`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. IPR2024-00964
`Patent No. 11,041,852
`_________________________
`
`
`
`DECLARATION OF PAUL T. SPELLMAN, PH.D.
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`TABLE OF CONTENTS
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`IPR2024-00964
`U.S. Patent No. 11,041,852
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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 ............................................................................. 5
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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 .................................................................................12
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`V.
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`BACKGROUND AND STATE OF THE ART ............................................15
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`A.
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`First Generation Sequencing ...............................................................15
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`B. Next Generation Sequencing Techniques Available as of
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`December 2010 ....................................................................................18
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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 .........................................19
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`Basic Principles Behind Illumina and Roche 454
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`Sequencing ................................................................................21
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`Immobilization of Nucleic Acids of Interest ............................25
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`Amplification of Nucleic Acids of Interest ...............................26
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`Generation of Sequence Reads and Sequences of Nucleic
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`Acids of Interest ........................................................................29
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`6.
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`Use of Sequence Identifiers in Multiplex Sequencing .............30
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`i
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`7.
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`Resources available to a POSA before December 2010 ...........39
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`VI. SUMMARY OF THE PRIOR ART ..............................................................40
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`A.
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`Parameswaran (EX1004) ...................................................................40
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`B. Gloor (EX1005) ..................................................................................46
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`C.
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`Bentley (EX1006) ................................................................................51
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`VII. THE ’852 PATENT .......................................................................................53
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`A. Overview .............................................................................................53
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`B.
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`The Claims of the ’852 Patent .............................................................54
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`C. Disclosures of the ’852 Patent .............................................................56
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`D.
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`E.
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`Prosecution History of the ’852 Patent ...............................................62
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`Claim Construction..............................................................................64
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`VIII. CHALLENGED CLAIMS ARE UNPATENTABLE OVER THE
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`PRIOR ART ...................................................................................................65
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`A. Ground 1: Claims 1-8 Would Have Been Obvious Over
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`Parameswaran and Gloor ...................................................................65
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`1.
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`Claim 1 Would Have Been Obvious Over Parameswaran
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`and Gloor ..................................................................................65
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`2.
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`A POSA Would Have Been Motivated to Combine
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`Parameswaran and Gloor .........................................................81
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`ii
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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 Gloor ....................87
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`4.
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`Dependent Claims 2-8 Would Have Been Obvious Over
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`the Combination of Parameswaran and Gloor ........................92
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`B. Ground 2: Claims 1-8 Would Have Been Obvious Over
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`Parameswaran and Bentley ...............................................................101
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`1.
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`Claim 1 Would Have Been Obvious Over Parameswaran
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`and Bentley ..............................................................................102
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`2.
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`A POSA Would Have Been Motivated to Combine
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`Parameswaran and Bentley ....................................................106
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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 Bentley ...............106
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`4.
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`Dependent Claims 2-8 Would Have Been Obvious Over
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`Parameswaran and Bentley ....................................................109
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`C. No Unexpected Results or Other Evidence of Nonobviousness .......112
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`IX. CONCLUSION ............................................................................................112
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`iii
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`I.
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`IPR2024-00964
`U.S. Patent No. 11,041,852
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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.
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`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.
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`6.
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`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.
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`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
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`Consortium.
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`12.
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`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.
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`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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`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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`5
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`
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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.
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`17.
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`In forming my opinions, I have considered the ’852 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.
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`undertaken from the perspective of a person of ordinary skill in the art (“POSA”) as
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`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 ’852 patent is December
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`23, 2010.
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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.
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`20.
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`In my opinion, a POSA would have had (i) a Ph.D. in molecular
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`biology, genetics, bioinformatics, or a related field and at least two years of
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`6
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`
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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
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`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
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`experience to provide an opinion as to what a POSA would have known and
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`concluded as of December 2010. Supra § I.A.
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`III. SUMMARY OF OPINIONS
`I understand that this declaration accompanies a Petition for IPR
`21.
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`involving U.S. Patent No. 11,041,852 (“the ’852 patent”) (EX1001). I understand
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`that
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`the earliest possible “effective filing date” for
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`the ’852 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 ’852 patent is December 23, 2010.
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`22. The claims of the ’852 patent are directed to a “method for reducing
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`template cross-over error introduced during sequencing workflow.” As discussed
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`below, the sequencing workflow typically involved several steps, including
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`extracting template nucleic acids from samples, preparing those template nucleic
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`acids for sequencing, and sequencing the products. It was often advantageous to use
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`polymerase chain reaction (PCR) or other amplification methods to replicate the
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`template during the sequencing workflow to increase the number of molecules
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`available for sequencing. In the case of multiplex sequencing where multiple
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`7
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`
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`samples were sequenced simultaneously, sequencing products were combined
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`(“pooled”) at various steps during the sequencing workflow to gain efficiencies and
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`reduce costs, including for PCR or other forms of DNA amplification.
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`23. Numerous different types of errors, however, could occur during the
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`amplification steps. One prominent type of error was where two different template
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`nucleic acids create a hybrid product during amplification (usually through
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`incomplete extension and mispriming at a subsequent step), resulting in new
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`molecules, the sequences of which were not present in the initial mixture of samples
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`prior to amplification. These incorrect molecules were commonly referred to as
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`“chimeric” molecules because they appear as the fusion of two pre-existing
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`molecules. These chimeric molecules could occur between templates derived from
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`a single sample or templates from two different samples.
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`24. The ’852 patent purports to reduce sequencing errors derived from
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`chimeric molecules resulting from the fusion of two templates from two samples,
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`which the ’852 Patent calls “crossover” errors. Specifically, the claims of the
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`’852 patent recite incorporating a distinct pair of identifier sequences (also referred
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`to as “barcodes” or “indexes”) to template nucleic acids from different samples,
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`amplifying the templates on a surface of a flow cell, and discarding any sequence
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`that contains a pair of identifier sequences that was not originally assigned to any of
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`8
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`the template nucleic acids—including chimeric sequences that arose through
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`“crossover” error.
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`25. Based on my knowledge and experience and my review of the
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`information described in this declaration, it is my opinion that claims 1-8 of the
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`’852 patent would have been obvious to a person of ordinary skill in the art
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`(“POSA”).
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`26. As of December 2010, it was well known that chimeric sequences,
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`including those resulting from cross-over errors, were problematic and could lead
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`to, among other problems, misassignment of sequences. By then, it was also well
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`known that using two identifier sequences per sample’s template nucleic acids (i.e.,
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`“dual-indexing”) would be helpful to identify and discard sequences arising from
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`cross-over errors. For example, Parameswaran et al., Nucleic Acids Research
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`(2007) 35:e130 (“Parameswaran”; EX1004) disclosed multiplex sequencing
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`methods where each sample’s template nucleic acids were combined with a pair of
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`identifier sequences. In Parameswaran, every identifier sequence was unique to
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`template nucleic acids from a single sample—a method commonly referred to as
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`“unique” dual-indexing. Using unique dual-indexing, Parameswaran identified
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`erroneous sequences containing incorrect pairs of identifiers, i.e., pairs of identifier
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`sequences that had not been assigned to any sample. Parameswaran explains that
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`these sequences are a “false-discovery,” constituting “misassignment” of sequences.
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`9
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`27.
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`It was also known that dual-indexing methods could be performed
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`using any major next-generation sequencing platform,
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`including Illumina
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`sequencers where template amplification occurs on a surface of a flow cell. While
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`performing the sequencing experiments using a Roche 454 sequencer (where
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`amplification does not occur on a surface of a flow cell), Parameswaran predicted
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`that its methods will be “compatible with other sequencing platforms,” including
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`Illumina sequencers. EX1004 (Parameswaran), 8, 1.
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`28. A few years
`
`later, Gloor et al., Cornell University Library
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`arXiv:1007.5075v1 (2010) (“Gloor”; EX1005) empirically demonstrated that dual-
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`indexing methods are platform agnostic and can be done using Illumina sequencing
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`platforms. Gloor also disclosed multiplex sequencing methods where templates
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`from each sample were tagged with a pair of identifier sequences. While Gloor
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`repeated individual identifier sequences across different samples, Gloor used a
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`distinct pair of forward and reverse identifier sequences per sample—a method
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`commonly referred to as “non-unique” dual-indexing. Using non-unique dual-
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`indexing, Gloor successfully identified erroneous sequences, including those
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`recognized as caused by cross-over errors.
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`29.
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`In addition, Bentley et al., Nature (2008) 456:53-59 (“Bentley”;
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`EX1006) disclosed detailed guidance for how to perform sequencing using Illumina
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`10
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`
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`sequencers, allowing researchers in the field of sequencing to perform dual-indexing
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`methods, like those disclosed in Parameswaran, using Illumina sequencers.
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`30. Thus, each and every element of the claims of the ’852 patent is found
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`in each of the following combinations: (1) Parameswaran and Gloor and
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`(2) Parameswaran and Bentley. As discussed below, a POSA would have been
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`motivated to combine the prior art with a reasonable expectation of success. Indeed,
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`to improve the accuracy of assigning sequences to samples, a POSA would have
`
`been motivated to use Parameswaran’s unique dual-indexing method, which
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`identified chimeric sequences. A POSA would have been motivated to apply
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`Parameswaran’s method to known next generation sequencing platforms including
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`Illumina sequencing, which was known to be cheaper and more accurate than the
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`platform used in Parameswaran. A POSA would have had a reasonable expectation
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`of success in doing so because Bentley provided detailed guidance for Illumina
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`sequencing and Gloor already demonstrated that Illumina sequencing was
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`compatible with dual-indexing methods, while not disclosing any non-obvious
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`technical hurdle that needed to be overcome. Thus, claims 1-8 of the ’852 patent
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`would have been obvious to a POSA as of December 23, 2010. I am currently not
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`aware of any objective evidence of non-obviousness for the claims of the ’852
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`patent. I reserve the right to consider and comment on any such evidence after I sign
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`this declaration.
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`11
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`IV. LEGAL STANDARDS
`31. 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 ’852 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
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`well as those for assessing obviousness of a patent claim.
`
`32.
`
`I have been informed that, in IPR proceedings such as this one, the party
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`challenging the patent bears the burden of proving unpatentability by a
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`preponderance of the evidence. I understand that a preponderance of the evidence
`
`means “more likely than not.”
`
`33.
`
`I understand that patentability must be analyzed from the perspective
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`of a POSA in the same field as the challenged patent as of the “effective filing date”
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`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.
`
`34.
`
`I understand that the Patent Trial and Appeal Board interprets claims
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`based on their ordinary meaning as understood by a POSA at the time of the effective
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`filing date in view of the claim language, patent specification, and prosecution
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`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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`12
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`35.
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`I understand that a patent claim is invalid if it would have been obvious
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`to a person of ordinary skill in the art as of the effective filing date. I understand that
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`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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`36.
`
`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,
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`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
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`success in combining the prior art to achieve the claimed subject matter. I also
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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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`37.
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`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
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`or unsolved problem in the field, or market demand.
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`13
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`38.
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`I also understand that a claim may be obvious if a POSA would have
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`found it obvious to try combining a finite number of predictable solutions known in
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`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
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`may have found it obvious to try that same technique on similar devices.
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`39.
`
`I understand
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`that, when present, evidence of “secondary
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`considerations” must be considered along with the other factual evidence relevant to
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`obviousness. Such secondary considerations could include unexpected results,
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`commercial success of products or processes using the invention, long-felt but unmet
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`need for the invention, failure of others to make the invention, industry acceptance
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`of the invention, or copying of the invention by others. I understand that, in order
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`for such evidence to support nonobviousness, it must have a “nexus” to the features
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`of the invention as claimed.
`
`40.
`
`I have been informed that a dependent claim is a patent claim that refers
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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.
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`14
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`V. BACKGROUND AND STATE OF THE ART
`First Generation Sequencing
`A.
`41. 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,
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`there are four types of nucleotides, which respectively contain adenine (A),
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`guanine (G), thymine (T), and cytosine (C) bases. The two strands of a DNA
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`molecule are held together by hydrogen bond formation between complementary
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`base pairs. EX1031 (Garland), 3. Specifically, guanine (G) is complementary to and
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`pairs with cytosine (C) and adenine (A) is complementary to and pairs with
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`thymine (T). EX1031 (Garland), 3. Each nucleotide has a 5’ phosphate (“5’” or
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`“5’-P”) end and a 3’ hydroxide (“3’” or “3’-OH”) end that links to the 5’ end of the
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`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
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`reaction of a deoxynucleotide triphosphate (dNTP) being used to add the nucleotide
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`releasing a diphosphate in the reaction. EX1031 (Garland), 38.
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`42. Knowing the sequence of a given DNA can be important for any
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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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`15
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`
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`sequencing techniques have been developed, most of which are based on the
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`U.S. Patent No. 11,041,852
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`complementarity of the two strands in DNA.
`
`43.
`
`In 1977, Dr. Frederick Sanger published his method of DNA
`
`sequencing, now known as “Sanger-based sequencing,” which uses a technique
`
`known as non-reversible termination to determine the order of nucleotides in a strand
`
`of DNA. EX1009 (Sanger 1977). The traditional Sanger-based sequencing workflow
`
`began by amplifying a sequence of interest into millions of copies through in vivo
`
`cloning. EX1012 (Shendure), 2. Genomic DNA of a target sequence was
`
`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
`
`bacterial colony was picked and plasmid DNA is isolated. EX1012 (Shendure), 2.
`
`44. Following amplification and isolation, reverse strand synthesis was
`
`performed on these copies using a known priming sequence upstream of the region
`
`to be sequenced. EX1010 (Kircher), 2-3; EX1011 (Metzker), 1-2; EX1012
`
`(Shendure), 1. Reverse strand synthesis used four pools of polymerization reactions,
`
`each with a mixture of dNTPs (i.e., dATP, dGTP, dCTP, and dTTP), plus one
`
`chemically labeled dideoxynucleoside triphosphate, a dNTP missing a hydroxyl
`
`group at the ’3-end (i.e., ddATP, ddGTP, ddCTP, or ddTTP). EX1012 (Shendure),
`
`1; EX1010 (Kircher), 2. In other words, four reactions were performed (consisting
`
`of dNTPs plus ddATP, ddGTP, ddCTP, or ddTTP), wherein polymerization of the
`
`16
`
`
`
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`nucleotides occurred by incorporation of dNTPs. EX1012 (Shendure), 1.
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`IPR2024-00964
`U.S. Patent No. 11,041,852
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`Polymerization was
`
`stochastically
`
`terminated wherever
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`the
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`labeled
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`dideoxynucleotides happened to be incorporated. EX1010 (Kircher), 2-3; EX1011
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`(Metzker), 1-2; EX1012 (Shendure), 1. Incorporation of a dideoxynucleotide into
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`the growing strand terminated DNA polymerization because dideoxynucleotides
`
`lack the 3’-OH group necessary for the phosphodiester bond formation that would
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`have allowed polymerization to continue. EX1010 (Kircher), 2-3; EX1011
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`(Metzker), 1-2; EX1012 (Shendure), 1. The dNTPs/ddNTP mixture thus causes
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`random, non-reversible termination of the extension reaction, creating different
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`copies of molecules extended to different lengths. EX1010 (Kircher), 2-3; EX1011
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`(Metzker), 1-2; EX1012 (Shendure), 1-2.
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`45. The resulting molecules were sorted by molecular weight via
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`electrophoresis (corresponding to the point where a labeled dideoxynucleotide
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`terminated polymerization), and signals generated from labels attached to the
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`terminating dideoxynucleotides were detected to determine which nucleotides were
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`added. EX1010 (Kircher), 2-3; EX1011 (Metzker), 1-2; EX1012 (Shendure), 1-2.
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`46. Although significant improvements were made to the technique after
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`1977, Sanger-based sequencing remained time-consuming and expensive compared
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`to later-developed techniques (“next generation sequencing”), described below.
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`EX1010 (Kircher), 2. Among other disadvantages, Sanger-based sequencing
`
`17
`
`
`
`
`required isolating every molecule to be sequenced separately and thus was limited
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`IPR2024-00964
`U.S. Patent No. 11,041,852
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`to sequencing only one target sequence in each reaction.
`
`47. Consequently, Sanger-based sequencing was ill suited for sequencing
`
`thousands of target sequences in parallel. For example, Sanger-based sequencing
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`was used in the first project of the Cancer Genome Atlas. See EX1035 (TCGA), 6.
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`But it required massive amplification of DNA from more than one hundred
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`specimens and then further amplification of thousands of individual targets to be
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`sequenced in hundreds of samples. See EX1035 (TCGA), 1, 6.
`
`B. Next Generation Sequencing Techniques Available as of December
`2010
`48. Starting in the mid-2000s, “next-generation sequencing” techniques
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`became commercially available, which greatly increased efficiency compared to
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`traditional Sanger-based sequencing. See EX1022 (Li), Abstract. Among other
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`advantages, “next generation sequencing” techniques facilitated first parallel
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`sequencing (sequencing millions of molecules under one set of reaction conditions)
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`and later “multiplex sequencing”—that is, the simultaneous sequencing of templates
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`from multiple samples during a single run. See infra § V.B.2; EX1023 (Choi), 1
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`(reporting “massively parallel DNA sequencing” using next-generation sequencing).
`
`49. As of December 2010, Illumina and Roche 454 were two of the major
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`“next-generation” sequencing platforms known
`
`in
`
`the art. See EX1004
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`18
`
`
`
`
`(Parameswaran), 1. Here, I provide a brief overview of the basic principles and the
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`IPR2024-00964
`U.S. Patent No. 11,041,852
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`sequencing workflow behind Illumina and Roche 454 sequencing.
`
`1. Overview of Sequencing Workflow
`50. Shortly after launching, “next-generation sequencing” techniques such
`
`as Roche 454 and Illumina “outperform[ed] the older Sanger-sequencing
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`technologies by a factor of 100-1,000” and simultaneously “reduc[ed] the cost of
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`sequencing.” EX1010 (Kircher), 1, 3, 5, 11. While the sequencing biochemistries
`
`used for Roche 454 and Illumina platforms differ, “their work flows are conceptually
`
`similar.” EX1012 (Shendure), 2.
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`19
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`
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`IPR2024-00964
`U.S. Patent No. 11,041,852
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`
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`Figure 1. General Sequencing Workflow
`(Revised from EX1012 (Shendure), 2 (Figure 1(b)))
`51. As shown in Figure 1, both platforms start with sample preparation.
`
`This involved extracting DNA from a sample (or extracting RNA and generating
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`cDNA). EX1012 (Shendure), 2 (Figure 1b). Sample preparation also involved
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`20
`
`
`
`
`attaching sequencer-specific adapters, which were used to immobilize copies of the
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`IPR2024-00964
`U.S. Patent No. 11,041,852
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`DNA on a solid substrate during sequencing. Both platforms commonly used PCR
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`to amplify the adapter-bound DNA molecules prior to sequencing. EX1012
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`(Shendure), 2 (Figure 1b); EX1010 (Kircher), 4. While Roche 454 and Illumina
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`platforms used their own platform-specific adapters, they shared many principles of
`
`operation and conf
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