USOO9738929B2
`
`(12) United States Patent
`US 9,738,929 B2
`(10) Patent N0.:
`Turner et al.
`
`(45) Date of Patent: *Aug. 22, 2017
`
`(54) NUCLEIC ACID SEQUENCE ANALYSIS
`
`USPC ........................................................... 435/61
`See application file for complete search history.
`
`(71) Applicant: Pacific Biosciences of California, Inc.,
`Menlo Park, CA (US)
`
`(56)
`
`References Clted
`
`US. PATENT DOCUMENTS
`
`(72)
`
`Inventors: Stephen Turner, Seattle, WA (US); Jon
`'
`Sorenson, Alameda, CA (US); Kenneth
`Mark Martha-In, Redwood .City, CA
`(US); John Eid, San Franc1sco, CA
`(US); Cheryl Heiner, La Honda, CA
`(US); Kevin Travers; Menlo Park, CA
`(US)
`
`_
`(73) Ass1gnee: Pacific Biosciences of California, Inc.,
`M61110 Park, CA (US)
`
`.
`.
`..
`( "‘ ) Notice.
`
`~
`.
`.
`.
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`.
`.
`.
`.
`.
`Th1s patent is subject to a terminal dis-
`Claimer.
`
`.
`(21) Appl' No" 15/383965
`,
`(22) Flledi
`
`(65)
`
`000- 19, 2016
`.
`,
`,
`Prior Publication Data
`
`Us 2017/0121764 A1
`
`May 4, 2017
`
`EP
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`
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`(Continued)
`
`
`
`
`FORilGN PAT7N1 DOCUMENTS
`1225234 B1
`11/2007
`1907573 B1
`1/2010
`
`Related US. Application Data
`
`(Continued)
`
`(63) Continuation of application No. 14/708,603, filed on
`May 11, 2015, now Pat. No. 9,556,480, which is a
`contmuatmn 0f application NO‘ 14/091796 17 filed. on
`NOV: 27: .2013: nowl’at. NO' 9,057,102, Wthh 15 a
`continuat1on of application No. 12/982,029, filed on
`Dec. 30, 2010, 110W Pat. No. 8,628,940, which is a
`continuation-in-part of application No. 12/413,226,
`11ch on Mar. 27, 2009, now Pat. No. 8,143,030.
`
`(60) Provisional application No. 61/099,696. filed on Sep.
`%45 2008’ pfOVlSlOllal application NO' 61/139,402,
`filed on Dec. 19, 2008.
`
`(51)
`
`Illt- CL
`(7121’ 19/34
`C12Q 1/68
`G01N 33/487
`
`(2006.01)
`(2006.01)
`(2006.01)
`
`(52) U'S' Cl'
`CPC ..... C12Q 1/6869 (2013.01); G01N33/48721
`(2013.01)
`
`(58) Field of Classification Search
`CPC ............ C12Q 1/6869; C12Q 2533/101; C12Q
`2537/149; C112Q 2565/6311; ClZQ
`2525/301; G01N 2021/7786; GOIN
`2021/6439; G01N 21/6428; GOIN
`21/6486; C12N 9/1252; G06F 19/22;
`Y10T 436/143333
`
`OTHER PUBLICATIONS
`Bashir, A. et al., “Evaluation of paired-end sequencing strategies for
`detection of genome rearrangements in cancer” Plos CompBiol
`(2003) 4(4);1_14.
`'
`
`(Continued)
`.
`.
`_
`_
`PWWJ’ Exam/W 2 Cynthla B Wlldér
`(74) Attorney, Agent, or Firm 7 Dav1d C. Scherer
`
`ABSTRACT
`(57)
`Methods, devices, and systems for performing intermittent
`.
`,
`,
`,
`,
`detect1on during analyt1cal reactions are prOVided. Such
`
`methods facilitate collection of reaction data from disparate
`t'
`t'
`F rtl
`1
`t1
`(1
`lef
`d
`.
`reac 1011 irnes. u 1er, sucime 10 sare use i orre ucmg
`photo—induced damage of one or more reactants in an
`
`illuminated analytical reaction at a given reaction time. In
`;
`.
`.
`.
`.
`.
`prelerred embodiments, the reaction mixture is subjected to
`at least one illuminated and non-illuminated period and
`allowed to proceed such that the time in which the reaction
`mixture is illuminated is less than a photo-induced damage
`threshold period.
`
`17 Claims, 26 Drawing Sheets
`
`Oxford, Exh. 1001, p. 1
`
`Oxford, Exh. 1001, p. 1
`
`

`

`US 9,738,929 B2
`
`Page 2
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`Oxford, Exh. 1001, p. 3
`
`Oxford, Exh. 1001, p. 3
`
`

`

`U.S. Patent
`
`Aug. 22, 2017
`
`Sheet 1 of 26
`
`US 9,738,929 B2
`
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`Oxford, Exh. 1001, p. 4
`
`Oxford, Exh. 1001, p. 4
`
`
`

`

`U.S. Patent
`
`Aug. 22, 2017
`
`Sheet 2 of 26
`
`US 9,738,929 B2
`
`Figure 28
`
`
`
`Reactionnumber
`
`Figure 2A
`Amwhmmw
`
`1s
`15 -
`14
`13
`12
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`
`Template nucieic acid sequence
`
`Oxford, EXh. 1001, p. 5
`
`Oxford, Exh. 1001, p. 5
`
`

`

`U.S. Patent
`
`Aug. 22, 2017
`
`Sheet 3 of 26
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`US 9,738,929 B2
`
`Figwe 3A
`
`310
`
`Figure 3B
`
`330
`
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`
`Figure BC
`
`380
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`
`Oxford, Exh. 1001, p. 6
`
`Oxford, Exh. 1001, p. 6
`
`

`

`U.S. Patent
`
`Aug. 22, 2017
`
`Sheet 4 of 26
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`US 9,738,929 B2
`
`Figure 4A
`
`
`
`Figure 4B
`
`
`
`Oxford, EXh. 1001, p. 7
`
`Oxford, Exh. 1001, p. 7
`
`

`

`U.S. Patent
`
`Aug. 22, 2017
`
`Sheet 5 of 26
`
`US 9,738,929 B2
`
`Figure 5A
`530
`
`520
`
`Figure 58
`
`550
`
`
`
`Figure 5D
`
`Oxford, Exh. 1001, p. 8
`
`Oxford, Exh. 1001, p. 8
`
`

`

`U.S. Patent
`
`Aug. 22, 2017
`
`Sheet 6 of 26
`
`US 9,738,929 B2
`
`
`
`
`
`
`
`Reversereference
`
`reference
`
`Forward
`
`pear peouenbas
`
`Figure6
`
`Oxford, Exh. 1001, p. 9
`
`Oxford, Exh. 1001, p. 9
`
`

`

`U.S. Patent
`
`Aug. 22, 2017
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`Sheet 7 of 26
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`US 9,738,929 B2
`
`Figure7
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`Oxford, Exh. 1001, p. 10
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`Oxford, Exh. 1001, p. 10
`
`

`

`U.S. Patent
`
`Aug. 22, 2017
`
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`Oxford, Exh. 1001, p. 11
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`

`

`U.S. Patent
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`Aug. 22, 2017
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`

`U.S. Patent
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`Aug. 22, 2017
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`

`

`U.S. Patent
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`Aug. 22, 2017
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`U.S. Patent
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`Aug. 22, 2017
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`

`

`U.S. Patent
`
`Aug. 22, 2017
`
`Sheet 13 of 26
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`

`

`U.S. Patent
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`Aug. 22, 2017
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`Aug. 22, 2017
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`Aug. 22, 2017
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`U.S. Patent
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`Aug. 22, 2017
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`Aug. 22, 2017
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`Aug. 22, 2017
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`U.S. Patent
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`Aug. 22, 2017
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`Aug. 22, 2017
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`

`U.S. Patent
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`Aug. 22, 2017
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`

`

`US 9,738,929 B2
`
`1
`NUCLEIC ACID SEQUENCE ANALYSIS
`
`
`
`CROSS—REFER 4 NC 2 TO R AI ATED
`
`APPLICATIONS
`
`
`
`This application is a continuation application of US.
`patent application Ser. No. 14/708,603, iled May 11, 2015,
`which is a continuation application of U.S. patent applica-
`tion Ser. No. 14/091,961, filed Nov. 27, 2013, now US. Pat.
`No. 9,057,102, which is a continuation application of US.
`patent application Ser. No. 12/982,029, filed Dec. 30, 2010,
`now US. Pat. No. 8,628,940, which (1) claims the benefit of
`US. Provisional Application No. 61/099,696, filed Sep. 24,
`2008; (2) claims the benefit of US. Provisional Application
`No. 61/139,402, filed Dec. 19, 2008; and (3) is a continu-
`ation-in-part application of US. patent application Ser. No.
`12/413,226, filed Mar. 27, 2009, now US. Pat. No. 8,143,
`030, the full disclosures of all of which are incorporated
`herein by reference in their entireties for all purposes.
`This application is also related to US. Provisional Appli-
`cation No. 61/072,160, filed Mar. 28, 2008, US. patent
`application Ser. No. 12/383,855, filed Mar. 27, 2009, now
`US. Pat. No. 8,236,499, and US. patent application Ser. No.
`12/413,258, filed Mar. 27, 2009, now US. Pat. No. 8,153,
`375, all of which are incorporated herein by reference in '
`their entireties for all purposes.
`STATEMENT REGARDING FEDERALLY
`SPONSORED RESEARCH
`
`10
`
`30
`
`Not Applicable.
`
`
`
`BACKGROUND OF THE INVENTION
`
`The use of optically detectable labeling groups, and
`particularly those groups having high quantum yields, e.g.,
`fluorescent or chemiluminescent groups,
`is ubiquitous
`throughout the fields of analytical chemistry, biochemistry,
`and biology. In particular, by providing a highly visible
`signal associated with a given reaction, one can better
`monitor that reaction as well as any potential effectors of that
`reaction. Such analyses are the basic tools of life science
`research in genomics, diagnostics, pharmaceutical research,
`and related fields.
`Such analyses have generally been performed under con-
`ditions where the amounts of reactants are present far in
`excess of what is required for the reaction in question. The
`result of this excess is to provide ample detectability, as well
`as to compensate for any damage caused by the detection
`system and allow for signal detection with minimal impact
`on the reactants. For example, analyses based on fluorescent
`labeling groups generally require the use of an excitation
`radiation source directed at the reaction mixture to excite the
`fluorescent labeling group, which is then separately detect-
`able. However, one drawback to the use of optically detect-
`able labeling groups is that prolonged exposure of chemical
`and biochemical reactants to such light sources, alone, or
`when in the presence of other components, e.g., the fluo-
`rescent groups, can damage such reactants. The traditional
`solution to this drawback is to have the reactants present so
`far in excess that the number of undamaged reactant mol-
`
`
`ecules far outnurnbers the damaged reactant molecules, thus
`
`
`minimizing or negating the e ects of the photo-induced
`damage.
`techniques currently being
`A variety of analytical
`explored deviate from the traditional techniques. In particu-
`lar, many reactions are based on increasingly smaller
`
`35
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`45
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`50
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`55
`
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`65
`
`
`
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`in microfluidic or nanofluidic
`amounts of reagents, e.g.,
`reaction vessels or channels, or in “single molecule” analy—
`ses. Such low reactant volumes are increasingly important in
`many high throughput applications, such as microarrays.
`The use of smaller reactant volumes oflers challenges to the
`use of optical detection systems. When smaller reactant
`volumes are used, damage to reactants, such as from expo—
`sure to light sources for fluorescent detection, can become
`problematic and have a dramatic impact on the operation of
`a given analysis. In other cases, other reaction conditions
`may impact the processivity, rate, fidelity, or duration of the
`reaction, including salt or buffer conditions, pH,
`tempera—
`
`ture, or even immobilization of reaction components within
`observable reaction regions. In many cases, the e ects of
`these dilferent reaction or environmental conditions can
`degrade the performance of the system over time. This can
`be particularly detrimental, for example, in real—time analy—
`sis of reactions that include fluorescent reagents
`iat can
`expose multiple different reactions components to optical
`energy. In addition, smaller reactant volumes can lead to
`limitations in the amount of signal generated upon applica—
`tion of optical energy.
`Further, in the case of sequencing-by-synthesis applica-
`tions, an additional challenge has been to develop ways to
`clfectively sequence noncontiguous portions of a template
`nucleic acid on a single molecule. This challenge is exac—
`erbated in template nucleic acids that contain highly repeti-
`tive sequence and/or are hundreds or thousands of nucleo-
`tides in length, such as certain genomic DNA fragments. The
`difficulty in generating such noncontiguous reads from a
`single template has hampered efforts to construct consensus
`sequences for long templates,
`for example,
`in genome
`sequencing projects.
`As such, methods and systems that result in enhanced
`reaction performance, such as an increase in processivity,
`rate, fidelity, or duration of a reaction of interest, would
`provide useful improvements to the methods and composi-
`tions currently available. For example, methods, devices,
`and systems that increase reaction performance by, e.g.,
`mitigating to some extent photo-induced damage in a reac-
`tion of interest and/or increasing various other performance
`metrics for the reaction would be particularly useful.
`
`BRIEF SUMMARY OF THE INVENTION
`
`In a general sense, the methods provided herein imple-
`ment
`intennittent detection of analytical reactions as a
`means to collect reliable data from times during the reaction
`that are less or not able to be analyzed if detection is constant
`throughout
`the reaction.
`In particular, certain detection
`methods can cause damage to reaction components, and
`such intermittent detection allows the damage to be avoided
`or at
`least delayed,
`thereby facilitating detection of the
`reaction at later stages. For example, if a detection method
`causes a reduction in processivity of a polymerase enzyme,
`then intermittent detection would allow data collection at
`noncontiguous regions of a template nucleic acid that extend
`farther from the initial binding site of the polymerase on the
`template than would be achievable under constant detection.
`Further, some detection methods have limits on how much
`data or for how long a time data may be generated in a single
`reaction, and intermittent detection of such a reaction can
`allow this data to be collected from various stages of a
`reaction, thereby increasing the flexibility of the investigator
`to spread out the data collection over multiple stages of a
`reaction. In certain aspects, the present invention is particu-
`larly suitable to characterization of analytical reactions in
`
`Oxford, EXh. 1001, p. 30
`
`Oxford, Exh. 1001, p. 30
`
`

`

`US 9,738,929 B2
`
`3
`real time, that is, during the course of the reaction. In certain
`aspects,
`the present
`invention is particularly suitable to
`characterization of single molecules or molecular complexes
`monitored in analytical
`reactions,
`for example,
`single
`enzymes, nucleotides, polynucleotides,
`and complexes
`thereof.
`the present invention is directed to
`In certain aspects,
`methods, devices, and systems for obtaining sequence data
`from discontiguous portions of single nucleic acid tem-
`plates. The methods generally comprise providing a moni—
`torable sequencing reaction comprising a polymerase, tem—
`plate, and primer sequence, as well as the various types of
`nucleotides or nucleotide analogs that are to be incorporated
`by the polymerase enzyme in the template-directed primer
`extension reaction. Typically, at least one or more or all of
`the nucleotides or nucleotide analogs are embodied with a
`detectable property that permits their identification upon or
`following incorporation. In the context of the present inven-
`tion,
`the sequence data for a first portion of a template
`nucleic acid is acquired during a first stage of the reaction
`under a first set of reaction conditions that includes at least
`one reaction condition that results in degraded performance
`of the reaction, but that may contribute to the detectability
`of the nucleotides being incorporated. During a second stage
`of the reaction. the degradative influence is eliminated or
`reduced, which may result in an inability or a reduced ability
`to obtain sequence data from a second portion of the
`template nucleic acid, but where the second portion of the
`template nucleic acid is contiguous with the first portion.
`Subsequently, the reaction condition resulting in degraded
`performance is reinstated and sequence data is obtained for
`a third portion of the template nucleic acid during a third
`stage of the reaction, but where the third portion of the
`sequence is not contiguous with the first portion of the
`sequence, but is contiguous with the second portion. The
`elimination or reduction of the degradative influence during
`the second stage of the reaction may be accomplished by
`changing or shortening one or more reaction conditions
`underlying degradative reaction performance,
`e.g., by
`changing one or more reaction conditions (e.g., temperature,
`pH, exposure to radiation, physical manipulation, etc.), and
`in particular may involve altering a reaction condition
`related to detection of one or more aspects or products of the
`reaction. However, in preferred embodiments, nucleotides
`or nucleotide analogs having the detectable property are
`present
`in the reaction mixture during all stages of the
`reaction, including stages in which the degradative influence
`is eliminated or reduced; as such,
`the reaction condition
`changed in stage two of such an embodiment would not
`comprise removal or dilution of such detectable nucleotides
`or nucleotide analogs.
`invention is generally
`the present
`In certain aspects,
`directed to methods, devices, and systems for enhancing the
`performance of illuminated reactions. The term “illuminated
`reactions” as used herein refers to reactions which are
`exposed to an optical energy source. In certain preferred
`embodiments, illuminated reactions comprise one or more
`fluorescent or fluorogenic reactants. Typically, such illumi-
`nation is provided in order to observe the generation and/or
`consumption of reactants or products that possess a particu—
`lar optical characteristic indicative of their presence, such as
`a shift in the absorbance spectrum and/or emission spectrum
`of the reaction mixture or its components. In some aspects,
`enhancing the performance of an illuminated reaction means
`increasing the processivity, rate, fidelity, and/or duration of
`the reaction. For example, enhancing the performance of an
`illuminated reaction can involve reducing or limiting the
`
`5
`
`10
`
`15
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`20
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`25
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`30
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`55
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`eflects of photo—induced damage during the reaction. The
`term “photo—induced damage” refers generally to any direct
`or indirect impact of illumination on one or more reagents in
`a reaction resulting in a negative impact upon that reaction.
`In certain aspects, methods of the invention useful for
`characterizing an analytical reaction comprise preparing a
`reaction mixture and initiating the analytical
`reaction
`therein, subjecting the reaction mixture to at
`least one
`detection period and at least one non-detection period during
`the course of the analytical reaction, collecting data during
`both the detection period(s) and the