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`(if, APPLICATIONS OF NEXT-GENERATION SEQUENCING
`
`
`Sequencing technologies —
`the next generation
`
`Michael L. Metzker *1
`
`Abstract | Demand has never been greater for revolutionary technologies that deliver
`
`last. inexpensive and accurate genome information. This challenge has catalysed the
`
`development of next-generation sequencing (NOS) technologies. The inexpensive
`
`production of large volumes of sequence data is the primary advantage over conventional
`
`methods. Here. I present a technical review of template preparation, sequenclng and
`
`imaging, genome alignment and assembly approaches, and recent advances in current
`
`and neat—term commercially available NCS instruments. I also outline the broad range of
`
`applications for N68 technologies, in addition to providing guidelines for platform
`
`selection to address biological questions of interest.
`
`
`
`Automated Sanger
`sequencing
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`
`‘ Human Genome Sequencing
`Center and Department of
`Molecular 61 Human
`Generlcs, Ball/or College of
`A'laclicine, One Bay/a! Plaza,
`N l 409. Houston. flexes
`77030, USA
`‘Laselc'e/i, lnc., 8052 El Rio
`Street. Houston. Texas
`77054, USA.
`amall. Inlnstzheru’mcm Pr'ltl
`tint
`it’t. tritJtSln-Qflfiflr’.
`i’ttl’alisited mime
`i" [‘t'ttlltlflt‘t T‘tt"l
`
`Over the past four years. there has been a fundamental
`shift away from the application of automated Sanger
`sequencing for genome analysis. Prior to this depar~
`lure, the automated Sanger method had dominated the
`industry for almost two decades and led to a number of
`monumental accomplishments. including the comple—
`tion of the only fimshedtgrade human genome sequence].
`Despite many technical improvements during this era,
`the limitations of automated Sanger sequencingshowed
`a need for new and improved technologies for sequencv
`ing large numbers of human genomes. Recent efforts
`have been directed towards the development of new
`methods, leaving Sanger sequencing with fewer reported
`advances. As such, automated Sanger sequencing is not
`covered here. and interested readers are directed to
`previous articles“.
`The automated Sanger method is considered as
`a ‘lirst-generation’ technology. and newer methods
`are referred to as next—generation sequencing (NGS).
`These newer technologies constitute various strategies
`that rely on a combination oftcrnptate preparation,
`sequencing and imaging. and genome alignment and
`assembly methods. The arrival of NGS technologies in
`the marketplace has changed the way we think about
`scientific approaches in basic. applied and clinical
`research. In some respects, the potential ofNGS is akin
`to the early days ofPCR, with one’s imagination being
`the primary limitation to its use. The major advance
`offered by NGS is the ability to produce an enormous
`volume of data cheaply — in some cases in excess of
`one billion short reads per instrument l'LU‘t. This feature
`expands the realm of experimentation beyond just
`
`determining the order of bases. For example, in
`gene-expression studies micro-arrays are now being
`replaced by seq based methods, which can identify and
`quantify rare transcripts without prior knowledge ofa
`particular gene and can provide information regarding
`alternative splicingand sequence variation in identified
`genes”. The ability to sequence the whole genuine of
`many related organisms has allowed large—scale com-
`parative and evolutionary studies to be performed that
`were unimaginable just a few years ago. The broadest
`application ofNGS may be the rescq uencing ofhumnn
`genomes to enhance our Luiderstanding of how genetic
`differences affect health and disease. The variety of
`NGS features makes it likely that multiple platforms
`will coexist in the marketplace, with some having clear
`advantages for particular applications over others.
`This Review focuses on commercially available tech-
`nologies from Roche/454, lllumina/Solexa, Life/AVG
`and Helicos BioSciences, the Polortator instrument and
`
`the near<term technology of Pacific Biosciences. who
`aim to bring their sequencing device to the market in
`2010. Nanoporc sequencing is not covered. although
`interested readers are directed to an article by Brenton
`and colleagues“, who describe the advances and remain-
`ing challenges for this technology. Here,I present a tech-
`nical review of template preparation, sequencing and
`imaging, genome alignment and assembly, and current
`NGS platform perfommnce to provide guidance on how
`these technologies work and how they may be applied
`to important biological questions. i highlight the appli-
`cations of human genome resequencing using targeted
`and whole‘genome approaches, and discuss the progress
`
`
`
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`
`and limitations of these methods, as well as upcoming
`advances and the impact they are expected to have over
`the next few years.
`
`Next-generation sequencing technologies
`Sequencing technologies include a number of methods
`that are grouped broadly as template preparation,
`sequencing and imaging, and data analysis. The unique
`combination of specific protocols distinguishes one
`technology from another and determines the type of
`data produced from each platform. These differences
`in data output present challenges when comparing plat-
`forms based on data quality and cost. Although qualv
`ity scores and accuracy estimates are provided by each
`manufacturer, there is no consensus that a ‘quality base'
`from one platform is equivalent to that from another
`platform. Various sequencing metrics are discussed later
`in the article.
`
`In the Following sections, stages of template prepara»
`tion and sequencing and imaging are discussed as they
`apply to existing and near-term commercial platforms.
`There are two methods used in preparing templates
`for N65 reactions: clonally amplified templates origi-
`nating from single DNA molecules, and single DNA-
`molecule templates. The term sequencing by synthesis.
`which is used to describe numerous DNA polymer-
`ase-dependent methods in the literature, is not used
`in this article because it fails to delineate the different
`
`mechanisms involved in sequencing”. Instead. these
`methods are classified as cyclic reversible termination
`(CRT), single—nucleotide addition (SNA) and real—time
`sequencing. Sequencing by ligation (SBL), an approach
`in which DNA polymerase is replaced by DNA ligase,
`is also described imaging methods coupled with these
`sequencing strategies range from measuring biolumines-
`cent signals to four-colour imaging of single molecular
`events. The voluminous data produced by these NGS
`platforms place substantial demands on informa-
`tion technology in terms of data storage, tracking and
`quality control (see R E, {t for details).
`
`Template preparation
`The need for robust methods that produces representative.
`non-biased source of nucleic acid material from the
`
`genome under investigation cannot be overemphasized.
`Current methods generally involve randomly breaking
`genomic DNA into smaller sizes from which either frag
`ment templates or mate pair templates are created A com-
`mon theme amongNGS technologies is that the template
`is attached or immobilized to a solid surface or support
`The immobilization of spatially separated template sites
`allows thousands to billions of sequencing reactions to
`be performed simultaneously.
`
`Clonally amplified templates. Most imaging systems have
`not been designed to detect single fluorescent events, so
`amplified templates are required. The two most common
`methods are emulsion PCR (emPCRY’ and solid~phase
`amplification”. EmPCR is used to prepare sequencing
`templates in a cell-free system, which has the advantage
`of avoiding the arbitrary loss of genomic sequences — a
`
`problem that is inherent in bacterial cloning methods. A
`library of fragment or matepair targets is created, and
`adaptors containing universal priming sites are ligated to
`the target ends, allowing complex genomes to be ampli—
`fied with common PCR primers. After ligation, the
`DNA is separated into single strands and captured onto
`beads under conditions that favour one DNA molecule
`
`per head it to l at. After the successful amplification and
`enrichment ofeml’CR beads, millions can be immobi-
`lized in 21 polyacrylamide gel on a standard microscope
`slide (Polonator “, chemically crosslinked to an amino-
`coated glass surface (Life/AFC; Polonator)” or deposited
`into individual PicoTiterPl-ate (PTP) wells (Roche/454)13
`in which the N63 chemistry can be performed.
`Solid—phase amplification can also be used to produce
`randomly distributed. clonally amplified clusters from
`fragment or mate-pair templates on a glass slide the, i m.
`Higlrdcnsity forward and reverse primers are covalently
`attached to the slide, and the ratio of the primers to the
`template on the support defines the surface density of
`the amplified clusters. Solid-phase amplification can
`produce 100-200 million spatially separated template
`clusters (Illumina/Solexa). providing free ends to which
`a universal sequencing primer can be hybridized to
`initiate the NGS reaction.
`
`Single-molecule templates. Although clonally amplified
`methods offer certain advantages over bacterial cloning,
`some of the protocols are cumbersome to implement
`and require a large amount of genomic DNA material
`(3—20 pg). The preparation of single—molecule tem-
`plates is more straightforward and requires less start-
`ing material (<1 pg). More importantly, these methods
`do not require l’CR, which creates mutations in clon-
`ally amplified templates that masquerade as sequence
`variants. AT<rich and (EC-rich target sequences may
`also show amplification bias in product yield, which
`results in their underrepresentation in genome align-
`ments and assemblies. Quantitative applications, such as
`RNA-scqf', perform more effectively with non-amplified
`template sources. which do not alter the representational
`abundance of mRNA molecules.
`
`Before the N63 reaction is carried out, single-
`molecule templates are usually immobilized on solid sup-
`ports using one of at least three different approaches. In
`the li rst approach, spatially distributed individual primer
`molecules are covalently attached to the solid support“.
`The template, which is prepared by randomly fragment-
`ing the starting material into small sizes (for example,
`~200-250 bp) and adding common adaptors to the frag-
`ment ends, is then hybridized to the immobilized primer
`it i .3.
`l C], In the second approach, spatially distributed sin-
`gle-molecule templates are covalently attached to the solid
`support ‘ " by pruning and extending single-stranded, sin -
`gle-molecule templates from immobilizedprimers ll V:
`i r).
`A common primer is then hybridized to the template
`lfiC. lot. in either approach. DNA polymerase can bind
`to the immobilized primed template configuration to
`initiate the NOS reaction. Both ofthe above approaches
`are used by Helicos BioSciences. in a third approach,
`spatially distributed single polymerase molecules
`
`Finished grade
`A quality measure. iota
`sequenced genome A
`t‘ivttslwd grade gunman
`t‘U‘lltllilC‘i‘l y referred lL a: a
`, higher
`'ftnizl led genome.
`
`IILlf:
`
`quality than a drar
` ft
`
`.. tune with mot
`
`coverage and lower ._ our} and
`ill: human
`
`gap. [for camp
`s‘nnme reference ‘omains
`
`3% ol‘ the
`h, covers
`
`
`llutllv} Willi 5—H gaps, and
`has or. error rate of l
`in every
`t’ fl‘flt‘vlt
`lZ‘l‘Jl
`
`Template
`Tl‘it: rel" ntwiaiii DNA
`
`mnlgzttli l, marl: Ill} of Ft
`m 'thl H gum. usually a vertm
`u‘l adaptir ;-:t.toeittftiit Wllltll
`a traversal primer ran Hurt
`and the tallest seqtiettte. which
`is typically an ttnlmowri putt ion
`tr he ,eutufiwmd.
`
`Seq~ba sed methods
`ASS-3th? llltnl use
`
`l‘lGAl gt. Gtallutt setttlt‘l‘itdtig
`technologies May include
`
`me livlduful (letwiiiiim the
`scout int: content and
`.ilnitut’tnl‘tt'n: oi mftNAI‘,
`in: limiting RNAn (ll ml small
`RNA; [rollertwely called
`RNA m'wt‘til and metl‘tmt;. int
`”trawling genuine wide
`pmllla; rtl llill‘flttlllillill‘Et.‘ll.lliF)lr'—.Cl
`DNA
`tielll tit.)lfll‘/lr..v.e5
`
` lilill? . qtmetltvlulien
`mite: lmelliyl sealanrl
`[\anc l liyoersmisttmtly
`:ltca [Div/assaeqt
`
`Polonator
`int. ézsview mostly describes
`technology platforms tliatare
`associated with a respective
`.ot'ittmv, hill the f’olrmalot
`t' v.17 ll'lall'tlll‘lr‘l‘lli which lit
`mam itartured and LllEl't'll‘ltlled
`ov t'nnnl‘tev lVlo‘tietui [a Utmul’
`Murmur V]
`is 3' Llclfll71Ulll'Lr
`plv’tlt‘v’trrri with freely available
`LLtllth—ll'éiill‘cll1lilldthl5 Uf-rtri
`lllrftl‘llllUCltllC lllc‘ll“ ‘JWll tangents
`tum-vi on l‘ittltllSl‘Wll retort; ‘lt'
`by ticllat‘votal mg thlt George.
`thumbs and rt
`' orother
`per}
`lHrliiltvlngy lll‘l/[
`
`Fragment templates
`A lmflliir‘lll
`lllalaw l;« plepntect
`luv randomly;
`ring genomic
`
`UNA ll’llt‘t ,mnll 2‘ .
`t'l < l
`l-zl‘i.
`
`and require. lair f‘l‘lA l'lliitl
`Would he needed tor a
` (lair ultra-try.
`
`
`
`32] lANL‘ARY 2010 l \IOLL‘ME l]
`
`@2010 Macmillan Publishers Limited.All rights reserved
`
`www.nature.camlre’8‘16%s§§]/geneti cs
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`
`a Roche/454. Life/APE. Polonator
`Emulsion PCR
`One DNA molecule per bead Clonal ainpliiiation to thousands of copies occurs in ITIicroreactors in an emulsion
`100—200 million beads
`
`j
`
`'
`
`.
`
`'
`
`
`.
`.
`
`template,
`Primei
`dNTFs and polymerase
`
`‘
`
`PCR
`amplification
`
`Breal
`emulmon
`
`Template
`dissocvatlon
`
`
`‘
`E
`i .
`i
`
`‘
`
`.
`
`’
`
`.
`
`'
`
`#5plate
`
`Chemically cross»
`linked to a glass slide
`
`5 lllumina/Solexa
`Solid-phase amplification
`One DNA molecule pei cluster
`
`
`Sample preparation
`
`DNA (5 pg)
`
`
`
`
`Template
`dNTPs
`and
`polymerase
`
`
`
`c Helicos BioSciences: one-pass sequencing
`Single molecule: primer immobilized
`
`Bridge amplification
`
`Billions of primed, single-molecule templates
`
`1! Helicos BioSciences: two-pass sequencing
`Single molecule: template immobilized
`
`a Pacific Biosciences Life/Visigen. Ll-COR Biosciences
`Single molecule. polymerase immobilized
`.
`
`15—
`
`I
`
`i
`
`.Au._ya
`
`.—,
`
`
` ———v
`ill.
`
`lei-"ll“IJl like
`
`.
`
`i
`
`A
`
`Billions of primed, singlemolecule templates
`
`Thousands of primed, Single—molecule templates
`
`Figure 1 | Template immobilization strategies. In emulsion PCR (emF’CR) (a). a reaction mixture consisting of
`an oil—aqUEous emulsion is created to encapsulate bead-DNA complexes into single aqueous droplets. PCR
`amplification is performed within these droplets to create beads containing several thousand copies of the same
`template sequence. EmPCR beads can be chemically attached to a glass slide or deposited into PicoTiterPlate
`wells iii: Se'i. Solid-phase amplification (b) is composed of two basic steps: initial priming and extending of the
`single—stranded. single—molecule template, and bridge amplification ofthe immobilized template with immediately
`adjacent primers to form clusters. Three approaches are shown for lmrnobilizlng single-molecule templates to a solid
`support: immobilization by a primerlc); immobilization by atemplate (d); and immobilization of a polymerase is),
`d NTP. 2'—deo,xyribonu cleoside triphosphate.
`
`Mate'pair templates
`A genomic Home: l: prepares
`ivy ‘lii’Liii‘ii‘lEiiig sheared DNA
`Ilml has i)
`’
`JEN/ill :-
`
`i‘iri‘r‘u-‘i‘: liiiiitli‘iti ili'r "Win
`‘l‘m My” *"':‘“"‘f"‘*f‘l’ ”MW“
`
`1:11“(W‘i‘lwiyn‘j:If‘1‘; lil:
`: 5,
`"m War WA WWW“
`.‘i'mii‘; maim loinniciiex
`
`Sequencing and imaging
`are attached to the solid support” to which a primed
`template molecule is bound [th iai. This approach15 There are fundamental differences in sequencing
`used by Pacific Biosciences’5 and15 described111 patents
`clonally amplified and singleinolecule templates. Clonal
`from Life/VisiGen‘" and Ll-C-OR Biosciences”. Larger
`amplification results in a population of identical lem-
`DNA molecules (up to tens oflhousands ofbase pairs)
`plates, each of which has undergone the sequencing
`can be used with this technique and, unlike the first two
`reaction. Upon imaging, the observed signal is a con-
`approaches, the third approach can be used with real-time
`sensus ofthe nucleotides or probes added to the iden-
`methods, resulting in potentially longer rcadlengths
`tical templates for a given cycle. This places a greater
`
`
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`
`demand on the efficiency of the addition process, and
`incomplete extension of the template ensemble results
`in lagging-strand dephasing. The addition of multiple
`nucleotides or probes can also occur in a given cycle.
`resulting in leading-strand dephasing. Signal dephas-
`ing increases fluorescence noise, causing base-calling
`errors and shorter reads”. Because dephasing is not an
`issue with single-molecule templates, the requirement
`for cycle efficiency is relaxed Single molecules. ho wever.
`are susceptible to multiple nucleotide or probe additions
`in any given cycle. Here, deletion errors will occur owing
`to quenching effects between adjacent dye molecules or
`no signal will be detected because of the incorporation
`ofdaik nucleotides or probes. In the following sections,
`sequencing and imagingstrategies that use both clonally
`amplified and singlevmolecule templates are discussed.
`
`Cyclic reversible termination. As the name implies, CRT
`uses reversible terminators in a cyclic method that com»
`prises nucleotide incorporation, fluorescence imaging
`and cleavagel. in the first step, a DNA polymerase,bound
`to the primed template, adds or incorporates just one tlu-
`orescently modified nucleotide [BOX 1 ;, which represents
`the complement of the template base. The termination of
`DNA synthesis after the addition ofa single nucleotide is
`an important feature of CRT. Following incorporation,
`the remaining unincorporated nucleotides are washed
`away. Imagingis then performed to determine the idenv
`tity of the incorporated nucleotide. This is followed by
`a cleavage step, which removes the terminating/inhibit-
`ing group and the fluorescent dye. Additional washing
`is performed before starting the next incorporation step.
`t
`it? is depicts a four~colour CRT cycle used by [llumina/
`Solexa, and MC 2:; illustrates a one-colour CRT cycle
`used by Ilelicos BioSciences.
`The key to the CRT method is the reversible ter-
`minator, of which there are two types: 3’ blocked and
`3’ unblocked [BOX t
`l- The use ofa dideoxynucleotide.
`which acts as a chain terminator in Sanger sequenc-
`ing, provided the basis for the initial development
`of reversible blocking groups attached to the 3’ end of
`iiucleotides'“'3". Blocking groups, such as 3'—O-allyl—
`2'»deoxyribonucleoside triphosphates (dN'l‘Ps)-" and
`3’vO—azidomethyLdNTPs’l, have been successfully used
`in CRT. 3’—blocked terminators require the cleavage of
`two chemical bonds to remove the lluorophore from the
`nucleobase and restore the 3’-Oll group.
`Currently, the lllumina/Solexa Genome Analyzer
`((3A)-'3 dominates the N68 market. It uses the clonally
`amplified template method illustrated in ac t o, coupled
`with the four—colour CRT method illustrated in Fla: is.
`
`The four colours are detected by total internal reflection
`fluorescence (TIRF) imaging using two lasers, the output
`of which is depicted in an 11:. The slide is partitioned
`into eight channels, which allows independent sam-
`ples to be run simultaneously. malt l shows the cur»
`rent sequencing statistics of the lllumina/Solexa GAu
`platform operating at the Baylor College of Medicine
`lluman Genome Sequencing Center (BCM-HGSC;
`l). Muzny, personal communication). Substitutions are
`the most common error type, with a higher portion of
`
`errors occurring when the previous incorporated
`nucleotide is a ‘G’ base“. Genome analysis ofIllumina/
`Solexa data has revealed an underrepresentation of
`AT<rich-’““ and GC-rich regionsl‘r’“, which is probably
`due to amplification bias during template preparation”.
`Sequence variants are called by aligning reads to a refer-
`ence genome using bioinformatics tools such as M AQl7
`or ELANIP‘. Bentley and colleagues reported high con-
`cordance (>99,S%) ofsingle—nucleotide variant (SNV)m
`calls with standard genotyping arrays using both align-
`ment tools, and a false-positive rate of 2.5% with novel
`SN Vs”. Other reports have described a higher false—
`positive rate associated with novel SNV detection usingthese
`alignment toolsl‘u".
`The difficulty involved in identifying a modified
`enzyme that efficiently incorporates 3'~blocked termi-
`nators — a process that entails screening large libraries
`of mutant DNA polymerases — has spurred the develop—
`ment of3’—unblocked reversible terminators. LaserGen,
`
`Inc. was the first group to show that a small terminating
`group attached to the base ofa 3’-unblocked nucleotide
`can act as an effective reversible terminator and be effi-
`
`ciently incorporated by wild<type DNA polymerases“.
`This led to the development of Lightning Terminators”
`iitox l
`l. Helicos BioSciences has reported the develop-
`ment of Virtual Terminators. which are 3’-unblockcd
`
`terminators with a second nucleoside analogue that
`acts as an inhibitor“. The challenge for 3’—unblockud
`terminators is creating the appropriate modifications
`to the terminating (Lightning Terminators)“ or inhib—
`iting (Virtual 'l‘emiinators)” groups so that DNA syn-
`thesis is terminated after a single base addition. This
`is important because an unblocked 3'—Oll group is the
`natural substrate for incorporating the next incoming
`nucleotide. Cleavage of only a single bond is required
`to remove both the terminating or inhibiting group and
`the fluorophore group from the nucleobase, which is a
`more efficient strategy than 3’-blocked terminators for
`restoring the nucleotide for the next CRT cycle.
`Helicos BioSciencc-s was the first group to commer-
`cialize a single~molecu1c sequencer. the HeliScope, which
`was based on the work of Quake and colleagues“. The
`lleliScope uses the single~molecu1e template methods
`shown in in:
`l r and HG.
`'
`tr coupled with the one—colour
`(CyS dye) CRT method shown in lilo, 2:. incorporation
`ofa nucleotide results in a fluorescent signal. The
`HeliScope also uses TIRF to image the CyS dye“, the
`imaging output ofwhich is shown in —lC Ed. Harris and
`colleaguesH used CyS- ilss—dNTPs. which are earlier ver-
`sions of their Virtual Terminators that ladc the inhibiting
`group, and reported that deletion errors in homopoly-
`meric repeat reg'ons were the most common error type
`(~S% frequency) when using the primerhnmobilized
`strategy shown in HE.
`is. This is likely to be related
`to the incorporation of two or more CyS—llss'dNTl’s
`in a given cycle. These errors can be greatly reduced
`with two-pass sequencing, which provides ~25vbase
`consensus reads using the template-immobilized strat~
`egy shown in l' C.
`i it. At the 2009 Advances in Genome
`Biology and Technology (AGBT) meeting, the HelicOs
`group reported their recent progress in sequencing the
`
`Dephasing
`Thu.
`l?\'.t‘.tlt1,Wllll Ltlt—ll'Wlnt
`attitit ion methods when
`étfiWlllgl‘it‘llllt‘f’wlYlt'th till :il
`«Vllci'ltlnltllv lot any given
`ry: ye, Lagging stl’alii‘l‘s {for
`
`:Afllltlllliilli l
`llulil llll‘
`:‘XQL‘tltid cyclel l“\3‘.iblll from
`incomplete e (tension, and
`leaning strands [tot example
`It
`7
`l
`‘ result from the audit tor
`ul‘ multiple ntitleotirlcs w
`probes in a Dotttllailcn at
`identical template;
`
`Dark nucleotides or probes
`A m :lL‘D‘l ll'fll.‘ tll' portly that
`lion 'nt mnlnln a lititiietzeni
`label
`lt can be generated (mm
`H
`rim/nee amt carry ovei
`tum the pun/toll: cycle tJl
`livr‘lrr‘vlysntl ill nil/l lmm it:
`dl‘yd'lctbellflllittlllllrfifllzill lll
`the sLllltJl‘ll c‘yt‘ltu
`
`lit:
`
`Total internal reflection
`fluorescence
`Attila interns lFllL‘L'll ”l
`littovemgaum it naginp i'lEl ics
`tent
`provinces an eva-
`
`wate e that
`iii. Wield
`
`nation wot/e with
`not ltl'lEfi‘V e
`.tll ult'
` ity that tlnr‘teasEfw
`t‘,\.l ‘iienliolly away irrim l'l'te
`jtil’th’t‘ This wave propagate};
`mifi‘m a boundary ,‘rtilli'ltfti
`sllt l" a: a glass altos res tiling
` in 'llE excnation oltttio ascent
`
`KEEN l<2tltt rim] or
`
`
`action at they
`
`:llllrfl 'Jll
` try a detectoii
`Libraries of mutant DNA
`polymerases
`tang: numbers [tiger-enmity
`#el‘etl DNA pelt/tn: ..
`
`
`ealed by Littliigr
`tat
`l.
`‘Zwllmllt‘p
`ted in" tantll‘ltn
`
`lllttlagaflfiblii which leads
`to one at more Htl‘lll’tfi acid
`atlltttllltllltjllb. ltlE-t‘l’llctllb and/ct
`deletions in the polymerase
`The: goal ot this approach is
`to llltltll’l’tiilfilr n‘itiditied
`ittltsleui ides more ulticieiitly
`litiniigtile sentiet‘it'lilg re’it'tl‘?"
`
`Consensus reads
`lites: we t,\l’tlyll1-»Hllll lair
`silli'llE iiitiletltile lecliiiitluos and
`5th prt'titlilt'ed l‘ly setitienring
`lillr‘ mint: liillltllriiti molecule
`mull: than once, The data are
`ll'lt‘ll tillgnwtl tit tittu'ltll‘c Pi
` '
`~c‘l‘5Llelf‘clILl‘
`lEtllltfl‘lLJ
` instir errors that may
`t’ilILlll‘l'
`ill a £1th ll
`:«L‘tjlllt‘llr‘tl it‘élil.
`
`
`
`34] lANt‘ARY 2010 l \IOLL'ME l1
`
`@2010 Macmillan Publishers Limited.All rights reserved
`
`www.notura.camlrefi‘/6§§§lgenetics
`
`FM1616-00510670
`
`A0533
`
`

`

`Case 1:20-cv-01580-LPS Document 39-4 Filed 03/05/21 Page 6 of 101 PageID #: 8037
`Case 1:20-cv-01580-LPS Document 39-4 Filed 03/05/21 Page 6 of 101 Pagfiev FQWS
`
`r
`
`Box 1 | Modified nucleotides used in next-generation sequencing methods
`
`
`
`a 3'-blocked reversible terminators
`
`|
`
`Fl
`
`l’
`
`i0
`
`U
`
`I: 3'-unblocked reversibleterminators
`
`LiOl
`
`PI
`
`U
`N
`
`® 0
`llN
`
`Hm
`
`i
`
`l
`
`Illumina/Soiexa
`\NH'
`ti
`
`\
`/
`..
`
`Lightning Terminator
`(LaserGen. inc)
`
`i)
`
`MN
`A l
`
`ill ixl‘lga‘ "I l\ i")
`/ \
`.' \
`r \
`n ‘0 (‘l \0 fl \0
`
`0
`
`i
`OJ!
`
`.
`
`i
`
`0
`jl J]
`
`l
`
`l
`
`‘
`
`'
`
`_
`-
`‘
`Ls. Ill
`/l
`’~
`T l
`
`0
`
`«it
`
`_
`
`'
`
`JYH
`, Nu
`
`i.)
`
`.
`
`.
`Iu at oi.
`
`V
`
`/
`
`i“
`
`iIN
`
`l
`
`inf/kn
`
`i I.‘ , _
`
`N;
`
`‘
`
`;
`‘
`
`HH
`A l
`N’
`
`l i
`
`l
`
`I
`
`
`
`
`
`Hik I’li\ ["1 ‘I In
`\
`\
`r
`HI \O u i/ \I I (i
`\t‘)
`
`H
`
`-"
`
`Hi I\ P/H\fi"”\‘1-"L‘
`‘1 \\
`, \\
`, \\
`V) O 0 H U i‘)
`
`DAN
`
`u
`
`on
`
`g
`g
`.
`“We” Terminator
`(Hakim Biobqences)
`
`4+
`I
`«g:
`l
`_ u
`
`l‘
`
`.7
`i
`in i
`
`7
`
`l
`
`‘
`
`'
`
`,
`
`r,
`
`- '
`c Real time "I‘d“"des
`
`’
`
`i
`
`‘
`
`im
`A l
`N
`
`n
`
`l
`
`l—ile/ViSIGen
`
`At the core of most
`next-generation sequencing
`(NGS) methods is the use of
`dye~labelled modified nucleotides. Ideally these nucleotides are
`incorporated specifically. cleaved efficiently during or following
`fluorescence imaging, and extended as modified or natural bases
`in ensuing cycles. In the figure, red chemical structures denote
`terminating functional groups. except in the Helicos BioSciences
`structure, which is characterized by an inhibitory function“
`Arrows indicate the site of cleavage separating the fluorophore
`.
`.
`from the nucleotide. and the blue chemical structures denote
`residual linker structures or molecularscars That are attached to
`the base and accumulate with subsequent cycles. DNA synthesis
`Is terminated by reversible terminators following the
`incorporationolonemodifiednucleotidebyDNApolymerase. ./l‘l\[1AKVU‘VJ
`
`H
`
`7‘
`
`HN'
`A i
`N
`
`n
`
`0
`
`DH
`
`LI-COR Bioscienms
`
`”-
`
`OH
`
`o
`
`0
`
`0
`
`HUD"
`
`Nil
`
`Fgwothgllnked nucleotides
`ac: K iOSCiefices)
`1
`>
`(U; ,0
`u .7 7k ’0 ’t ._
`U
`, g), ("g
`, "Kr ,r'\\
`7 {Pg 7 W“ 7
`” ‘
`'
`' ” ‘
`U H " ‘
`’
`‘
`'
`U
`
`0
`
`'N
`
`II\
`)\
`
`n
`
`i
`
`a
`0H
`
`,0/ \lw ‘/ \in ‘l \}i
`
`H
`
`n
`
`Two types of reversible terminators have been described:
`3’-blocked terminators. which contain a cleavable group
`attached to the 3’voxygen ofthe 2’-deoxyribose sugar, and
`3’-unblocl<ed terminatorst
`Several blocking groups have been described
`(see the figure. part 23). including 3'«O~allyl"’-""m
`L}
`5
`(Ju8 colleagues.who exclusivelylicensedtheir w illHAL
`,p\\
`,v\\
`technology lo Intelligent Bio-Systems) and 3'-O-
`3 SA, \S/\/ 1““
`azidomethylWH‘”(lllumina/Solexa).Theblocking
`7
`'
`"’ ""“ ""‘ ‘7‘
`group attached to the 3' end causes a bias against
`incorporation with DNA polymerase. Mtrtagenesis of DNA polymerase is required to lacilitate
`the incorporation of 3’-blocked terminators.
`3'—unblocked reversible terminators (part b) show more favourable enzymatic incorporation
`and. in some cases. can be Incorporated as well as a natural nucleotide using wild~type DNA
`polymerases“. Other groups. including Church and colleagues'°" and Tur catti and colleagues'°'.
`have described 3'vunbloclted terminators that rely on steric hindrance of the bulky dye group to
`inhibit incorporation afterthe addition of the first nucleotide.
`With real-time nucleotides (part c). the fluorophore is attached to the terminal phosphate
`group (Life/VisiCien” and Pacific Biosciences") rather than the nucleobase, which also reduces
`bias against incorporation with DNA polymerase. In additionto labelling the terminal phosphate
`group. LI~COR Biosciences' nucleotides attach a quencher moleculetothe base“. Gamma
`labelled 2'-deoxyribonucleoside triphosphates (dNTPs) were first described in 1979 by Yarbrough
`etali'“. and more recently. Kumar etal. described theirten‘ninally labelled polyphosphate
`nucleotides“".With the exception of Ll-COR Biosciences' nucleotides.whichleave the quencher
`group attached. natural bases are incorporated into the growing primer strand.
`
`
`NA‘I'UREREV‘IEWS GENETOCS
`VOLUME. ll AN M Y 2010 35
`|
`I l
`hog?“
`I
`
`@2010 Macmillan Publishers Limited.All rights reserved
`
`FM1616-0051067‘l
`
`A0534
`
`

`

`Case 1:20-cv-01580-LPS Document 39-4 Filed 03/05/21 Page 7 of 101 PageID #: 8038
`R gfifFElW§v-01580-LPS Document 39-4 Filed 03/05/21 Page 7 of 101 PagelD #: 8038
`
`a lllumina/Solexa — Reversible terminators
`
`c Helicos BioSciences — Reversible terminators
`
`63
`
`incorporate
`all four
`nucleotides.
`each label
`With a
`.‘lii‘ierent dye
`
`Wash Four-
`colour imaging
`
`SW
`
`Each Cid?»
`
`
`add a
`different
`"
`
`dye-labelled
`clNTP
`
`Incorporate
`single.
`dye-labelled
`nucleotide:
`
`Wash, one
`
`colour imaging
`
`
`Cleave dye
`and Terminating
`groups. wash
`
`Cleave dye
`and inhibiting
`groups. cap.
`
`wash
` Top' CATCGT
`Bottom: CCCCCC Top CTAGTG
`
`§~’
`
`Re mat cycles
`
`~~’
`
`Bottom CAGCTA
`
`Figure 2| Four-colour and one-colour cyclic reversible termination methods. a l The four-colour cyclic reversible
`termination (CRT) method uses Illumina/Solexa's 3'-Orazidomethyl reversible terminator chemistry” ”" il%t"!)(
`l
`i using
`solid-phaseamplified template clusters (Fit:
`in. shown as single templates for illustrative purposes). Following
`imaging. a cleavage step removes the fluorescent dyes and regenerates the 3’-OH group using the reducing agent
`tris(2-carboxyethyl)phosp hine (TCEPV’. b |The four-colour images highlightthe sequencing data from two clonally
`amplified templates. c| Unlike Illumina/Solexa's terminators. the Helicos Virtual Terminators‘J are labelled with the
`same dye and dispensed individually in a predetermined order. analogous to a sing|e~nucleotide addition method.
`Following total internal reflection fluorescence imaging. a cleavage step removes the fluorescent dye and inhibitory
`groups using TCEP to permit the addition of the next CyS-2'-deoxyribonucleoside triphosphate (d NTP) analogue. The
`free sulphhydryl groups are then capped with iodoacetamicle before the next nucleotide addition” (step not shown).
`
`C! | The one»colour images highlight the sequencing data from two single-molecule templates.
`
`One-base—encoded probe
`Ant
`l ,nnucleriiirle sequence ir
`
`7
`
`
`
`
`
`Fl VIE li.‘
`gramme A in the first
`twwtwn i’t‘l‘”c‘:ljii_iiid: in a
`the An exnnprt gt :1 on» hast:
`
`rlwgmerjte probe ..
`l cum ‘. Willtll lilill :. w:
`ham llwlll‘gl tuitltmliil, lb lllL‘
`inn rueollnri iti‘wi‘:
`lili‘
` re dining!
`i‘Jl
`.2 NH l
`i.- li'rl :rp-‘vsgihl:
`all ll‘
` liq”). w .
`in (“mil it'r.‘
`.
`
`Cavimrl'iabdilis elcgum genome. From a single HellScope
`run using only 7 ol'ihe instmment’s 50 channels, approx
`imately 2.8 (lb ofhigliquality data were generated in
`8 days from >2S~base consensus reads with 0,
`1 or 2
`errors. Greater than 99% coverage of the