned their resolution to single base precision. The sequencing of RNA also has transitioned and now includes full-length cDNA analyses, serial analysis of gene expression (SAGE)-based methods, and noncoding RNA dis- Click here for quick links to Annual Reviews content online, including: • Other articles in this volume • Top cited articles • Top downloaded articles • Our comprehensive search FurtherANNUAL REVIEWS Annu.Rev.Genom.HumanGenet.2008.9:387-402.Downloadedfromarjournals.annualreviews.org byUniversidadNacionalAutonomadeMexicoon11/17/09.Forpersonaluseonly. Annu. Rev. Genomics Hum. Genet. 2008. 9:387–402 First published online as a Review in Advance on June 24, 2008 The Annual Review of Genomics and Human Geneti is online at genom.annualreviews.org This article’s doi: 10.1146/annurev.genom.9.081307.164359 Copyright c⃝ 2008 by Annual Reviews. All rights reserved 1527-8204/08/0922-0387$20.00 Annu.Rev.Genom.HumanGenet.2008.9:3 byUniversidadNacionalAutonoma• Open Access Review Papers • http://www.microbialinformaticsj.com/content/2/1/3/ • http://www.hindawi.com/journals/bmri/2012/251364/abs/ • http://m.cancerpreventionresearch.aacrjournals.org/content/ 5/7/887.full
  4. 4. 17 Approaching to NGS Discovery of DNA structure (Cold Spring Harb. Symp. Quant. Biol. 1953;18:123-31) 1953 Sanger sequencing method by F. Sanger (PNAS ,1977, 74: 560-564) 1977 PCR by K. Mullis (Cold Spring Harb Symp Quant Biol. 1986;51 Pt 1:263-73) 1983 Development of pyrosequencing (Anal. Biochem., 1993, 208: 171-175; Science ,1998, 281: 363-365) 1993 1980 1990 2000 2010 Single molecule emulsion PCR 1998 Human Genome Project (Nature , 2001, 409: 860–92; Science, 2001, 291: 1304–1351) Founded 454 Life Science 2000 454 GS20 sequencer (First NGS sequencer) 2005 Founded Solexa 1998 Solexa Genome Analyzer (First short-read NGS sequencer) 2006 GS FLX sequencer (NGS with 400-500 bp read lenght) 2008 Hi-Seq2000 (200Gbp per Flow Cell) 2010 Illumina acquires Solexa (Illumina enters the NGS business) 2006 ABI SOLiD (Short-read sequencer based upon ligation) 2007 Roche acquires 454 Life Sciences (Roche enters the NGS business) 2007 NGS Human Genome sequencing (First Human Genome sequencing based upon NGS technology) 2008 Miseq Roche Jr Ion Torrent PacBio Oxford From Slideshare presentation of Cosentino Cristian http://www.slideshare.net/cosentia/high-throughput-equencing Sequencing Technology Timeline
  5. 5. Evolution of Sequencing 18
  6. 6. Evolution of Sequencing 18
  7. 7. Evolution of Sequencing 18
  8. 8. Evolution of Sequencing 18 Gen 0: Proto Sequencing
  9. 9. Evolution of Sequencing 18 Gen 0: Proto Sequencing
  10. 10. Evolution of Sequencing 18 Gen 0: Proto Sequencing
  11. 11. Evolution of Sequencing 18 Gen 0: Proto Sequencing
  12. 12. Evolution of Sequencing 18 Gen 0: Proto Sequencing
  13. 13. Gen 0: Proto-Sequencing 19
  14. 14. Evolution of Sequencing 20 Gen 0: Proto Sequencing
  15. 15. Evolution of Sequencing 20 Gen 0: Proto Sequencing
  16. 16. Gen 1: Manual Sequencing 21 Gen 0: Proto Sequencing
  17. 17. Gen 1: Manual Sequencing 21 Gen 0: Proto Sequencing
  18. 18. Gen 1: Manual Sequencing 21 Gen 0: Proto Sequencing
  19. 19. Gen 1: Manual Sequencing 21 Gen 0: Proto Sequencing Sanger Maxam-Gilbert Gen 1: Manual Sequencing
  20. 20. Maxam-Gilbert Sequencing 22 From http://www.pnas.org/content/74/2/560.full.pdf
  21. 21. Sanger Sequencing of PhiX174 23 http://www.ncbi.nlm.nih.gov/ pmc/articles/PMC431765/
  22. 22. Sanger Sequencing 24
  23. 23. Sanger Sequencing 25
  24. 24. Nobel Prize 1980: Berg, Gilbert, Sanger 26 http://www.nobelprize.org/nobel_prizes/chemistry/laureates/1980/
  25. 25. 27 Gen 0: Proto Sequencing Sanger Maxam-Gilbert Gen 1: Manual Sequencing Gen 1: Manual Sequencing
  26. 26. 27 Gen 0: Proto Sequencing Sanger Maxam-Gilbert Gen 1: Manual Sequencing Gen 1: Manual Sequencing
  27. 27. 27 Gen 0: Proto Sequencing Sanger Maxam-Gilbert Gen 1: Manual Sequencing Gen 1: Manual Sequencing
  28. 28. Gen 2: Automated Sanger 28 Gen 0: Proto Sequencing Sanger Maxam-Gilbert Gen 1: Manual Sequencing
  29. 29. Gen 2: Automated Sanger 28 Gen 0: Proto Sequencing Sanger Maxam-Gilbert Gen 1: Manual Sequencing
  30. 30. Gen 2: Automated Sanger 28 Gen 0: Proto Sequencing Sanger Maxam-Gilbert Gen 1: Manual Sequencing
  31. 31. Gen 2: Automated Sanger 28 Gen 0: Proto Sequencing Sanger Maxam-Gilbert Gen 1: Manual Sequencing Gen 2: Automated Sanger
  32. 32. Gen 2: Automated Sanger 28 Gen 0: Proto Sequencing Sanger Maxam-Gilbert Gen 1: Manual Sequencing Gen 2: Automated Sanger
  33. 33. Gen 2: Automated Sanger 28 Gen 0: Proto Sequencing Sanger Maxam-Gilbert Gen 1: Manual Sequencing Gen 2: Automated Sanger
  34. 34. Gen 2: Automated Sanger 28 Gen 0: Proto Sequencing Sanger Maxam-Gilbert Gen 1: Manual Sequencing Gen 2: Automated Sanger
  35. 35. Automation of Sanger Part I 29 Sanger method with labeled dNTPs The Sanger mehtods is based on the idea that inhibitors can terminate elongation of DNA at specific points
  36. 36. Many Systems for Sanger Automation 30 Thanks to Robin Coope and Dale Yazuki for comments on 2014 talk ABI 3700 ABI 3730 ABI 3730xl Megabase 1000 Megabase 4000 LiCor
  37. 37. Automation of Sanger Part II 31
  38. 38. Some Automated Sanger Highlights • 1991: ESTs by Venter • 1995: H. influenzae shotgun genome • 1996: Yeast, archaeal genomes • 1998: 1st animal genome - C. elegans • 1999: Drosophila shotgun genome • 2000: Arabidopsis genome • 2000: Human genome • 2004: Shotgun metagenomics 32 Thanks to Keith Bradnam for C. elegans suggestion
  39. 39. Gen 3: Clusters not Clones (NextGen) 33 Gen 0: Proto Sequencing Sanger Maxam-Gilbert Gen 1: Manual Sequencing Gen 2: Automated Sanger
  40. 40. Gen 3: Clusters not Clones (NextGen) 33 Gen 0: Proto Sequencing Sanger Maxam-Gilbert Gen 1: Manual Sequencing Gen 2: Automated Sanger
  41. 41. Gen 3: Clusters not Clones (NextGen) 33 Gen 0: Proto Sequencing Sanger Maxam-Gilbert Gen 1: Manual Sequencing Gen 2: Automated Sanger
  42. 42. Gen 3: Clusters not Clones (NextGen) 33 Gen 0: Proto Sequencing Sanger Maxam-Gilbert Gen 1: Manual Sequencing Gen 2: Automated Sanger Gen 3: Clusters Not Clones
  43. 43. Gen 3: Clusters not Clones (NextGen) 33 Gen 0: Proto Sequencing Sanger Maxam-Gilbert 454- Roche Gen 1: Manual Sequencing Gen 2: Automated Sanger Gen 3: Clusters Not Clones
  44. 44. Gen 3: Clusters not Clones (NextGen) 33 Gen 0: Proto Sequencing Sanger Maxam-Gilbert 454- Roche Gen 1: Manual Sequencing Gen 2: Automated Sanger Gen 3: Clusters Not Clones
  45. 45. Gen 3: Clusters not Clones (NextGen) 33 Gen 0: Proto Sequencing Sanger Maxam-Gilbert Solexa- Illumina 454- Roche Gen 1: Manual Sequencing Gen 2: Automated Sanger Gen 3: Clusters Not Clones
  46. 46. Gen 3: Clusters not Clones (NextGen) 33 Gen 0: Proto Sequencing Sanger Maxam-Gilbert Solexa- Illumina 454- Roche Gen 1: Manual Sequencing Gen 2: Automated Sanger Gen 3: Clusters Not Clones
  47. 47. Gen 3: Clusters not Clones (NextGen) 33 Gen 0: Proto Sequencing Sanger Maxam-Gilbert ABI- Solid Solexa- Illumina 454- Roche Gen 1: Manual Sequencing Gen 2: Automated Sanger Gen 3: Clusters Not Clones
  48. 48. Gen 3: Clusters not Clones (NextGen) 33 Gen 0: Proto Sequencing Sanger Maxam-Gilbert ABI- Solid Solexa- Illumina 454- Roche Gen 1: Manual Sequencing Gen 2: Automated Sanger Gen 3: Clusters Not Clones
  49. 49. Gen 3: Clusters not Clones (NextGen) 33 Gen 0: Proto Sequencing Sanger Maxam-Gilbert ABI- Solid Solexa- Illumina 454- Roche Ion Torrent Gen 1: Manual Sequencing Gen 2: Automated Sanger Gen 3: Clusters Not Clones
  50. 50. Gen 3: Clusters not Clones (NextGen) 33 Gen 0: Proto Sequencing Sanger Maxam-Gilbert ABI- Solid Solexa- Illumina 454- Roche Ion Torrent Gen 1: Manual Sequencing Gen 2: Automated Sanger Gen 3: Clusters Not Clones
  51. 51. Generation III: Clusters not Clones 34
  52. 52. Generation III = “NextGen” 35
  53. 53. NextGen Sequencing Outline 36 Next-generation sequencing platforms Isolation and purification of target DNA Sample preparation Library validation Cluster generation on solid-phase Emulsion PCR Sequencing by synthesis with  3’-blocked reversible terminators Pyrosequencing Sequencing by ligation Single colour imaging Sequencing by synthesis with  3’-unblocked reversible terminators AmplificationSequencingImaging Four colour imaging Data analysis Roche 454Illumina GAII ABi SOLiD Helicos HeliScope From Slideshare presentation of Cosentino Cristian http://www.slideshare.net/cosentia/high-throughput-equencing
  54. 54. 37 Pyrosequencing Sanger method - ABi SOLiD HeliScope Nanopore Roche 454 lumina GAII From Slideshare presentation of Cosentino Cristian http://www.slideshare.net/cosentia/high- throughput-equencing NextGen #1: 454
  55. 55. 38 Pyrosequencing Sanger method - ABi SOLiD HeliScope Nanopore Roche 454 lumina GAII From Slideshare presentation of Cosentino Cristian http://www.slideshare.net/cosentia/high- throughput-equencing NextGen #1: Roche 454
  56. 56. 39 Pyrosequencing Sanger method - ABi SOLiD HeliScope Nanopore Roche 454 lumina GAII From Slideshare presentation of Cosentino Cristian http://www.slideshare.net/cosentia/high- throughput-equencing NextGen #1: Roche 454
  57. 57. 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 40
  58. 58. 41 Roche 454 Wokflow From http://acb.qfab.org/acb/ws09/presentations/Day1_DMiller.pdf http://www.slideshare.net/AGRF_Ltd/ngs-technologies-platforms-and-applications
  59. 59. a b DNA library preparation Emulsion PCR A A A B B B 4.5 hours 8 hours Ligation Selection (isolate AB fragments only) •Genome fragmented by nebulization •No cloning; no colony picking •sstDNA library created with adaptors •A/B fragments selected using avidin-biotin purification gDNA sstDNA library gDNA fragmented by nebulization or sonication Fragments are end- repaired 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. Roche 454 Step 1: Libraries 42
  60. 60. 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 b c Emulsion PCR Sequencing A B 8 hours 7.5 hours using avidin-biotin purification gDNA sstDNA library sstDNA library Bead-amplified sstDNA library Roche 454 Step 2: Emulsion PCR From Mardis 2008. Annual Rev. Genetics 9: 387. 43
  61. 61. DNA capture beads reagents in water-in-oil microreactors inside microreactors enrich for DNA-positive beads Amplified sstDNA library beads Quality filtered bases c Sequencing 7.5 hours sstDNA library Bead-amplified sstDNA library •Well diameter: average of 44 µm •400,000 reads obtained in parallel •A single cloned amplified sstDNA bead is deposited per well 390 Mardis Roche 454 Step 3: Pyrosequencing From Mardis 2008. Annual Rev. Genetics 9: 387. 44
  62. 62. Pyrosequencing 44 µm Pyrosequecning Reads are recorded as flowgrams Annu. Rev. Genomics Hum. Genet., 2008, 9: 387-402 Nature Reviews genetics, 2010, 11: 31-46 Sanger method - ABi SOLiD HeliScope Nanopore Roche 454 Illumina GAII From Slideshare presentation of Cosentino Cristian http://www.slideshare.net/cosentia/high-throughput-equencing Roche 454 Step 3: Pyrosequencing 45
  63. 63. Roche 454 Key Issues • Number of repeated nucleotides estimated by amount of light ... many errors • Reasonable number of failures in EM- PCR and other steps 46
  64. 64. Roche 454 Evolution 47 http://www.slideshare.net/AGRF_Ltd/ngs-technologies-platforms-and-applications
  65. 65. NextGen #2: Solexa 48 Sequecning by synthesis with reversible terminator anger ethod he 454 SOLiD iScope nopore - From Slideshare presentation of Cosentino Cristian http://www.slideshare.net/cosentia/high-throughput-equencing
  66. 66. NextGen #2: Solexa Illumina Sequecning by synthesis with reversible terminator anger ethod he 454 SOLiD iScope nopore - From Slideshare presentation of Cosentino Cristian http://www.slideshare.net/cosentia/high-throughput-equencing 49
  67. 67. NextGen #2: Illumina Accessories 50 Cluster station Genome Analyzer IIxPaired-end module Linux server Bioanalyzer 2100 Instrumentation mple aration sters fication ncing by thesis alysis eline duction na GAII igh ughput From Slideshare presentation of Cosentino Cristian http://www.slideshare.net/cosentia/high-throughput-equencing
  68. 68. Illumina Outline 51 Clusters amplification Clusterstation Wash cluster station Clustergeneration Linearization, Blocking and primer Hybridization Read 1 Prepare read 2 Read 2 GAIIx&PE SBSsequencing Pipeline base call Data analysis Sample preparation and library validation Analysis Sequencing workflow Sample preparation Clusters amplification Sequencing by synthesis Analysis pipeline Introduction Illumina GAII High throughput From Slideshare presentation of Cosentino Cristian http:// www.slideshare.net/ cosentia/high- throughput-equencing
  69. 69. Illumina Step 1: Prep & Attach DNA 52 termined by user-defined instrument settings, which permits discrete read lengths of 25–35 the Illumina data from each run, removing poor-quality sequences. Adapter DNA fragment Dense lawn of primers Adapter Attached DNA Adapters Prepare genomic DNA sample Randomly fragment genomic DNA and ligate adapters to both ends of the fragments. Attach DNA to surface Bind single-stranded fragments randomly to the inside surface of the flow cell channels. Nucleotides a From Mardis 2008. Annual Rev. Genetics 9: 387. Step 1: Sample Preparation The DNA sample of interest is sheared to appropriate size (average ~800bp) using a compressed air device known as a nebulizer. The ends of the DNA are polished, and two unique adapters are ligated to the fragments. Ligated fragments of the size range of 150-200bp are isolated via gel extraction and amplified using limited cycles of PCR
  70. 70. Illumina Step 2: Clusters by Bridge PCR 53 Attached Prepare genomic DNA sample Randomly fragment genomic DNA and ligate adapters to both ends of the fragments. Attach DNA to surface Bind single-stranded fragments randomly to the inside surface of the flow cell channels. Bridge amplification Add unlabeled nucleotides and enzyme to initiate solid- phase bridge amplification. Denature the double stranded molecules Nucleotides Figure 2 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 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. 392 Mardis • From : http://seqanswers.com/forums/showthread.php?t=21. Steps 2-6: Cluster Generation by Bridge Amplification. In contrast to the 454 and ABI methods which use a bead-based emulsion PCR to generate "polonies", Illumina utilizes a unique "bridged" amplification reaction that occurs on the surface of the flow cell. The flow cell surface is coated with single stranded oligonucleotides that correspond to the sequences of the adapters ligated during the sample preparation stage. Single-stranded, adapter-ligated fragments are bound to the surface of the flow cell exposed to reagents for polyermase-based extension. Priming occurs as the free/distal end of a ligated fragment "bridges" to a complementary oligo on the surface. Repeated denaturation and extension results in localized amplification of single molecules in millions of unique locations across the flow cell surface. This process occurs in what is referred to as Illumina's "cluster station", an automated flow cell processor. From Mardis 2008. Annual Rev. Genetics 9: 387.
  71. 71. Clusters 54
  72. 72. 55 25–35 bp, and each sequencing run yields be- tween 2–4 Gb of DNA sequence data. Once generation read type has a unique error model different from that already established for b Laser 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 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. Sequence read over multiple chemistry cycles Repeat cycles of sequencing to determine the sequence Before initiating the next chemistry cycle The blocked 3' terminus and the fluorophore from each incorporated base are removed. GCTGA... From Mardis 2008. Annual Rev. Genetics 9: 387. Illumina Step 3: Sequencing by Synthesis From : http://seqanswers.com/forums/showthread.php?t=21. Steps 7-12: Sequencing by Synthesis. A flow cell containing millions of unique clusters is now loaded into the 1G sequencer for automated cycles of extension and imaging. The first cycle of sequencing consists first of the incorporation of a single fluorescent nucleotide, followed by high resolution imaging of the entire flow cell. These images represent the data collected for the first base. Any signal above background identifies the physical location of a cluster (or polony), and the fluorescent emission identifies which of the four bases was incorporated at that position. This cycle is repeated, one base at a time, generating a series of images each representing a single base extension at a specific cluster. Base calls are derived with an algorithm that identifies the emission color over time. At this time reports of useful Illumina reads range from 26-50 bases.
  73. 73. 56 SBS technology Sample eparation Clusters mplification quencing by synthesis Analysis pipeline troduction umina GAII High roughput From Slideshare presentation of Cosentino Cristian http://www.slideshare.net/cosentia/high-throughput-equencing Illumina Step 3: Sequencing by Synthesis
  74. 74. 57 Laser terminators, primers, and DNA polymerase enzyme to the flow cell. Image of first 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. Sequence read over multiple chemistry cycles Repeat cycles of sequencing to determine the sequence of bases in a given fragment a single base at a time. Before initiating the next chemistry cycle The blocked 3' terminus and the fluorophore from each incorporated base are removed. GCTGA... Figure 2 (Continued ) www.annualreviews.org • Next-Generation DNA Sequencing Methods 393 From Mardis 2008. Annual Rev. Genetics 9: 387. Illumina Step 3: Cycling
  75. 75. Illumina Evolution 58 http://www.slideshare.net/AGRF_Ltd/ngs-technologies-platforms-and-applications
  76. 76. MiSeq Dx 59
  77. 77. HiSeq x Ten 60
  78. 78. HiSeq x Ten 61
  79. 79. NextGen #3: 454: ABI Solid 62 Sequecning by ligation Sanger method Roche 454 - HeliScope Nanopore ABi SOLiD Illumina GAII From Slideshare presentation of Cosentino Cristian http:// www.slideshare.net/ cosentia/high- throughput-equencing
  80. 80. ABI Solid Details 63 3-GG09-20 ARI 25 July 2008 14:57 A C G T 1stbase 2nd base A C G T 3'TAnnnzzz5' 3'TCnnnzzz5' 3'TGnnnzzz5' 3'TTnnnzzz5' Cleavage site Di base probesSOLiD™ substrate 3' TA AT Universal seq primer (n) 3' P1 adapter Template sequence POH Universal seq primer (n–1) Ligase Phosphatase + 1. Prime and ligate 2. Image 4. Cleave off fluor 5. Repeat steps 1–4 to extend sequence 3' Universal seq primer (n–1) 1. Melt off extended sequence 2. Primer reset3' AA AC G G GG C C C T AA A GG CC T TTT 6. Primer reset 7. Repeat steps 1–5 with new primer 8. Repeat Reset with , n–2, n–3, n–4 primers TA AT AT 3' TA AT 3' Excite Fluorescence Cleavage agent P HO TA AA AG AC AAAT TT TC TG TT AC TG CG GC 3' 3. Cap unextended strands 3' PO4 1 2 3 4 5 6 7 ... (n cycles)Ligation cycle 3' 3'1 μm bead 1 μm bead 1 μm bead –1 Universal seq primer (n) 3' 1 Primer round 1 Template Primer round 2 1 base shift Glass slide 3'5' Template sequence 1 μm bead P1 adapter Read position 35343332313029282726252423222120191817161514131211109876543210 a The ligase-mediated sequencing approach of the Applied Biosystems SOLiD sequencer. In a manner similar to Roche/454 emulsion PCR amplification, DNA fragments for SOLiD sequencing are amplified on the surfaces of 1-μm magnetic beads to provide sufficient signal during the sequencing reactions, and are then deposited onto a flow cell slide. Ligase-mediated sequencing begins by annealing a primer to the shared adapter sequences on each amplified fragment, and then DNA ligase is provided along with specific fluorescent- labeled 8mers, whose 4th and 5th bases are encoded by the attached fluorescent group. Each ligation step is followed by fluorescence detection, after which a regeneration step removes bases from the ligated 8mer (including the fluorescent group) and concomitantly prepares the extended primer for another round of ligation. (b) Principles of two-base encoding. Because each fluorescent group on a ligated 8mer identifies a two-base combination, the resulting sequence reads can be screened for base-calling errors versus true polymorphisms versus single base deletions by aligning the individual reads to a known high-quality reference sequence. From Mardis 2008. Annual Rev. Genetics 9: 387.
  81. 81. ABI Solid Evolution 64 http://www.slideshare.net/AGRF_Ltd/ngs-technologies-platforms-and-applications
  82. 82. Complete Genomics 65 REVIEW ased the number of false positive gene ely reduced the number gene candidates sitosterolemia phenotype were determined after comparison of the patient’s genome to a collection of reference genomes. Ultimately, it was determined that the patient failed the standard 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. gure 3. Schematic of Complete Genomics’ DNB array generation and cPAL technology. (A) Design of sequencing fragments, subsequent DN nthesis, and dimensions of the patterned nanoarray used to localize DNBs illustrate the DNB array formation. (B) Illustration of sequencing with a common probes corresponding to 5 bases from the distinct adapter site. Both standard and extended anchor schemes are shown. Reprinted w 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. From Niedringhaus et al. Analytical Chemistry 83: 4327. 2011.
  83. 83. Comparison in 2008 66 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 7 h 4 days 5 days Read length 250 bp 32-40 bp 35 bp Cost per run (total) $8439 $8950 $17447 Cost per Mb $84.39 $5.97 $5.81 From “Introduction to Next Generation Sequencing” by Stefan Bekiranov prometheus.cshl.org/twiki/pub/Main/CdAtA08/ CSHL_nextgen.ppt 66
  84. 84. Comparison in 2012 67 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 7 h 4 days 5 days Read length 250 bp 32-40 bp 35 bp Cost per run (total) $8439 $8950 $17447 Cost per Mb $84.39 $5.97 $5.81 From “Introduction to Next Generation Sequencing” by Stefan Bekiranov prometheus.cshl.org/twiki/pub/Main/CdAtA08/ CSHL_nextgen.ppt 67
  85. 85. 68 Gen 3+: Faster w/ Clusters
  86. 86. Ion Torrent PGM 69
  87. 87. Applied Biosystems Ion Torrent PGM 70
  88. 88. Applied Biosystems Ion Torrent PGM 71 Workflow similar to that for Roche/454 systems. Not surprising, since invented by people from 454.
  89. 89. Ion Torrent pH Based Sequencing 72 Mardis ER. Next-generation sequencing platforms. Annu Rev Anal Chem 2013;6:287-303.
  90. 90. Ion Torrent Evolution 73
  91. 91. Bells and Whistles 74
  92. 92. Multiplexing 75 From http://www.illumina.com/technology/multiplexing_sequencing_assay.ilmn
  93. 93. Multiplexing 76 http://res.illumina.com/documents/products/datasheets/datasheet_sequencing_multiplex.pdf
  94. 94. Small Amounts of DNA 77 http://www.epibio.com/docs/default-source/protocols/nextera-dna-sample-prep-kit-(illumina--compatible).pdf?sfvrsn=4
  95. 95. Capture Methods 78 High throughput sample preparation Sample preparation Clusters amplification Sequencing by synthesis Analysis pipeline Introduction Illumina GAII High throughput Nature Methods, 2010, 7: 111-118 RainDance Microdroplet PCR Roche Nimblegen Salid-phase capture with custom- designed oligonucleotide microarray Reported 84% of capture efficiency Reported 65-90% of capture efficiency From Slideshare presentation of Cosentino Cristian http://www.slideshare.net/cosentia/high-throughput-equencing
  96. 96. 79 High throughput sample preparation Sample preparation Clusters amplification Sequencing by synthesis Analysis pipeline Introduction Illumina GAII High throughput Agilent SureSelect Solution-phase capture with streptavidin-coated magnetic beads Reported 60-80% of capture efficiency From Slideshare presentation of Cosentino Cristian http://www.slideshare.net/cosentia/ high-throughput-equencing Capture Methods
  97. 97. Illumina Paired Ends 80 Paired-end technology Paired-end sequencing works into GA and uses chemicals from the PE module to perform cluster amplification of the reverse strandSample preparation Clusters amplification Sequencing by synthesis Analysis pipeline Introduction Illumina GAII High throughput From Slideshare presentation of Cosentino Cristian http://www.slideshare.net/ cosentia/high-throughput- equencing
  98. 98. Moleculo 81
  99. 99. Moleculo 81 DNA
  100. 100. Moleculo 81 Large fragments DNA
  101. 101. Moleculo 81 Large fragments DNA
  102. 102. Moleculo 81 Large fragments DNA Isolate and amplify
  103. 103. Moleculo 81 Large fragments DNA Isolate and amplify
  104. 104. Moleculo 81 Large fragments DNA Isolate and amplify Sublibrary w/ unique barcodes
  105. 105. Moleculo 81 Large fragments DNA Isolate and amplify CACC GGAA TCTC ACGT AAGG GATC AAAA Sublibrary w/ unique barcodes
  106. 106. Moleculo 81 Large fragments DNA Isolate and amplify CACC GGAA TCTC ACGT AAGG GATC AAAA Sublibrary w/ unique barcodes Sequence w/ Illumina
  107. 107. Moleculo 81 Large fragments DNA Isolate and amplify CACC GGAA TCTC ACGT AAGG GATC AAAA Sublibrary w/ unique barcodes Sequence w/ Illumina
  108. 108. Moleculo 81 Large fragments DNA Isolate and amplify CACC GGAA TCTC ACGT AAGG GATC AAAA Sublibrary w/ unique barcodes Sequence w/ Illumina
  109. 109. Moleculo 81 Large fragments DNA Isolate and amplify CACC GGAA TCTC ACGT AAGG GATC AAAA Sublibrary w/ unique barcodes Sequence w/ Illumina Assemble seqs w/ same codes
  110. 110. Moleculo 81 Large fragments DNA Isolate and amplify CACC GGAA TCTC ACGT AAGG GATC AAAA Sublibrary w/ unique barcodes Sequence w/ Illumina Assemble seqs w/ same codes
  111. 111. HiC 82 Suggested by Carlos Bustamante and Keith Bradnam
  112. 112. HiC Crosslinking & Sequencing Beitel CW, Froenicke L, Lang JM, Korf IF, Michelmore RW, Eisen JA, Darling AE. (2014) Strain- and plasmid- level deconvolution of a synthetic metagenome by sequencing proximity ligation products. PeerJ 2:e415 http://dx.doi.org/10.7717/peerj.415 Table 1 Species alignment fractions. The number of reads aligning to each replicon present in the synthetic microbial community are shown before and after filtering, along with the percent of total constituted by each species. The GC content (“GC”) and restriction site counts (“#R.S.”) of each replicon, species, and strain are shown. Bur1: B. thailandensis chromosome 1. Bur2: B. thailandensis chromosome 2. Lac0: L. brevis chromosome, Lac1: L. brevis plasmid 1, Lac2: L. brevis plasmid 2, Ped: P. pentosaceus, K12: E. coli K12 DH10B, BL21: E. coli BL21. An expanded version of this table can be found in Table S2. Sequence Alignment % of Total Filtered % of aligned Length GC #R.S. Lac0 10,603,204 26.17% 10,269,562 96.85% 2,291,220 0.462 629 Lac1 145,718 0.36% 145,478 99.84% 13,413 0.386 3 Lac2 691,723 1.71% 665,825 96.26% 35,595 0.385 16 Lac 11,440,645 28.23% 11,080,865 96.86% 2,340,228 0.46 648 Ped 2,084,595 5.14% 2,022,870 97.04% 1,832,387 0.373 863 BL21 12,882,177 31.79% 2,676,458 20.78% 4,558,953 0.508 508 K12 9,693,726 23.92% 1,218,281 12.57% 4,686,137 0.507 568 E. coli 22,575,903 55.71% 3,894,739 17.25% 9,245,090 0.51 1076 Bur1 1,886,054 4.65% 1,797,745 95.32% 2,914,771 0.68 144 Bur2 2,536,569 6.26% 2,464,534 97.16% 3,809,201 0.672 225 Bur 4,422,623 10.91% 4,262,279 96.37% 6,723,972 0.68 369 Figure 1 Hi-C insert distribution. The distribution of genomic distances between Hi-C read pairs is shown for read pairs mapping to each chromosome. For each read pair the minimum path length on the circular chromosome was calculated and read pairs separated by less than 1000 bp were discarded. The 2.5 Mb range was divided into 100 bins of equal size and the number of read pairs in each bin was recorded for each chromosome. Bin values for each chromosome were normalized to sum to 1 and plotted. E. coli K12 genome were distributed in a similar manner as previously reported (Fig. 1; (Lieberman-Aiden et al., 2009)). We observed a minor depletion of alignments spanning the linearization point of the E. coli K12 assembly (e.g., near coordinates 0 and 4686137) due to edge eVects induced by BWA treating the sequence as a linear chromosome rather than circular. 10.7717/peerj.415 9/19 Figure 2 Metagenomic Hi-C associations. The log-scaled, normalized number of Hi-C read pairs associating each genomic replicon in the synthetic community is shown as a heat map (see color scale, blue to yellow: low to high normalized, log scaled association rates). Bur1: B. thailandensis chromosome 1. Bur2: B. thailandensis chromosome 2. Lac0: L. brevis chromosome, Lac1: L. brevis plasmid 1, Lac2: L. brevis plasmid 2, Ped: P. pentosaceus, K12: E. coli K12 DH10B, BL21: E. coli BL21. reference assemblies of the members of our synthetic microbial community with the same alignment parameters as were used in the top ranked clustering (described above). We first Figure 3 Contigs associated by Hi-C reads. A graph is drawn with nodes depicting contigs and edges depicting associations between contigs as indicated by aligned Hi-C read pairs, with the count thereof depicted by the weight of edges. Nodes are colored to reflect the species to which they belong (see legend) with node size reflecting contig size. Contigs below 5 kb and edges with weights less than 5 were excluded. Contig associations were normalized for variation in contig size. typically represent the reads and variant sites as a variant graph wherein variant sites are represented as nodes, and sequence reads define edges between variant sites observed in the same read (or read pair). We reasoned that variant graphs constructed from Hi-C data would have much greater connectivity (where connectivity is defined as the mean path length between randomly sampled variant positions) than graphs constructed from mate-pair sequencing data, simply because Hi-C inserts span megabase distances. Such Figure 4 Hi-C contact maps for replicons of Lactobacillus brevis. Contact maps show the number of Hi-C read pairs associating each region of the L. brevis genome. The L. brevis chromosome (Lac0, (A), Chris Beitel @datscimed Aaron Darling @koadman
  113. 113. Dovetail “Chicago” Libraries 84 http://arxiv.org/abs/1502.05331 Suggested by
  114. 114. Single Cell Sequencing 85 Suggested by Keith Robison
  115. 115. Gen 4: Single Molecule 86 Gen 0: Proto Sequencing Sanger Maxam-Gilbert ABI- Solid Solexa- Illumina 454- Roche Ion Torrent Gen 1: Manual Sequencing Gen 2: Automated Sanger Gen 3: Clusters Not Clones
  116. 116. Gen 4: Single Molecule 86 Gen 0: Proto Sequencing Sanger Maxam-Gilbert ABI- Solid Solexa- Illumina 454- Roche Ion Torrent Gen 1: Manual Sequencing Gen 2: Automated Sanger Gen 3: Clusters Not Clones
  117. 117. Gen 4: Single Molecule 86 Gen 0: Proto Sequencing Sanger Maxam-Gilbert ABI- Solid Solexa- Illumina 454- Roche Ion Torrent Gen 1: Manual Sequencing Gen 2: Automated Sanger Gen 3: Clusters Not Clones
  118. 118. Gen 4: Single Molecule 86 Gen 0: Proto Sequencing Sanger Maxam-Gilbert ABI- Solid Solexa- Illumina 454- Roche Ion Torrent Gen 1: Manual Sequencing Gen 2: Automated Sanger Gen 3: Clusters Not Clones Gen 4: Single Molecule
  119. 119. Helicos Gen 4: Single Molecule 86 Gen 0: Proto Sequencing Sanger Maxam-Gilbert ABI- Solid Solexa- Illumina 454- Roche Ion Torrent Gen 1: Manual Sequencing Gen 2: Automated Sanger Gen 3: Clusters Not Clones Gen 4: Single Molecule
  120. 120. Pacbio Helicos Gen 4: Single Molecule 86 Gen 0: Proto Sequencing Sanger Maxam-Gilbert ABI- Solid Solexa- Illumina 454- Roche Ion Torrent Gen 1: Manual Sequencing Gen 2: Automated Sanger Gen 3: Clusters Not Clones Gen 4: Single Molecule
  121. 121. Oxford NanoPore Pacbio Helicos Gen 4: Single Molecule 86 Gen 0: Proto Sequencing Sanger Maxam-Gilbert ABI- Solid Solexa- Illumina 454- Roche Ion Torrent Gen 1: Manual Sequencing Gen 2: Automated Sanger Gen 3: Clusters Not Clones Gen 4: Single Molecule
  122. 122. 87 Single Molecule I: Helicos 3rd Generation Sequencing y w for cular ent_sequencing
  123. 123. 88 Single Molecule II: Pacific Biosciences
  124. 124. 89 Single Molecule II: Pacific Biosciences Mardis ER. Next-generation sequencing platforms. Annu Rev Anal Chem 2013;6:287-303.
  125. 125. 90 Analytical Chemistry REVIEW Φ29 polymerase. Each amplified product of a circularized fragment is called a DNA nanoball (DNB). DNBs are selectively attached to a hexamethyldisilizane (HMDS) coated silicon chip that is photolithographically patterned with aminosilane active sites. Figure 3A illustrates the DNB array design. The use of the DNBs coupled with the highly patterned array offers several advantages. The production of DNBs increases signal intensity by simply increasing the number of hybridization sites available for probing. Also, the size of the DNB is on the same length scale as the active site or “sticky” spot patterned on Each hybridization and ligation cycle is followed by fluorescent imaging of the DNB spotted chip and subsequently regeneration of the DNBs with a formamide solution. This cycle is repeated until the entire combinatorial library of probes and anchors is examined. This formula of the use of unchained reads and regeneration of the sequencing fragment reduces reagent con- sumption and eliminates potential accumulation errors that can arise in other sequencing technologies that require close to completion of each sequencing reaction.19,52,53 Complete Genomics showcased their DNB array and cPAL 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 temporal fluorescence detection with respect to each of the five incorporation steps is shown below. Reprinted with permission from ref 39. Copyright 2009 American Association for the Advancement of Science. 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 temporal fluorescence detection with respect to each of the five incorporation steps is shown below. From Niedringhaus et al. Analytical Chemistry 83: 4327. 2011. Single Molecule II: Pacific Biosciences
  126. 126. Why Finish Genomes? 91 The Value of Finished Bacterial Genomes Why Are Finished Genomes So Important? When Sanger sequencing was the only available sequencing technique, it was expensive — but not unusual — to improve genome drafts until they were good enough to be considered finished. With the availability of short-read sequencing technologies, draft genomes became cheap and easy to produce, and the majority of researchers skipped the more labor- and time-intensive task of finishing genomes, with the realization that critical data may be missing (Figure 3). Finished genomes are crucial for understanding microbes and advancing the field of microbiology3 because: • Functional genomic studies demand a high-quality, complete genome sequence as a starting point • Comparative genomics is meaningful only in terms of complete genome sequences • Understanding genome organization provides biological insights • Microbial forensics requires at least one complete reference genome sequence • Finished genomes aid in microbial outbreak source identification and phylogenetic analysis • A complete genome is a permanent scientific resource Figure 3: History of drafted vs. finished genomes (adapted from ref. 2). Microbial Genetics Using SMRT Sequencing 0 2000 4000 6000 8000 10000 12000 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 Numberofgenomes Drafted Bacterial Genomes Finished Bacterial Genomes
  127. 127. Why Finish Genomes 92 JOURNAL OF BACTERIOLOGY, Dec. 2002, p. 6403–6405 Vol. 184, No. 23 0021-9193/02/$04.00ϩ0 DOI: 10.1128/JB.184.23.6403–6405.2002 Copyright © 2002, American Society for Microbiology. All Rights Reserved. DIALOG The Value of Complete Microbial Genome Sequencing (You Get What You Pay For) Claire M. Fraser,* Jonathan A. Eisen, Karen E. Nelson, Ian T. Paulsen, and Steven L. Salzberg The Institute for Genomic Research, Rockville, Maryland 20850 Since the publication of the complete Haemophilus influen- zae genome sequence in July 1995 (4), the field of microbiology has been one of the largest beneficiaries of the breakthroughs in genomics and computational biology that made this accom- plishment possible. When the 1.8-Mbp H. influenzae project began in 1994, it was not certain that the whole-genome shot- gun sequencing strategy would succeed because it had never been attempted on any piece of DNA larger than an average lambda clone (ϳ40 kbp) (9). During the past 7 years, progress in DNA sequencing tech- nology, the design of new vectors for library construction for use in shotgun sequencing projects, significant improvements in closure and finishing strategies, and more sophisticated and robust methods for gene finding and annotation have dramat- ically reduced the time required for each stage of a genome project and the cost per base pair while at the same time organisms to be sampled because of the cost savings that would come from not taking each project to completion. While this strategy does achieve a cost savings, today it is only approxi- mately 50%, and this comes at a cost in terms of the quality and utility of the finished product. A complete genome sequence represents a finished product in which the order and accuracy of every base pair have been verified. In contrast, a draft sequence, even one of high cov- erage, represents a collection of contigs of various sizes, with unknown order and orientation, that contain sequencing errors and possible misassemblies. As stated by Selkov et al. in a 2000 paper on a draft sequence of Thiobacillus ferrooxidans, “It is clear that such sequencing. . .produces more errors than com- plete genome sequencing. . . . The current error rate is esti- mated to be 1 per 1,000 to 2,000 base pairs vs. 1 in 10,000 base pairs for complete sequencing” (10). In fact, the difference is http://jb.asm.org/Downloadedfrom http://jb.asm.org/content/184/23/6403.full
  128. 128. HGAP Assembly from PacBio 93 PacBio assembly CDC assembly Illumina assemblySanger validation HGAP Assembler for PacBio Data http://www.pacificbiosciences.com/pdf/ microbial_primer.pdf
  129. 129. Detecting Modified Bases 94 Page 2 www.pacb.com/basemod processes. The potential benefits of detecting base modification, using SMRT sequencing, include: Single-base resolution detection of a wide two successive base incorporations, are altered by the presence of a modified base in the DNA template 3 . This is observable as an increased space between fluorescence pulses, which is called the interpulse duration (IPD), as shown in Figure 2. Figure 2. Principle of detecting modified DNA bases during SMRT sequencing. The presence of the modified base in the DNA template (top), shown here for 6-mA, results in a delayed incorporation of the corresponding T nucleotide, i.e. longer interpulse duration (IPD), compared to a control DNA template lacking the modification (bottom). 3 http://www.pacificbiosciences.com/pdf/microbial_primer.pdf
  130. 130. Pink Berry Genomics PB-PSB1 (Purple sulfur bacteria) PB-SRB1 (Sulfate reducing bacteria) (sulfate) (sulfide) Wilbanks, E.G. et al (2014). Environmental Microbiology Lizzy Wilbanks @lizzywilbanks
  131. 131. 99 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. From Oxford Nanopores Web Site Single Molecule III: Oxford Nanopores
  132. 132. • 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). 100 nalytical Chemistry REVIEW etected across a metal oxide-silicon layered structure. The translocated through a solid-state nanopore. In addition, sever igure 6. Biological nanopore scheme employed by Oxford Nanopore. (A) Schematic of RHL protein nanopore mutant depicting the positions of t yclodextrin (at residue 135) and glutamines (at residue 139). (B) A detailed view of the β barrel of the mutant nanopore shows the locations he arginines (at residue 113) and the cysteines. (C) Exonuclease sequencing: A processive enzyme is attached to the top of the nanopore to cleave sing ucleotides from the target DNA strand and pass them through the nanopore. (D) A residual current-vs-time signal trace from an RHL protein nanopo hat shows a clear discrimination between single bases (dGMP, dTMP, dAMP, and dCMP). (E) Strand sequencing: ssDNA is threaded through rotein nanopore and individual bases are identified, as the strand remains intact. Panels A, B, and D reprinted with permission from ref 91. Copyrig 009 Nature Publishing Group. Panels C and E reprinted with permission from Oxford Nanopore Technologies (Zoe McDougall). Figure6. Biological nanopores cheme employed by Oxford Nanopore.(A) Schematic of RHL protein nano pore mutant depicting the positions of the 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). Single Molecule III: Oxford Nanopores From Niedringhaus et al. Analytical Chemistry 83: 4327. 2011.
  133. 133. 101 Analytical Chemistry REVIEW would result, in theory, in detectably altered current flow through the pore. Theoretically, nanopores could also be designed to measure tunneling current across the pore as bases, each with a distinct tunneling potential, could be read. The nanopore ap- lipid bilayer, using ionic current blockage method. The autho predicted that single nucleotides could be discriminated as lon as: (1) each nucleotide produces a unique signal signature; (2 the nanopore possesses proper aperture geometry to accomm Figure 5. Nanopore DNA sequencing using electronic measurements and optical readout as detection methods. (A) In electronic nanopore scheme signal is obtained through ionic current,73 tunneling current,78 and voltage difference79 measurements. Each method must produce a characteristic sign to differentiate the four DNA bases. Reprinted with permission from ref 83. Copyright 2008 Annual Reviews. (B) In the optical readout nanopore desig each nucleotide is converted to a preset oligonucleotide sequence and hybridized with labeled markers that are detected during translocation of the DN fragment through the nanopore. Reprinted from ref 82. Copyright 2010 American Chemical Society. Nanopore DNA sequencing using electronic measurements and optical readout as detection methods.(A)In electronic nanopore schemes, signal is obtained through ionic current,73 tunneling current, and voltage difference measurements. Each method must produce a characteristic signal to differentiate the four DNA bases. (B) In the optical readout nanopore design, each nucleotide is converted to a preset oligonucleotide sequence and hybridized with labeled markers that are detected during translocation of the DNA fragment through the nanopore. Single Molecule III: Oxford Nanopores From Niedringhaus et al. Analytical Chemistry 83: 4327. 2011.
  134. 134. 102 Oxford Nanopores MinIon “It’s kind of a cute device,” says Jaffe of the MinION, which is roughly the size and shape of a packet of chewing gum. “It has pretty lights and a fan that hums pleasantly, and plugs into a USB drive.” But his technical review is mixed. From http://www.nature.com/news/data-from-pocket-sized-genome-sequencer-unveiled-1.14724
  135. 135. Disclosure: Not Huge Fan of Nanopore … 103
  136. 136. Yet, Must Accept They Are Not a Myth 104 Pics Provided by Nick Loman
  137. 137. Yet, Must Accept They Are Not a Myth 104 Pics Provided by Nick Loman
  138. 138. minION prep (via Nick Loman) 105 End-repair (15mins) dA-tailing (15mins) Fragment (2mins) Clean up (5mins) Clean up (5mins) Ligation (10mins) Tether annealing (10mins) HP motor incubation (30mins) Total: 92 minutes Slide Provided by Nick Loman
  139. 139. Real Time Sequencing? 106 Slide Provided by Nick Loman
  140. 140. 107 Slide Provided by Nick Loman
  141. 141. 108 Slide Provided by Nick Loman

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