`UNITED STATES PATENT AND TRADEMARK OFFICE
`_________________________
`BEFORE THE PATENT TRIAL AND APPEAL BOARD
`_________________________
`
`
`ILLUMINA INC.,
`Petitioner,
`v.
`MOLECULAR LOOP BIOSCIENCES, INC.,
`Patent Owner.
`_________________________
`
`Case No. IPR2024-00965
`Patent No. 11,768,200
`_________________________
`
`
`
`DECLARATION OF PAUL T. SPELLMAN, PH.D.
`
`
`
`
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`
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`TABLE OF CONTENTS
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`IPR2024-00965
`U.S. Patent No. 11,768,200
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`Page
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`I.
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`INTRODUCTION ........................................................................................... 1
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`A. Qualifications and Experience .............................................................. 1
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`B. Materials Considered ............................................................................. 6
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`II.
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`LEVEL OF ORDINARY SKILL IN THE ART ............................................. 6
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`III. SUMMARY OF OPINIONS ........................................................................... 7
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`IV. LEGAL STANDARDS .................................................................................13
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`V.
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`BACKGROUND AND STATE OF THE ART ............................................16
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`A.
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`First Generation Sequencing ...............................................................16
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`B. Next Generation Sequencing Techniques Available as of
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`December 2010 ....................................................................................19
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`1.
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`2.
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`3.
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`4.
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`5.
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`Overview of Sequencing Workflow .........................................20
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`Basic Principles Behind Illumina and Roche 454
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`Sequencing ................................................................................22
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`Immobilization of Nucleic Acids of Interest ............................26
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`Amplification of Nucleic Acids of Interest ...............................27
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`Generation of Sequence Reads and Sequences of Nucleic
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`Acids of Interest ........................................................................30
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`6.
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`Use of Sequence Identifiers in Multiplex Sequencing .............32
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`i
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`7.
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`Resources available to a POSA before December 2010 ...........40
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`VI. SUMMARY OF THE PRIOR ART ..............................................................41
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`A.
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`Parameswaran (EX1004) ...................................................................41
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`B. Gloor (EX1005) ..................................................................................47
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`C.
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`D.
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`Binladen (EX1029)..............................................................................51
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`Bentley (EX1006) ................................................................................54
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`VII. THE ’200 PATENT .......................................................................................57
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`A. Overview .............................................................................................57
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`B.
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`The Claims of the ’200 Patent .............................................................57
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`C. Disclosures of the ’200 Patent .............................................................59
`
`D.
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`Prosecution History .............................................................................65
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`1.
`
`2.
`
`Prosecution History of the ’281 Patent .....................................67
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`Prosecution History of the ’200 Patent .....................................68
`
`E.
`
`Claim Construction..............................................................................69
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`VIII. THE CHALLENGED CLAIMS ARE UNPATENTABLE OVER
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`THE PRIOR ART ..........................................................................................69
`
`A. Ground 1: Claims 1-6 Are Anticipated by Gloor ...............................69
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`1.
`
`2.
`
`Claim 1 is Anticipated by Gloor ...............................................69
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`Dependent Claims 2-6 Are Anticipated by Gloor ....................78
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`B. Ground 2: Claims 1-6 Are Anticipated by Parameswaran ................81
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`ii
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`1.
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`2.
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`Claim 1 Is Anticipated by Parameswaran ................................81
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`Dependent Claims 2-6 Are Anticipated by
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`Parameswaran ..........................................................................91
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`C. Ground 3: Claims 1-6 Would Have Been Obvious Over
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`Parameswaran and Gloor ...................................................................94
`
`1.
`
`Claim 1 Would Have Been Obvious Over Parameswaran
`
`and Gloor ..................................................................................95
`
`2.
`
`A POSA Would Have Been Motivated to Combine
`
`Parameswaran and Gloor .........................................................98
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`3.
`
`A POSA Would Have Had a Reasonable Expectation of
`
`Success in Combining Parameswaran and Gloor ..................103
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`4.
`
`Dependent Claims 2-6 Would Have Been Obvious Over
`
`the Combination of Parameswaran and Gloor ......................107
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`D. Ground 4: Claims 1-6 Would Have Been Obvious Over
`
`Parameswaran and Binladen ............................................................109
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`1.
`
`Claim 1 Would Have Been Obvious Over Parameswaran
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`and Binladen ...........................................................................110
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`2.
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`A POSA Would Have Been Motivated to Combine
`
`Parameswaran and Binladen ..................................................117
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`iii
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`3.
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`A POSA Would Have Had a Reasonable Expectation of
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`Success in Combining Parameswaran and Binladen .............119
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`4.
`
`Dependent Claims 2-6 Would Have Been Obvious Over
`
`the Combination of Parameswaran and Binladen ..................120
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`E.
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`Ground 5: Claims 6 Would Have Been Obvious Over
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`Parameswaran, Binladen, and Bentley .............................................122
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`F.
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`No Unexpected Results or Other Evidence of Nonobviousness .......125
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`IX. CONCLUSION ............................................................................................126
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`iv
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`I.
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`IPR2024-00965
`U.S. Patent No. 11,768,200
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`INTRODUCTION
`I, Dr. Paul T. Spellman, have been retained as an independent expert in
`1.
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`the field of genetics and DNA sequencing. I submit this declaration on behalf of
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`Petitioner Illumina, Inc., in the above-captioned inter partes review (“IPR”).
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`2.
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`I am being compensated for my time in connection with this IPR at my
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`standard hourly consulting rate of $500/hour. I do not have any personal or financial
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`stake or interest in the outcome of this proceeding and my compensation is in no
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`way contingent on the nature of my analysis or the outcome of this IPR or any other
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`proceeding.
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`3.
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`I am over 21 years of age and, if I am called upon to do so, I would be
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`competent to testify as to the matters set forth herein.
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`A. Qualifications and Experience
`I believe that I am qualified to serve as a technical expert in this matter
`4.
`
`based upon my qualifications, discussed in detail below. A copy of my curriculum
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`vitae is attached as Appendix A to this declaration.
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`1
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`
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`5.
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`I am a Professor of Medicine in the Division of Hematology and
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`Oncology as well as in the Department of Human Genetics at the University of
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`California Los Angeles (“UCLA”) David Geffen School of Medicine. My research
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`focuses on bioinformatics and genome sequencing, including developing improved
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`sequencing techniques and applying genomic and computational technologies to
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`improve human health. My research encompasses all phases of genomic research
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`and sequencing, from technology and method development to application of
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`technologies to answer critical questions in cancer biology, to population studies to
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`understand the impact of genetic variation of disease, and to implementation trials
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`that directly impact health. I have more than 25 years of experience in nucleic acid
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`sequencing and have first-hand experience in designing multiplex sequencing
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`assays.
`
`6.
`
`I graduated from the Massachusetts Institute of Technology in 1995
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`with a Bachelor of Science in Biology. I then earned my Ph.D. in Genetics from the
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`Stanford University School of Medicine in 2000.
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`2
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`
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`7.
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`Following my Ph.D., I conducted post-doctoral research from 2000 to
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`2003 at the University of California Berkley Department of Molecular and Cellular
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`Biology, studying gene regulation and genomics. From 2003 to 2011, I was a
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`Scientist and Staff Scientist at the Lawrence Berkeley Lab, Life Science Division.
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`In 2011, I also served as the Special Assistant to the Deputy Director of the National
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`Cancer Institute.
`
`8.
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`In 2011, I joined the Oregon Health & Science University (“OHSU”)
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`School of Medicine first serving as an Associate Professor, then Professor with
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`tenure, and then the Penny and Phil Knight Endowed Professor in Cancer Research
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`Innovation. There, I studied the use of population genetics to help determine who is
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`at risk for cancer, how to computationally analyze genomic data to identify early
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`changes in cancers, and how to accurately screen different populations for the
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`disease. My research also included using genetic and genomic approaches to
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`understand the processes by which cancer develops, monitor disease, and identify
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`therapeutic strategies.
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`3
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`
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`9.
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`I spent 12 years at the OHSU School of Medicine before joining the
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`faculty of UCLA in 2023. During that time at OHSU, I held multiple leadership roles
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`for programs relating to genomic sequencing, including serving as the Program
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`Leader of the Quantitative Oncology Program, the Co-Director of the Cancer Early
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`Detection Advanced Research (“CEDAR”) Center in the OHSU Knight Cancer
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`Institute, and the Interim Director for the Program in Computational Biology.
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`10. To date, I have authored about 200 publications, including 175 articles,
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`3 book chapters, 5 letters to the editor, 12 literary reviews, and 4 abstracts, many of
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`which relate to genomic research or apply genomic sequencing.
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`11. Since 2002, I have served on 25 grant review committees, 6 conference
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`planning committees, and over 30 project advisory, institutional, and editorial
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`committees. I am also active in related professional societies, including having
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`served as Treasurer and Board member for the Microarray Gene Expression Data
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`Society and Data Coordination and as a member of the Management Working Group
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`and Scientific Planning Committee for the International Cancer Genomics
`
`Consortium.
`
`12.
`
`I have served as a regular reviewer for journals relating to genome
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`sequencing and biotechnology, including Bioinformatics, Nucleic Acids Research,
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`Nature Communications, PLoS ONE, Cell Reports, Genome Research, and Genes,
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`among others.
`
`4
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`
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`13. Since 2004, I have been awarded dozens of grants, fellowships, and
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`other sources of funding for my work, including for studying topics such as
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`developing cost effective sequence-based technologies, studying genes relating to
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`breast and ovarian cancer, and analyzing genomic data. Currently, I have research
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`funding for a systematic analysis of genetic and gene regulation information in
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`clinical cohorts as part of the Genome Data Analysis Network and a clinical trial
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`implementing genetic health screening for hereditary breast and ovarian cancer and
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`Lynch syndromes.
`
`14. Over the years, my work has been highlighted by news media outlets,
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`including by the Salem Statesman Journal. I have given over 80 guest lectures on
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`my work, and I have taught over 50 courses, workshops, and seminars on topics
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`including genetics, genome
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`sequencing, biotechnology, and
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`sequencing
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`technologies. I have also served as an advisor to over 20 post-doctoral fellows and
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`pre-doctoral graduate students.
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`15. As high-throughput sequencing has come to dominate the technological
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`basis of my career, I have been well aware of, and my lab routinely uses, unique and
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`non-unique dual-index sequencing methods.
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`5
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`B. Materials Considered
`16. My analysis in this declaration is based on my education, personal
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`knowledge, and professional and academic experience in the areas of genetics and
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`DNA sequencing, including multiplex sequencing and dual-indexing methods. My
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`analysis is particularly focused on the state of the art in December of 2010.
`
`17.
`
`In forming my opinions, I have considered the ’200 patent and other
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`exhibits cited in my declaration, including those listed in Appendix B. I reserve the
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`right to rely on documents cited in the appendices to this declaration, and to
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`supplement my opinions in view of new materials and information that becomes
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`available to me during this proceeding.
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`II. LEVEL OF ORDINARY SKILL IN THE ART
`I understand that an assessment of a patent’s claims should be
`18.
`
`undertaken from the perspective of a person of ordinary skill in the art (“POSA”) as
`
`of the “effective filing date” of the patent claims. In performing my analysis, I have
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`been asked to assume that the “effective filing date” of the ’200 patent is December
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`23, 2010.
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`6
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`19.
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`I understand that the level of ordinary skill in the art is determined by
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`looking at; (1) the type of problems encountered in the art; (2) the prior-art solutions
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`to those problems; (3) the rapidity with which innovations are made; (4) the
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`sophistication of the technology; and (5) the educational level of active workers in
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`the field.
`
`20.
`
`In my opinion, a POSA would have had (i) a Ph.D. in molecular
`
`biology, genetics, bioinformatics, or a related field and at least two years of
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`experience in high-throughput sequencing technologies or (ii) a Master’s degree in
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`one of the same fields with at least four years of experience in high-throughput
`
`sequencing technologies. As explained above in Section I.A, I had at least these
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`qualifications by December 2010 and am qualified based on my education and
`
`experience to provide an opinion as to what a POSA would have known and
`
`concluded as of December 2010. Supra § I.A.
`
`III. SUMMARY OF OPINIONS
`I understand that this declaration accompanies a Petition for IPR
`21.
`
`involving U.S. Patent No. 11,768,200 (“the ’200 patent”) (EX1001). I understand
`
`that
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`the earliest possible “effective filing date” for
`
`the ’200 patent is
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`December 23, 2010. In performing my analysis, I have been asked to assume that
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`the “effective filing date” of the ’200 patent is December 23, 2010.
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`7
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`22. The claims of the ’200 patent are directed to a method for validating the
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`sequence of a nucleic acid analyte of interest in a multiplex sequencing reaction. As
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`discussed below, the sequencing workflow typically involved several steps,
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`including extracting template nucleic acids from samples, preparing those template
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`nucleic acids for sequencing, and sequencing the products. It was often
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`advantageous to use polymerase chain reaction (PCR) or other amplification
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`methods to replicate the template during the sequencing workflow to increase the
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`number of molecules available for sequencing. In the case of multiplex sequencing
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`where multiple samples were sequenced simultaneously, sequencing products were
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`combined (“pooled”) at various steps during the sequencing workflow to gain
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`efficiencies and reduce costs, including for PCR or other forms of DNA
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`amplification.
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`23. Numerous different types of errors, however, could occur during this
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`workflow. The original template could get damaged during extraction or
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`amplification. Primers used for DNA amplification could be erroneously
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`synthesized. Further, during DNA amplification, chimeric molecules consisting of
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`two template nucleic acids could be formed. When these errors occurred in multiplex
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`sequencing, the accuracy of sequencing could be significantly affected, resulting in
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`erroneous sequences that did not exist in the original samples or misassignments of
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`sequences to incorrect samples.
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`8
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`
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`24. The claims of the ’200 patent purport to address these known problems
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`by using a dual-indexing method. The claims recite that the sequence of a nucleic
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`acid analyte of interest is validated by detecting the presence of two or more
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`identifier sequences uniquely associated with the nucleic acid analyte of interest,
`
`sequencing the nucleic acid analyte of interest flanked with the two identifier
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`sequences, and validating the sequence by analyzing both identifier sequences and
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`excluding those sequences containing only one or an incorrect pair of identifier
`
`sequences.
`
`25. Based on my knowledge and experience and my review of the
`
`information described in this declaration, it is my opinion that claims 1-6 of the
`
`’200 patent are anticipated by two references, each of which disclosed every step of
`
`the claimed dual-indexing methods.
`
`26. First, Gloor et al., Cornell University Library arXiv:1007.5075v1
`
`(2010) (“Gloor”; EX1005), anticipates claims 1-6 of the ’200 patent. Gloor
`
`disclosed a multiplex sequencing method where nucleic acid analytes from multiple
`
`samples were tagged with a pair of identifier sequences. While Gloor repeated
`
`individual identifier sequences across multiple samples, the pairs of identifiers did
`
`not repeat, i.e., each pair of identifiers was uniquely associated with nucleic analytes
`
`of interest from a single sample. Using Illumina sequencing, Gloor sequenced
`
`nucleic acid analytes from multiple samples simultaneously. Gloor also validated
`
`9
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`
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`the sequences of the analytes by analyzing the two identifiers present in each
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`sequence, and excluding sequences that contained only one identifier or pairs of
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`incorrect identifiers. Because Gloor discloses each limitation of the challenged
`
`claims, Gloor anticipates those claims.
`
`27. Parameswaran et al., Nucleic Acids Research (2007) 35:e130
`
`(“Parameswaran”; EX1004) also anticipates claims 1-6 of the ’200 patent. Like
`
`Gloor, Parameswaran disclosed multiplex sequencing methods where nucleic acid
`
`analytes of interest from each sample were tagged with a unique pair of identifier
`
`sequences. Using Roche 454 sequencing, Parameswaran sequenced nucleic acid
`
`analytes from multiple samples simultaneously. Parameswaran validated the
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`sequences of the analytes by analyzing the two identifiers and separated sequences
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`with only one identifier from those with two correct identifiers. Among those
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`separated sequences, Parameswaran identified sequences containing incorrect pairs
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`of
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`identifiers and characterized
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`them as “false-discover[ies]” constituting
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`“misassignment[s]” of sequences. Because Parameswaran discloses each limitation
`
`of the challenged claims, Parameswaran anticipates those claims.
`
`28.
`
`In addition, it is my opinion that claims 1-6 of the ’200 patent would
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`have been obvious to a person of ordinary skill in the art (“POSA”) as of
`
`December 23, 2010, over the combination of Parameswaran and Gloor.
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`10
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`29. Each and every element of the claims of the ’200 patent is found in the
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`combination of Parameswaran and Gloor. As discussed below, a POSA would have
`
`been motivated to combine Parameswaran and Gloor with a reasonable expectation
`
`of success. Indeed, to maximize sequencing accuracy, cost, and efficiency, a POSA
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`would have been motivated to use Parameswaran’s dual-indexing method, which
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`identified different types of erroneous sequences produced during multiplex
`
`sequencing, with the Illumina sequencing used in Gloor. A POSA would have been
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`motivated to exclude those erroneous sequences from further analysis to achieve
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`“more accurate” Illumina sequencing. A POSA would have had a reasonable
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`expectation of success in doing so because both Gloor and Parameswaran
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`empirically demonstrated that erroneous sequences generated during a multiplex
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`sequencing reaction can be readily identified using dual-indexing methods and
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`excluded to improve accuracy.
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`30.
`
`It is also my opinion that claims 1-6 of the ’200 patent would have
`
`been obvious to a POSA in view of Parameswaran and Binladen et al., PLoS ONE
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`(2007) 2(2):e197 (“Binladen”; EX1029), another reference that used the Roche 454
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`platform to sequence multiple samples simultaneously, and identified and discarded
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`erroneous sequences using a dual-indexing method. To improve the accuracy of
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`multiplex sequencing, a POSA would have been motivated to use the dual-indexing
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`methods in Parameswaran and Binladen to identify and exclude erroneous
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`11
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`sequences. A POSA would have had a reasonable expectation of success in
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`combining Parameswaran and Binladen because both references demonstrated that
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`the presence or absence of dual indexes in sequences can be used to identify and
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`exclude erroneous sequences, thereby lowering sequence misassignment rates.
`
`31.
`
`Claim 6 of the ’200 patent recites that the nucleic acid analytes of
`
`interest are on “on the surface of a flow cell” during the multiplex sequencing
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`reaction. To the extent this requires direct attachment of the analytes to the surface
`
`of the flow cell, claim would have been also obvious to a POSA over the
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`combination of Parameswaran, Binladen, and Bentley et al., Nature (2008)
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`456:53-59 (“Bentley”; EX1006). Bentley teaches Illumina sequencing on the surface
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`of a flow cell. EX1006 (Bentley), 2 (Figure 1). While Parameswaran and Binladen
`
`used Roche 454 sequencing, a POSA would have been motivated to use Illumina
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`sequencing, as taught in Bentley, which was known to be faster, more efficient, and
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`less expensive than Roche 454 sequencing. A POSA would have a reasonable
`
`expectation of success in making that switch in view of Bentley’s detailed guidance
`
`for performing Illumina sequencing.
`
`32. Thus, claims 1-6 of the ’200 patent would have been obvious to a POSA
`
`as of December 23, 2010. I am currently not aware of any objective evidence of non-
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`obviousness for the claims of the ’200 patent. I reserve the right to consider and
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`comment on any such evidence after I sign this declaration.
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`12
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`IV. LEGAL STANDARDS
`33. The opinions I express in this declaration involve the application of my
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`technical knowledge and experience in evaluating certain prior art with respect to
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`the ’200 patent. In preparing this declaration, certain patent law concepts have been
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`explained to me by counsel, including the legal standard for interpreting claims as
`
`well as those for assessing obviousness of a patent claim.
`
`34.
`
`I have been informed that, in IPR proceedings such as this one, the party
`
`challenging the patent bears the burden of proving unpatentability by a
`
`preponderance of the evidence. I understand that a preponderance of the evidence
`
`means “more likely than not.”
`
`35.
`
`I understand that patentability must be analyzed from the perspective
`
`of a POSA in the same field as the challenged patent as of the “effective filing date”
`
`of the claims. I understand that a POSA is a hypothetical individual presumed to
`
`know the relevant art as of the effective filing date of the claims.
`
`36.
`
`I understand that the Patent Trial and Appeal Board interprets claims
`
`based on their ordinary meaning as understood by a POSA at the time of the effective
`
`filing date in view of the claim language, patent specification, and prosecution
`
`history. I also understand that the specification may reveal a special definition given
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`to a claim term by an inventor that differs from its ordinary meaning as understood
`
`by a POSA. In that case, I understand that the inventor’s definition governs.
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`13
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`37.
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`I understand that a patent claim is invalid as anticipated if the claimed
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`subject matter was described in a printed publication before the effective filing date.
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`I have been informed that a patent claim is anticipated if a single prior art reference
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`discloses all limitations of the claimed subject matter. I have been informed that the
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`anticipatory prior art reference need not use the very same terms used in the patent
`
`claim. I also understand that a reference disclosing a method of using one
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`composition within the scope of the claimed subject matter anticipates the claim.
`
`38.
`
`I understand that a patent claim is invalid if it would have been obvious
`
`to a person of ordinary skill in the art as of the effective filing date. I understand that
`
`assessing obviousness entails considering: (1) the scope and content of the prior art,
`
`(2) the differences between the prior art and the claimed invention, (3) the level of
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`ordinary skill in the art, and (4) any secondary considerations of non-obviousness.
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`39.
`
`I understand that a claim may be obvious based on a combination of
`
`multiple prior-art references, as well as based on the knowledge and skill of a POSA
`
`as of the effective filing date. I also understand that to combine prior-art references,
`
`there must have been a motivation that would have prompted a POSA to do so. In
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`other words, obviousness requires a motivation to combine the features of the prior
`
`art. I further understand that a POSA must have reasonably expected that the
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`combination would work. That is, obviousness requires a reasonable expectation of
`
`success in combining the prior art to achieve the claimed subject matter. I also
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`14
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`understand that, while obviousness requires a reasonable expectation of success, it
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`does not require absolute predictability of success in achieving the claimed subject
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`matter.
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`40.
`
`I understand that a motivation to combine prior-art references may arise
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`from a variety of sources, including, among other things, scientific literature, a need
`
`or unsolved problem in the field, or market demand.
`
`41.
`
`I also understand that a claim may be obvious if a POSA would have
`
`found it obvious to try combining a finite number of predictable solutions known in
`
`the art. For example, if one technique had been used to improve a method of using a
`
`device, and a POSA would recognize that the same technique was one of a limited
`
`number of solutions and would improve similar devices in the same way, a POSA
`
`may have found it obvious to try that same technique on similar devices.
`
`42.
`
`I understand
`
`that, when present, evidence of “secondary
`
`considerations” must be considered along with the other factual evidence relevant to
`
`obviousness. Such secondary considerations could include unexpected results,
`
`commercial success of products or processes using the invention, long-felt but unmet
`
`need for the invention, failure of others to make the invention, industry acceptance
`
`of the invention, or copying of the invention by others. I understand that, in order
`
`for such evidence to support nonobviousness, it must have a “nexus” to the features
`
`of the invention as claimed.
`
`15
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`
`
`43.
`
`I have been informed that a dependent claim is a patent claim that refers
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`IPR2024-00965
`U.S. Patent No. 11,768,200
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`
`
`back to another patent claim. I have been informed that a dependent claim includes
`
`all of the limitations of the claim to which it refers.
`
`V. BACKGROUND AND STATE OF THE ART
`First Generation Sequencing
`A.
`44. Deoxyribonucleic acid (“DNA”) carries genetic information. DNA
`
`consists of two “reverse complementary” strands made of deoxynucleotides where
`
`nucleotides are linked between their phosphate and deoxyribose portions. In DNA,
`
`there are four types of nucleotides, which respectively contain adenine (A),
`
`guanine (G), thymine (T), and cytosine (C) bases. The two strands of a DNA
`
`molecule are held together by hydrogen bond formation between complementary
`
`base pairs. EX1031 (Garland), 3. Specifically, guanine (G) is complementary to and
`
`pairs with cytosine (C) and adenine (A) is complementary to and pairs with
`
`thymine (T). EX1031 (Garland), 3. Each nucleotide has a 5’ phosphate (“5’” or
`
`“5’-P”) end and a 3’ hydroxide (“3’” or “3’-OH”) end that links to the 5’ end of the
`
`next nucleotide. During extension of a given DNA by enzymatic or chemical means,
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`nucleotides are added to the 3’ end of the existing chain through the catalyzed
`
`reaction of a deoxynucleotide triphosphate (dNTP) being used to add the nucleotide
`
`releasing a diphosphate in the reaction. EX1031 (Garland), 38.
`
`16
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`
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`45. Knowing the sequence of a given DNA can be important for any
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`IPR2024-00965
`U.S. Patent No. 11,768,200
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`
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`number of reasons, including disease diagnosis, identifying disease-causing
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`mutations, and confirming genetic identity. For the last five decades, many DNA-
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`sequencing techniques have been developed, most of which are based on the
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`complementarity of the two strands in DNA.
`
`46.
`
`In 1977, Dr. Frederick Sanger published his method of DNA
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`sequencing, now known as “Sanger-based sequencing,” which uses a technique
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`known as non-reversible termination to determine the order of nucleotides in a strand
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`of DNA. EX1009 (Sanger 1977). The traditional Sanger-based sequencing workflow
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`began by amplifying a sequence of interest into millions of copies through in vivo
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`cloning. EX1012 (Shendure), 2. Genomic DNA of a target sequence was
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`fragmented, cloned into plasmid vectors, and then transformed to E. coli for
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`amplification. EX1012 (Shendure), 2. For each sequencing reaction, a single
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`bacterial colony was picked and plasmid DNA is isolated. EX1012 (Shendure), 2.
`
`47. Following amplification and isolation, reverse strand synthesis was
`
`performed on these copies using a known priming sequence upstream of the region
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`to be sequenced. EX1010 (Kircher), 2-3; EX1011 (Metzker), 1-2; EX1012
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`(Shendure), 1. Reverse strand synthesis used four pools of polymerization reactions,
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`each with a mixture of dNTPs (i.e., dATP, dGTP, dCTP, and dTTP), plus one
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`chemically labeled dideoxynucleoside triphosphate, a dNTP missing a hydroxyl
`
`17
`
`
`
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`group at the ’3-end (i.e., ddATP, ddGTP, ddCTP, or ddTTP). EX1012 (Shendure), 1;
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`IPR2024-00965
`U.S. Patent No. 11,768,200
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`EX1010 (Kircher), 2. In other words, four reactions were performed (consisting of
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`dNTPs plus ddATP, ddGTP, ddCTP, or ddTTP), wherein polymerization of the
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`nucleotides occurred by incorporation of dNTPs. EX1012 (Shendure), 1.
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`Polymerization was
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`stochastically
`
`terminated wherever
`
`the
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`labeled
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`dideoxynucleotides happened to be incorporated. EX1010 (Kircher), 2-3; EX1011
`
`(Metzker), 1-2; EX1012 (Shendure), 1. Incorporation of a dideoxynucleotide into
`
`the growing strand terminated DNA polymerization because dideoxynucleotides
`
`lack the 3’-OH group necessary for the phosphodiester bond formation that would
`
`have allowed polymerization to continue. EX1010 (Kircher), 2-3; EX1011
`
`(Metzker), 1-2; EX1012 (Shendure), 1. The dNTPs/ddNTP mixture thus causes
`
`random, non-reversible termination of the extension reaction, creating different
`
`copies of molecules extended to different lengths. EX1010 (Kircher), 2-3; EX1011
`
`(Metzker), 1-2; EX1012 (Shendure), 1-2.
`
`48. The resulting molecules were sorted by molecular weight via
`
`electrophoresis (corresponding to the point where a labeled dideoxynucleotide
`
`terminated polymerization), and signals generated from labels attached to the
`
`terminating dideoxynucleotides were detected to determine which nucleotides were
`
`added. EX1010 (Kircher), 2-3; EX1011 (Metzker), 1-2; EX1012 (Shendure), 1-2.
`
`18
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`
`
`49. Although significant improvements were made to the technique after
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`IPR2024-00965
`U.S. Patent No. 11,768,200
`
`
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`1977, Sanger-based sequencing remained time-consuming and expensive compared
`
`to later-developed techniques (“next generation sequencing”), described below.
`
`EX1010 (Kircher), 2. Among other disadvantages, Sanger-based sequencing
`
`required isolating every molecule to be sequenced separately and thus was limited
`
`to sequencing only one target sequence in each reaction.
`
`50. Consequently, Sanger-based sequencing was ill suited for sequencing
`
`thousands of target sequences in parallel. For example, Sanger-based sequencing
`
`was used in the first project of the Cancer Genome Atlas. See
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