UNITED STATES PATENT AND TRADEMARK OFFICE
`___________________
`
`BEFORE THE PATENT TRIAL AND APPEAL BOARD
`___________________
`
`TWINSTRAND BIOSCIENCES, INC.
`Petitioner,
`v.
`GUARDANT HEALTH, INC.
`Patent Owner.
`
`___________________
`
`No. IPR2022-01400
`Patent No. 11,149,306
`___________________
`
`REPLY DECLARATION OF RAHUL SATIJA, D.PHIL IN SUPPORT OF
`PETITIONER’S REPLY
`
`Mail Stop "PATENT BOARD"
`Patent Trial and Appeal Board
`U.S. Patent and Trademark Office
`P.O. Box 1450
`Alexandria, VA 22313-1450
`
`TwinStrand EX1098
`TwinStrand Biosciences v. Guardant Health
`IPR2022-01400
`
`

`

`Case IPR2022-01400
`Reply Declaration of Rahul Satija, D.Phil
`
`
`TABLE OF CONTENTS
`
`B.
`C.
`
`D.
`
`
`I.
`INTRODUCTION ....................................................................................... 1
`II. MY BACKGROUND AND QUALIFICATIONS ....................................... 2
`III. LIST OF DOCUMENTS CONSIDERED .................................................... 4
`IV. PERSON OF ORDINARY SKILL IN THE ART ........................................ 7
`V.
`STATE OF THE ART ................................................................................. 8
`VI. LEGAL BASIS FOR MY ANALYSIS ........................................................ 8
`A. Obviousness ....................................................................................... 8
`VII. SCHMITT AND NARAYAN TEACH ALL OF THE ELEMENTS
`OF CLAIMS 1-3, 5, 7, 9-14, 17-27, AND 29 ............................................. 10
`A.
`Schmitt teaches “mapping at least a subset of the plurality of
`sequence reads to the reference sequence” as recited in step (e)
`of claim 1. .........................................................................................10
`Schmitt teaches tagging cfDNA molecules as recited in claim 1. ......16
`The ’306 patent claims encompass Schmitt’s hybrid
`embodiment. .....................................................................................18
`Schmitt teaches sorting sequence reads into families of paired
`and unpaired reads as recited in claims 17-28 and 29. .......................20
`VIII. A POSA WOULD HAVE BEEN MOTIVATED TO APPLY
`SCHMITT’S ERROR CORRECTION METHODS TO CFDNA .............. 24
`A.
`Schmitt’s methods demonstrate sensitivity in detecting
`mutations. .........................................................................................24
`The ’306 patent claims do not require detection of cancer
`mutations in a clinical setting. ...........................................................28
`Schmitt’s 3-mer hybrid SMI embodiment would not have been
`more error-prone compared to the 12-mer exogenous SMI. ..............29
`Contrary to Dr. Quackenbush’s opinions, Dr. Spellman
`identifies multiple reasons to select Schmitt’s 3-mer hybrid
`approach. ..........................................................................................33
`
`B.
`
`C.
`
`D.
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`Case IPR2022-01400
`Reply Declaration of Rahul Satija, D.Phil
`E.
`Kukita is relevant prior art. ...............................................................35
`IX. DR. SPELLMAN ESTABLISHED THAT ALL DEPENDENT
`CLAIMS OF THE ’306 PATENT WOULD HAVE BEEN OBVIOUS ..... 36
`A.
`Claim 3. ............................................................................................36
`B.
`Claim 5. ............................................................................................37
`C.
`Claim 7. ............................................................................................40
`D.
`Claim 14. ..........................................................................................41
`E.
`Claims 4 and 6. .................................................................................42
`F.
`Claim 8. ............................................................................................44
`G.
`Claims 15, 16, and 28. ......................................................................46
`
`
`
`
`
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`

` Case IPR2022-01400
` Reply Declaration of Rahul Satija, D.Phil
`
`
`I, Rahul Satija, hereby declare as follows.
`
`I.
`
`INTRODUCTION
`I have been retained on behalf of Twinstrand Biosciences, Inc.
`1.
`
`(“Twinstrand” or “Patent Owner”) for the above-captioned inter partes review
`
`proceeding to provide my expert opinions and expert knowledge. I understand that
`
`this proceeding involves U.S. Patent No. 11,149,306 (“the ’306 patent”) titled
`
`“Methods and Systems for Detecting Genetic Variants,” and that the ’306 patent is
`
`currently assigned to Guardant Health, Inc. (“Guardant” or “Patent Owner”).
`
`2.
`
`I understand that in response to a Petition submitted by Twinstrand, an
`
`inter partes review of claims 1-29 of the ’306 patent was instituted by the Patent
`
`Trial and Appeal Board (“the Board”) on February 8, 2023.
`
`3.
`
`In preparing this declaration, I have reviewed Dr. Spellman’s
`
`declaration (EX1002) and am familiar with all the documents cited therein. I also
`
`agree with Dr. Spellman’s opinions. I have also reviewed and am familiar with all
`
`of the documents cited herein. I confirm that to the best of my knowledge the
`
`accompanying exhibits are true and accurate copies of what they purport to be, and
`
`that an expert in the field would reasonably rely on them to formulate opinions
`
`such as those set forth in this declaration.
`
`4.
`
`I am being compensated at my customary rate of $750 per hour for
`
`my work on this case. My compensation is not dependent upon my opinions, my
`
`1
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` Case IPR2022-01400
` Reply Declaration of Rahul Satija, D.Phil
`
`
`testimony, or the outcome of this case.
`
`II. MY BACKGROUND AND QUALIFICATIONS
`I am a Core Faculty Member at the New York Genome Center, and an
`5.
`
`Associate Professor with tenure at the Center for Genomics and Systems Biology
`
`at New York University. I also hold an appointment as an Associate Faculty
`
`Member at the Institute for Systems Genetics at NYU Langone Medical Center.
`
`6.
`
`I was a postdoctoral fellow at the Broad Institute of Harvard and MIT
`
`from 2011-2014. My research focused on applying sequencing technologies to
`
`understand the behavior of immune cells, and I published articles describing my
`
`results in the journal Nature. I also conducted research on DNA sequencing
`
`technologies as a training fellow at the Medical Research Council in the United
`
`Kingdom from 2010-2011.
`
`7.
`
`I received my D.Phil in Statistics at the University of Oxford in 2010.
`
`I attended Oxford University on a Rhodes Scholarship, and my graduate thesis
`
`focused on the statistical analysis of DNA sequencing data. I graduated summa
`
`cum laude from Duke University in 2006 with a Bachelor of Science in Biology,
`
`Mathematics, and Music.
`
`8.
`
`The primary area of my research is the development and use of
`
`genomic technologies, such as technologies that involve DNA sequencing, as well
`
`as the computational analysis of data related to these technologies.
`
`2
`
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`

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` Case IPR2022-01400
` Reply Declaration of Rahul Satija, D.Phil
`
`
`9.
`
`I have a particular familiarity and expertise with genomic
`
`technologies for the analysis of nucleic acids present in miniscule samples,
`
`including DNA and RNA molecules that are present in single-cells. This includes
`
`the developments in molecular biology and microfluidics that enable the analysis
`
`of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) from single cells, as
`
`well as technologies that evaluate the ability of thousands of different cells to
`
`produce specific protein molecules. My research is also focused on the
`
`development of statistical techniques involved in the analysis of this data.
`
`10.
`
`I have over 15 years of experience in the field of molecular biology. I
`
`have taught undergraduate and graduate level courses at NYU in the area of
`
`sequencing technologies, computational analysis of genome sequencing data, and
`
`genetics.
`
`11. My work has been published in more than 80 peer-reviewed scientific
`
`articles that have been cited more than 46,000 times. Since commencing my
`
`faculty appointment, I have presented more than 150 invited lectures, seminars, or
`
`workshops on my research. I have also received grant funding to continually
`
`support my research from multiple organizations including the National Institutes
`
`of Health, Chan Zuckerberg Initiative, Medical Research Council, and the Rhodes
`
`Trust.
`
`12.
`
`I have received multiple awards and honors, including the “New
`3
`
`
`

`

` Case IPR2022-01400
` Reply Declaration of Rahul Satija, D.Phil
`
`Innovator Award” from the National Institutes of Health, and a selection as a Kavli
`
`Fellow from the National Academy of Sciences. In 2019, I was selected as a highly
`
`cited researcher by the Institute for Scientific Information. In 2020, I was selected
`
`to lead a Center of Excellence in Genomic Science by the National Human
`
`Genome Research Institute.
`
`13. My Curriculum Vitae is attached to this declaration (EX1101), and
`
`contains additional details on my education, honors and awards, publications,
`
`professional activities, and qualifications to deliver an expert opinion in this
`
`matter.
`
`III. LIST OF DOCUMENTS CONSIDERED
`In formulating my opinions, I have reviewed all the references and
`14.
`
`documents cited herein, including those listed below.
`
`Exhibit # Description
`
`1001
`
`Talasaz, A. and Mortimer, S.A.W., “Methods And Systems For
`Detecting Genetic Variants,” U.S. Patent No. 11,149,306 (filed July
`31, 2020; issued October 19, 2021)
`1002
`Declaration of Paul T. Spellman, Ph.D.
`1005 Meyer, M., et al., “Parallel tagged sequencing on the 454 platform,”
`Nature Protocols 3(2): 267-278 (2008)
`Kivioja, T., et al., “Counting absolute numbers of molecules using
`unique molecular identifiers,” Nature Methods 9(1): 72-76 (2012)
`Craig, D.W., et al., “Identification Of Genetic Variants Using
`Barcoded Multiplexed Sequencing”, Nature Methods 5:887–893
`(2008)
`
`1006
`
`1007
`
`4
`
`
`

`

` Case IPR2022-01400
` Reply Declaration of Rahul Satija, D.Phil
`
`
`Exhibit # Description
`
`Kukita, Y., et al., “Quantitative Identification of Mutant Alleles
`Derived from Lung Cancer in Plasma Cell-Free DNA via Anomaly
`Detection Using Deep Sequencing Data,” PLOS One 8(11): 1-31
`(2013)
`Schmitt, M., et al., “Method of Lowering the Error Rate of Massively
`Parallel DNA Sequencing Using Duplex Consensus Sequencing,”
`International Publication Number WO2013/142389 (filed on March
`15, 2003; published on September 26, 2013)
`Heitzer, E., et al., “Tumor-associated copy number changes in the
`circulation of patients with prostate cancer identified through whole-
`genome sequencing ,” Genome Medicine 5:30-46 (2013)
`Li, H. and Durbin, R., “Fast and accurate short read alignment with
`Burrows–Wheeler Transform,” Bioinformatics 25(14): 1754–1760
`(2009)
`Fan, H.C., et al., “Analysis of the Size Distributions of Fetal and
`Maternal Cell-Free DNA by Paired-End Sequencing,” Clinical
`Chemistry 56(8): 1279–1286 (2010)
`Schmitt, M.W., et al., “Detection of ultra-rare mutations by
`next-generation sequencing,” PNAS 109(36): 14508–14513 (2012)
`Perakis, S., et al., “Chapter Three - Advances in Circulating Tumor
`DNA Analysis,” Advances in Clinical Chemistry 80: 73-157 (2017)
`Schmitt, M.W, et al., “Sequencing small genomic targets with high
`efficiency and extreme accuracy,” Nature Methods 12(5): 423-4226
`with Suppl. Material (2015)
`Narayan et al., “Ultrasensitive Measurement of Hotspot Mutations in
`Tumor DNA in Blood Using Error-Suppressed Multiplexed Deep
`Sequencing,” Cancer Research, 72(14):3492-3498 (2012)
`U.S. Provisional Application No. 61/625,623, filed on April 17, 2012
`
`Deposition Transcript of Dr. Ian Hagemann, Aug. 3, 2023
`
`Deposition Transcript of John Quackenbush, Ph.D., Aug. 15, 2023
`
`Curriculum Vitae of Rahul Satija, D.Phil
`
`1008
`
`1009
`
`1032
`
`1038
`
`1059
`
`1064
`
`1067
`
`1068
`
`1082
`
`1083
`
`1096
`
`1097
`
`1101
`
`5
`
`
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` Case IPR2022-01400
` Reply Declaration of Rahul Satija, D.Phil
`
`
`Exhibit # Description
`
`1103
`
`1104
`
`1105
`
`2003
`
`2004
`
`2013
`
`2015
`
`2016
`
`2017
`
`2018
`
`2019
`
`2020
`
`2021
`
`2022
`
`2023
`
`Kirsch, S., Klein, C. A., “Sequence Error Storms and the Landscape of
`Mutations in Cancer,” PNAS, 109(36):14289-14290 (Sept. 2012)
`“Quality Scores for Next-Generation Sequencing: Assessing
`Sequencing Accuracy Using Phred Quality Scoring,” Illimina,
`Technical Note (2011)
`Sah, S., et al., “Functional DNA Quanitifaciton Guides Accurate Next-
`Generation Sequencing Mutation Detection in Formalin-fixed,
`Paraggrin-embedded, Tumor Biopsies,” Genome Medicine, 5:77
`(2012)
`Kennedy et al., “Detecting ultralow-frequency mutations by
`Duplex Sequencing,” Nat. Protoc. 9(11) (2014)
`Kinde et al., “Detection and quantification of rare mutations
`with massively parallel sequencing,” PNAS 108(23) (2011)
`Stoler et al., “Streamlined analysis of duplex sequencing data with Du
`Novo,” Genome Biology 17, 180 (2016)
`Declaration of Dr. John Quackenbush
`
`Declaration of Dr. Ian Hagemann
`
`Deposition Transcript of Dr. Paul Spellman
`
`Deposition Transcript of Dr. Rahul Satija
`
`Zhu et al., “Sensitivity, Specificity, Accuracy, Associated
`Confidence Interval and ROC Analysis with Practical SAS®
`Implementations” (2010)
`Kircher et al., “Addressing challenges in the production and
`analysis of Illumina sequencing data” (2011)
`Illumina Technical Note: Sequencing “Using a PhiX Control
`for HiSeq® Sequencing Runs” (2013)
`Dr. John Quackenbush CV
`
`Dr. Ian Hagemann CV
`
`6
`
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` Case IPR2022-01400
` Reply Declaration of Rahul Satija, D.Phil
`
`
`Exhibit # Description
`
`2025
`
`2026
`
`QIAGEN Sample & Assay Technologies – “QIAamp DNA
`Blood Mini Kit” Third Edition (2012)
`QIAGEN Sample & Assay Technologies – “QIAamp DNA
`Micro Kit” Second Edition (2010)
`Thavendiranathan et al., “Do Blood Tests Cause Anemia in
`Hospitalized Patients?” Journal of General Internal Medicine
`(2004)
`Salisbury et al., “Diagnostic Blood Loss From Phlebotomy
`and Hospital-Acquired Anemia During Acute Myocardial
`Infarction” American Medical Association (2011)
`2031 M. Levi, “Twenty-five million liters of blood into the sewer”
`Journal of Thrombosis and Haemostasis (2014)
`
`2029
`
`2030
`
`
`
`IV. PERSON OF ORDINARY SKILL IN THE ART
`I understand that a person of ordinary skill in the art (“POSA”) is a
`15.
`
`hypothetical person who is presumed to be aware of all pertinent art, thinks along
`
`conventional wisdom in the art and is a person of ordinary creativity.
`
`16.
`
`I understand that Dr. Spellman provided a definition of a POSA in his
`
`declaration. EX1002, ¶¶27-30. I agree with his POSA definition. A POSA relevant
`
`to the ’306 patent would have had knowledge of the scientific literature concerning
`
`methods of DNA manipulation and analysis, including DNA sample preparation
`
`for sequencing, amplification, methods of DNA sequencing (including NGS and
`
`related sequencing methods), and bioinformatics methods for raw data analysis.
`
`17. A POSA may have also worked as part of a multidisciplinary team
`
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` Reply Declaration of Rahul Satija, D.Phil
`
`
`and drawn upon not only his or her own skills, but also taken advantage of certain
`
`specialized skills of others on the team, e.g., to solve a given problem.
`
`18. A POSA would have had (i) a Ph.D., in molecular biology, genetics,
`
`bioinformatics, or a related field, and have at least about two years of experience in
`
`the use and development of sequencing technologies; or (ii) a Master’s degree in
`
`one of the same fields with at least about five years of the same experience. I
`
`understand that Dr. Quackenbush has applied this same definition of a POSA in his
`
`analysis. EX2015, ¶30.
`
`V.
`
`STATE OF THE ART
` I have reviewed and agree with Dr. Spellman’s summary of the state
`19.
`
`of the art as it pertains to the ’306 patent. EX1002, ¶¶31-106.
`
`VI. LEGAL BASIS FOR MY ANALYSIS
`A. Obviousness
`I understand that obviousness is ultimately a question of law based on
`20.
`
`a determination of a number of factual issues. Those factual issues relate to: (1) the
`
`scope and content of the prior art, (2) the differences between the prior art and the
`
`claimed invention as a whole, (3) the level of ordinary skill in the art at the time
`
`the invention was made, and (4) objective secondary considerations that reflect, for
`
`example, the contemporaneous response to the invention at the time, such as
`
`commercial success, long-felt need, failure of others, industry-wide praise, and
`
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` Case IPR2022-01400
` Reply Declaration of Rahul Satija, D.Phil
`
`
`unexpected results.
`
`21.
`
`I understand that combining or modifying the teachings of the prior
`
`art to achieve the claimed invention is another way to establish obviousness. I
`
`understand that where there is a reason to modify or combine the prior art to
`
`achieve the claimed invention, there must also be a reasonable expectation of
`
`success in doing so. I understand that the reason to combine prior art references
`
`can come from a variety of sources, not just the prior art itself or the specific
`
`problem the patentee was trying to solve. I further understand that the references
`
`themselves need not provide a specific hint or suggestion of the alteration needed
`
`to arrive at the claimed invention; the analysis may include recourse to logic,
`
`judgment, and common sense available to a POSA that does not need to be explicit
`
`in any reference.
`
`22.
`
`I understand that obviousness typically exists when a range of values
`
`in a claimed composition overlaps with the ranges disclosed in the prior art. I
`
`further understand that obviousness exists even when there is a slight overlap in
`
`ranges. I also understand that the obviousness of a claimed range can be rebutted if
`
`there is a showing that the claimed range is critical and provides unexpectedly
`
`superior results over the prior art range.
`
`23.
`
`I further understand that a patent must contain a written description of
`
`the claimed invention. The written description must convey clearly to those skilled
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`
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`in the art, that, as of the filing date sought, the applicant was in possession of the
`
`invention claimed. This involves an objective inquiry into the four corners of the
`
`specification from the perspective of a POSA in the art.
`
`VII. SCHMITT AND NARAYAN TEACH ALL OF THE ELEMENTS OF
`CLAIMS 1-3, 5, 7, 9-14, 17-27, AND 29
`Schmitt teaches “mapping at least a subset of the plurality of
`A.
`sequence reads to the reference sequence” as recited in step (e) of
`claim 1.
`24. Dr. Quackenbush alleges that Dr. Spellman failed to establish that
`
`Schmitt teaches step (e) of claim 1 because Dr. Spellman’s “claim mapping
`
`confuses and conflates” the terms consensus sequences and sequence reads.
`
`EX2015, ¶¶67-68. I disagree. As discussed below, a POSA would understand that
`
`claim 1(e) encompasses mapping raw sequence reads and mapping consensus
`
`sequence reads, and Schmitt teaches doing both.
`
`25. Claim 1 requires “reducing or tracking redundancy of a plurality of
`
`sequence reads,” which a POSA would understand to mean generating a consensus
`
`sequence. In fact, the ’306 patent equates “reducing or tracking redundancy” with
`
`“determining consensus sequence reads”:
`
`“Methods disclosed herein can comprise determining consensus
`sequence reads in sequence reads (e.g., as shown in step (108), FIG. 1),
`such as by reducing or tracking redundancy.”
`
`EX1001, 31:31-34, FIG. 1.
`
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` Case IPR2022-01400
` Reply Declaration of Rahul Satija, D.Phil
`
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`26. Dr. Quackenbush agrees with Dr. Spellman and does not dispute that
`
`Schmitt teaches reducing or tracking redundancy of sequence reads, i.e., generating
`
`consensus sequences. Indeed, Dr. Quackenbush testified that in Schmitt’s methods,
`
`“sequence reads for a single strand of an original double-stranded sample molecule
`
`are collapsed” to form a ‘single strand consensus sequence’ or ‘SSCS’” and then
`
`“paired with a strand-mate SSCS to generate a duplex consensus sequence
`
`(’DCS’).” EX2015, ¶331; EX1097, 44:12-22; EX1002, ¶¶173-177. Dr.
`
`Quackenbush further admitted that “Schmitt describes pairing single strand
`
`consensus sequences to form a duplex consensus sequence.” EX2015, ¶70. A
`
`POSA would understand that such a collapsing step in Schmitt involves generating
`
`consensus sequences. The ’306 patent itself discloses that “[m]ethods disclosed
`
`herein can comprise collapsing, e.g., generating a consensus sequence by
`
`comparing multiple sequence reads.” EX1001, 31:62-64. Thus, Schmitt teaches
`
`reducing or tracking redundancy in the set of sequence reads by generating
`
`consensus sequences.
`
`27.
`
` Despite claim 1 encompassing generating consensus sequence reads,
`
`Dr. Quackenbush now attempts to narrowly construe step (e) of claim 1 to be
`
`limited to mapping raw sequence reads and not consensus sequences, while
`
`
`1 Emphasis added unless otherwise noted.
`
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` Case IPR2022-01400
` Reply Declaration of Rahul Satija, D.Phil
`
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`opining that Schmitt “exclusively discuss[es] mapping consensus sequences to a
`
`reference sequence.” EX2015, ¶¶66-68. I disagree with Dr. Quackenbush. A POSA
`
`would understand that claim 1 encompasses mapping raw sequence reads and
`
`consensus sequence reads. In addition, Schmitt teaches mapping both raw
`
`sequence reads and consensus sequence reads.
`
`28. A POSA would understand that claim 1 encompasses mapping
`
`consensus reads because the ’306 patent uses the term “sequence reads” and
`
`“reads” to refer to both raw sequence reads and consensus sequence reads. For
`
`example, in the following disclosure, the ’306 patent states that consensus
`
`sequences “can be mapped” and then uses the term “reads” to refer to the mapped
`
`consensus sequence:
`
`“Then, the members of a family can be collapsed into a consensus
`sequence. The consensus sequence can be generated using any
`collapsing method disclosed herein. Then the consensus sequence can
`be mapped to locations in the genome. Reads mapped to a locus can
`be quantified (e.g., counted).”
`
`EX1001, 34:30-35.
`
`29. Throughout the specification, the ’306 patent repeatedly refers to
`
`reads in the context of “consensus sequence reads.” Indeed, the ’306 patent
`
`discloses “determining consensus reads that are representative of single strands of
`
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` Case IPR2022-01400
` Reply Declaration of Rahul Satija, D.Phil
`
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`the original DNA fragment.” EX1001, 4:2-4, 31:30. The ’306 also states that its
`
`disclosed method can “include reducing or tracking redundancy (as described
`
`above) in the sequence reads to determine consensus reads that are representative
`
`of single strands of the original DNA fragments.” EX1001, 41:18-20 (“determine
`
`consensus reads”), 41:52-53 (“quantifying the consensus reads”); EX1097, 29:4-
`
`12, 44:24-47:4.
`
`30. Additionally, the ’306 patent discloses in the following workflow
`
`depicted in Figure 1 the steps of first “reduc[ing] and/or track[ing] redundancy,”
`
`i.e. generating consensus sequences, in step 108, then mapping the consensus
`
`sequence “reads” from step 108 in step 110, followed by grouping the same
`
`consensus sequence “reads” generated in step 108 and mapped in step 110 in step
`
`112. EX1001, FIG. 1. A POSA would understand from these disclosures that ’306
`
`patent uses the term “reads” to refer to consensus sequence reads.
`
`EX1001, FIG. 1 (excerpted, annotated). I understand that Dr. Quackenbush offered
`
`
`
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` Case IPR2022-01400
` Reply Declaration of Rahul Satija, D.Phil
`
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`no opinion on the 306 patent’s Figure 1. EX1097, 84:12-85:12.
`
`31. Thus, a POSA would understand that the ’306 patent uses “reads”
`
`generically to encompass both raw sequence reads and consensus sequence reads.
`
`EX1001, 4:2-4, 34:30-35, 41:18-19, 41:52-53, FIG. 1. As such, in the context of
`
`independent claim 1 and claims 2-16 and 29 (that depend on claim 1), a POSA
`
`would understand that “mapping at least a subset of the plurality of sequence
`
`reads” in step (e) encompasses mapping both raw sequence reads and mapping
`
`consensus sequence reads.
`
`32. And, as Dr. Spellman correctly explains in his declaration, Schmitt
`
`teaches mapping both raw sequence reads and consensus sequence reads to a
`
`reference sequence. EX1002, ¶¶178-179; EX1083, ¶¶60, 66, 68. For example, in
`
`paragraph 60, Schmitt describes a data processing workflow by aligning “reads” to
`
`the human genome, which a POSA would understand to refer to both consensus
`
`sequence reads and raw sequence reads. EX1083, ¶60. Moreover, paragraph 68
`
`discusses aligning duplex consensus sequences with the human genome to
`
`determine mutation frequencies. EX1083, ¶68. Lastly, paragraph 66 of Schmitt
`
`teaches using “samtools rmdup,” which a POSA would understand is a software
`
`tool that can be used to align and map sequence reads. EX1083, ¶66; EX1038,
`
`1754, 1757; EX1002, ¶179.
`
`33. Furthermore, Dr. Quackenbush misconstrues the teachings in Schmitt
`14
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` Case IPR2022-01400
` Reply Declaration of Rahul Satija, D.Phil
`
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`as being limited to teaching only mapping consensus sequence reads. For example,
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`paragraph 66 of Schmitt discloses that “[m]utations were initially scored without
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`consideration of the SMI sequences,” which means that the first run-through of the
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`workflow in Schmitt involved mapping raw sequence reads to a reference
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`sequence. EX1083, ¶66; EX1002, ¶178. Dr. Quackenbush did not address this
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`disclosure in Schmitt.
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`34. Dr. Quackenbush also does not address Schmitt’s teaching in Table 1
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`of Example 4 in the Schmitt PCT application, that discloses the data yield from
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`Duplex Sequencing showing that “[i]nitial nucleotides represent raw reads that
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`contain the expected fixed adapter sequence following 12 degenerate nucleotides
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`and map to the reference genome.” EX1009, ¶128. Thus, a POSA would
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`understand that Schmitt teaches mapping raw sequence reads to the reference
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`genome. Dr. Quackenbush acknowledges that Schmitt at least suggests mapping
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`sequence reads. EX2015, ¶72 (“[t]o the extent Schmitt describes mapping
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`sequence reads….).
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`35. Notably, the ’306 patent explicitly discloses mapping both raw
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`sequence reads and consensus sequence reads to a reference sequence. As
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`discussed above, Figure 1 discloses first generating a consensus sequence
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`(“Reduce and/or track redundancy of reads”) in step 108, followed by mapping the
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`same consensus sequences in step 110, which the patent refers to as “mapping
`15
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` Case IPR2022-01400
` Reply Declaration of Rahul Satija, D.Phil
`
`
`reads” to a selected locus in the genome in step 110. EX1001, FIG. 1; EX1097,
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`84:12-85:12. Moreover, the ’306 patent discloses that “collapsing” to form
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`consensus sequences “can be performed before mapping.” EX1001, 34:25-26;
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`EX1097, 48:1-16.
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`36. Lastly, a POSA reading the ’306 patent claims would understand that
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`the use of the word “comprising” around the mapping step only further
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`demonstrates that the claims encompass generating consensus sequence reads
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`before mapping. I understand that in U.S. patent law, the term “comprising” is
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`considered an open-ended term, meaning that additional limitations or steps can be
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`present in the claimed step. Indeed, claim 1 recites that the “reducing or tracking
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`the redundancy of the plurality of sequence reads comprises mapping … to the
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`reference sequence,” which a POSA would understand to mean that the claim
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`allows an intermediate step of generating consensus sequence reads prior to the
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`mapping step. EX1001, 61:26-43.
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`37. Therefore, in view of the above, a POSA would understand that
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`Schmitt teaches all of the elements of claims 1-16 and 29, including the mapping
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`step recited in step (e).
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`Schmitt teaches tagging cfDNA molecules as recited in claim 1.
`B.
`38. Dr. Quackenbush alleges that Dr. Spellman fails to establish that
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`Schmitt teaches the tagging step of claim 1 because Dr. Spellman “improperly and
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` Case IPR2022-01400
` Reply Declaration of Rahul Satija, D.Phil
`
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`inconsistently” maps only the exogenous 3-mer portion of Schmitt’s hybrid SMI
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`embodiment in his discussion of the challenged claims despite acknowledging that
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`both SMIs and hybrid SMIs (combination of shear sequence and exogenous n-mer
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`sequence) are “molecular barcodes.” EX2015, ¶¶43-57. I disagree with Dr.
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`Quackenbush’s opinion because Schmitt’s SMI-tagged product meets the
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`definition of “duplex tag” in the ’306 patent.
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`39. Step (b) of claim 1 recites “tagging a plurality of the cfDNA
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`molecules in the population with duplex tags comprising molecular barcodes…”
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`EX1001, Claim 1. The ’306 patent defines “duplex” tag as “tags that differently
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`label the complementary strands (i.e., the ‘Watson’ and ‘Crick’ strands) of a
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`double-stranded molecule.” EX1001, 17:9-13. As Dr. Spellman correctly opined in
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`his Declaration, Schmitt teaches differently labeling the complementary strands
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`using the combination of the double stranded barcode portions and the
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`asymmetrical portions to distinguish the two strands. EX1002, ¶162; EX1009,
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`¶¶12, 38; EX1083, ¶¶11, 30; EX1097, 98:16-99:1.
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`40. Thus, a POSA would understand, that Schmitt’s tagged DNA product
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`meets the ’306 patent’s definition of DNA tagged with duplex tags whether it be
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`tagged with SMIs or hybrid SMIs containing a combination of shear sequence and
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`exogenous n-mer sequences.
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`17
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` Case IPR2022-01400
` Reply Declaration of Rahul Satija, D.Phil
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`C. The ’306 patent claims encompass Schmitt’s hybrid embodiment.
`41. Dr. Quackenbush argues that Dr. Spellman fails to demonstrate that
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`the