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Evolution of DNA Sequencing - talk by Jonathan Eisen for the Bodega Workshop in Applied Phylogenetics

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Published on March 10, 2014

Author: phylogenomics

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Talk by Jonathan Eisen on the Evolution of DNA Sequencing Methods
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Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics ! Evolution of Sequencing ! Workshop in Applied Phylogenetics March 9, 2014 Bodega Bay Marine Lab ! Jonathan A. Eisen UC Davis Genome Center

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Review Papers Mardis ER. Next-generation sequencing platforms. Annu Rev Anal Chem 2013;6:287-303. doi: 10.1146/annurev- anchem-062012-092628.

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Review Papers 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 k links to ontent online, his volume articles ve search ther 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 Annu.Rev.Genom.HumanGenet.2008.9: byUniversidadNacionalAutonom

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Open Access Papers of Interest • 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

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics 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

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Generation I: Manual Sequencing

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Maxam-Gilbert Sequencing http://www.pnas.org/content/74/2/560.full.pdf

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Sanger Sequencing of PhiX174 http://www.ncbi.nlm.nih.gov/ pmc/articles/PMC431765/

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Sanger Sequencing

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Sanger Sequencing

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Nobel Prize 1980: Berg, Gilbert, Sanger

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Generation II: Automated Sanger

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Automation of Sanger Part I Sanger method with labeled dNTPs The Sanger mehtods is based on the idea that inhibitors can terminate elongation of DNA at specific points

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Automation of Sanger Part II

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Automated Sanger Highlights • 1991: ESTs by Venter • 1995: Haemophilus influenzae genome • 1996: Yeast, archaeal genomes • 1999: Drosophila genome • 2000: Arabidopsis genome • 2000: Human genome • 2004: Shotgun metagenomics

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Generation III: Clusters not Clones

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Generation III = “NextGen”

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics NextGen Sequencing OutlineNext-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

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Pyrosequencing Sanger method - ABi SOLiD HeliScope Nanopore Roche 454 Illumina GAII From Slideshare presentation of Cosentino Cristian http://www.slideshare.net/cosentia/high- throughput-equencing NextGen #1: 454

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Pyrosequencing Sanger method - ABi SOLiD HeliScope Nanopore Roche 454 Illumina GAII NextGen #1: Roche 454 From Slideshare presentation of Cosentino Cristian http://www.slideshare.net/cosentia/high- throughput-equencing

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Pyrosequencing Sanger method - ABi SOLiD HeliScope Nanopore Roche 454 Illumina GAII NextGen #1: Roche 454 From Slideshare presentation of Cosentino Cristian http://www.slideshare.net/cosentia/high- throughput-equencing

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics 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

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics 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

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics 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 A B B 8 hours 7.5 hours only) with adaptors •A/B fragments selected using avidin-biotin purification gDNA sstDNA library sstDNA library Bead-amplified sstDNA library From Mardis 2008. Annual Rev. Genetics 9: 387. Roche 454 Step 2: Emulsion PCR

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics 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 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 From Mardis 2008. Annual Rev. Genetics 9: 387. Roche 454 Step 3: Pyrosequencing

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics 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

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics 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

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Roche 454 Evolution http://www.slideshare.net/AGRF_Ltd/ngs-technologies-platforms-and-applications

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics NextGen #2: Solexa Sequecning by synthesis with reversible terminator From Slideshare presentation of Cosentino Cristian http:// www.slideshare.net/ cosentia/high- throughput- equencing

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Sequecning by synthesis with reversible terminator From Slideshare presentation of Cosentino Cristian http:// www.slideshare.net/ cosentia/high- throughput-equencing NextGen #2: Solexa Illumina

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics NextGen #2: Illumina Accessories Cluster station Genome Analyzer IIxPaired-end module Linux server Bioanalyzer 2100 Instrumentation le tion ers ation ng by esis sis ne ction GAII h hput From Slideshare presentation of Cosentino Cristian http:// www.slideshare. net/cosentia/ high-throughput- equencing

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Illumina Outline 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

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Illumina Step 1: Prep & Attach DNA continues for a specific number of cycles, as de- termined 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. 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

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Illumina Step 2: Clusters by Bridge PCR 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 Mardis 2008. Annual Rev. Genetics 9: 387. 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.

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Clusters

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics tween 2–4 Gb of DNA sequence data. Once 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 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... 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.

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics SBS technology Sample reparation Clusters mplification quencing by synthesis Analysis pipeline troduction umina GAII High hroughput From Slideshare presentation of Cosentino Cristian http:// www.slideshare.net/ cosentia/high- throughput-equencing Illumina Step 3: Sequencing by Synthesis

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Laser 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 next chemistry cy The blocked 3' termi and the fluorophore from each incorpora 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

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Illumina Evolution http://www.slideshare.net/AGRF_Ltd/ngs-technologies-platforms-and-applications

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics MiSeq Dx

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics HiSeq x Ten

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics HiSeq x Ten

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics NextGen #3: 454: ABI SolidSequecning 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

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics ABI Solid Details G09-20 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)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.

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics ABI Solid Evolution http://www.slideshare.net/AGRF_Ltd/ngs-technologies-platforms-and-applications

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Complete Genomics 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. 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 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.

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics 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 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

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics 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 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

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Bells and Whistles

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Multiplexing From http://www.illumina.com/technology/multiplexing_sequencing_assay.ilmn

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Multiplexing http://res.illumina.com/documents/products/datasheets/datasheet_sequencing_multiplex.pdf

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Small Amounts of DNA http://www.epibio.com/docs/default-source/protocols/nextera-dna-sample-prep-kit-(illumina--compatible).pdf?sfvrsn=4

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Capture MethodsHigh 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

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics 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

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Illumina Paired Ends 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

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Moleculo 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

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Generation III+: Faster w/ Clusters

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Ion Torrent PGM

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Applied Biosystems Ion Torrent PGM

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Applied Biosystems Ion Torrent PGM Workflow similar to that for Roche/454 systems. ! Not surprising, since invented by people from 454.

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Ion Torrent pH Based Sequencing Mardis ER. Next-generation sequencing platforms. Annu Rev Anal Chem 2013;6:287-303.

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Ion Torrent Evolution

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Generation IV: Single Molecule

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Single Molecule I: Helicos 3rd Generation Sequencing by low for lecular scent_sequencing

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Single Molecule II: Pacific Biosciences

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Single Molecule II: Pacific Biosciences Mardis ER. Next-generation sequencing platforms. Annu Rev Anal Chem 2013;6:287-303.

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics 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

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Why Finish Genomes? 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

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Why Finish Genomes 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 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 http://jb.asm.orDownloadedfrom http://jb.asm.org/content/184/23/6403.full

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics HGAP Assembly from PacBio PacBio assembly CDC assembly Illumina assemblySanger validation HGAP Assembler for PacBio Data http://www.pacificbiosciences.com/pdf/ microbial_primer.pdf

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Detecting Modified Bases 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 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

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics 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

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics • 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). tical Chemistry RE 6. Biological nanopore scheme employed by Oxford Nanopore. (A) Schematic of RHL protein nanopore mutant depicting the position extrin (at residue 135) and glutamines (at residue 139). (B) A detailed view of the β barrel of the mutant nanopore shows the loca inines (at residue 113) and the cysteines. (C) Exonuclease sequencing: A processive enzyme is attached to the top of the nanopore to cleav tides from the target DNA strand and pass them through the nanopore. (D) A residual current-vs-time signal trace from an RHL protein na ows a clear discrimination between single bases (dGMP, dTMP, dAMP, and dCMP). (E) Strand sequencing: ssDNA is threaded th n nanopore and individual bases are identified, as the strand remains intact. Panels A, B, and D reprinted with permission from ref 91. Co 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 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 ar 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.

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics Analytical Chemistry REVIEW would result, in theory, in detectably altered current flow through he pore. Theoretically, nanopores could also be designed to measure tunneling current across the pore as bases, each with a istinct tunneling potential, could be read. The nanopore ap- lipid bilayer, using ionic current blockage method. The author 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 accommo igure 5. Nanopore DNA sequencing using electronic measurements and optical readout as detection methods. (A) In electronic nanopore schemes ignal is obtained through ionic current,73 tunneling current,78 and voltage difference79 measurements. Each method must produce a characteristic signa 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 and hybridized with labeled markers that are detected during translocation of the DNA ragment 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.

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics 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

Slides for Jonathan Eisen talk at UC Davis Bodega Bay Workshop in Applied Phylogenetics

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