UNITED STATES PATENT AND TRADEMARK OFFICE
`___________________
`
`BEFORE THE PATENT TRIAL AND APPEAL BOARD
`___________________
`
`TWINSTRAND BIOSCIENCES, INC.
`Petitioner,
`v.
`GUARDANT HEALTH
`Patent Owner.
`
`___________________
`
`Inter Partes Review Case No. IPR2022-01400
`
`U.S. Patent No. 11,149,306
`___________________
`
`DECLARATION OF PAUL T. SPELLMAN, Ph.D.
`
`Mail Stop "PATENT BOARD"
`Patent Trial and Appeal Board
`U.S. Patent and Trademark Office
`P.O. Box 1450
`Alexandria, VA 22313-1450
`
`EX1002
`
`

`

`Inter Partes Review of US Patent 11,149,306
`Declaration of Paul T. Spellman, Ph.D.
`
`TABLE OF CONTENTS
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`- i -
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`B.
`
`C.
`
`INTRODUCTION ....................................................................................... 1
`I.
`II. MY BACKGROUND AND QUALIFICATIONS ....................................... 2
`III.
`SUMMARY OF OPINIONS ....................................................................... 4
`IV. LIST OF DOCUMENTS CONSIDERED.................................................. 10
`V.
`PERSON OF ORDINARY SKILL IN THE ART...................................... 16
`VI. STATE OF THE ART ............................................................................... 17
`A.
`The protocols for DNA sequencing were well known in the art ........17
`1.
`Genetic information comprises the building blocks of life ..... 17
`2.
`DNA sequencing was commonly performed to determine
`the order of nucleotides in an individual’s DNA .................... 18
`Next-generation sequencing involved well-known steps........ 20
`a)
`Template preparation and tagging................................ 20
`b)
`Amplification............................................................... 31
`c)
`Enrichment .................................................................. 32
`d)
`Sequencing and detection............................................. 33
`e)
`Sequence alignment and assembly ............................... 34
`Prior to December 28, 2013, cell-free DNA in blood had
`attracted interest for the diagnosis of cancer, fetal gender, and
`many other inherited disorders and was commonly sequenced
`using NGS platforms ........................................................................35
`1.
`Cell-free tumor DNA was a well-known cancer
`biomarker............................................................................... 36
`Circulating cell-free fetal DNA was also a well-known
`biomarker for prenatal screening and diagnostics................... 42
`Commercially available kits were routinely used to
`extract and isolate cfDNA from blood samples ...................... 43
`Prior to December 28, 2013, Duplex Sequencing was developed
`to dramatically improve the accuracy of next-generation
`sequencing ........................................................................................44
`
`3.
`
`2.
`
`3.
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`

`

`D.
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`Inter Partes Review of US Patent 11,149,306
`Declaration of Paul T. Spellman, Ph.D.
`Tagging of DNA with adapters comprising molecular
`barcodes................................................................................. 46
`Amplification......................................................................... 51
`2.
`Target enrichment.................................................................. 52
`3.
`Sequencing ............................................................................ 53
`4.
`5. Mapping the sequence reads to a reference sequence ............. 53
`6.
`Grouping the sequenced strands into paired families.............. 54
`7.
`Generating a single-strand consensus sequences .................... 56
`8.
`Comparing the single strand consensus sequence to its
`complementary strand-mate (generating a Duplex
`Consensus)............................................................................. 57
`Prior to December 28, 2013, the art taught that Duplex
`Sequencing could be used to sequence cfDNA .................................58
`VII. THE ’306 PATENT SPECIFICATION AND CLAIMS ............................ 61
`VIII. PROSECUTION HISTORY...................................................................... 68
`IX. THE MEANING OF CLAIM TERMS ...................................................... 73
`X.
`LEGAL BASIS FOR MY ANALYSIS...................................................... 74
`XI. KEY PRIOR ART ..................................................................................... 75
`A. Narayan (EX1082)............................................................................76
`B.
`Schmitt (EX1009), Schmitt-623 (EX1083) .......................................76
`C. Meyer (EX1005)...............................................................................82
`D. Kivioja (EX1006) .............................................................................83
`E.
`Craig (EX1007) ................................................................................83
`XII. GROUND 1: CLAIMS 1-3, 5, 7, 9-14, 17-27, AND 29 WOULD
`HAVE BEEN OBVIOUS OVER NARAYAN AND SCHMITT............... 84
`A.
`Claim 1 .............................................................................................85
`1.
`A method, comprising: (a) providing a population of cell-
`free deoxyribonucleic acid (cfDNA) molecules having
`first and second complementary strands................................. 85
`(b) tagging a plurality of the cfDNA molecules in the
`population with duplex tags comprising molecular
`barcodes to produce tagged parent polynucleotides,
`
`1.
`
`2.
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`- ii -
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`

`

`3.
`
`4.
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`Inter Partes Review of US Patent 11,149,306
`Declaration of Paul T. Spellman, Ph.D.
`wherein the duplex tags are attached to both ends of a
`molecule of the plurality of the cfDNA molecules, ................ 86
`wherein the plurality of the cfDNA molecules are tagged
`with n different combinations of molecular barcodes,
`wherein n is at least 2 and no more than 100,000*z,
`wherein z is a mean of an expected number of duplicate
`molecules in the population of cfDNA molecules that
`map to identical start and stop positions on a reference
`sequence ................................................................................ 88
`(c) amplifying a plurality of the tagged parent
`polynucleotides to produce amplified progeny
`polynucleotides...................................................................... 91
`(d) sequencing at least a subset of the amplified progeny
`polynucleotides to produce a set of sequence reads; and ........ 92
`(e) reducing or tracking redundancy of a plurality of
`sequence reads from the set of sequence reads using at
`least sequencing information from the molecular
`barcodes of the duplex tags to determine distinct cfDNA
`molecules from among the tagged parent
`polynucleotides, wherein the distinct cfDNA molecules
`are determined based on (i) paired reads corresponding to
`sequence reads generated from a first tagged strand and a
`second tagged complementary strand derived from
`cfDNA molecules from among the tagged parent
`polynucleotides, or (ii) unpaired reads corresponding to
`sequence reads generated from a first tagged strand
`having no second tagged complementary strand derived
`from cfDNA molecules from among the tagged parent
`polynucleotides, wherein reducing or tracking the
`redundancy of the plurality of sequence reads comprises
`mapping at least a subset of the plurality of sequence
`reads to the reference sequence. ............................................. 92
`B. Motivation to combine......................................................................99
`1.
`A POSA would have been motivated to use Schmitt’s 3-
`mer hybrid tagging approach to tag cfDNA molecules..........102
`A POSA would have been motivated to use the number
`of different combinations of molecular barcodes that
`
`2.
`
`5.
`
`6.
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`- iii -
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`

`

`3.
`
`2.
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`3.
`
`C.
`
`D.
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`Inter Partes Review of US Patent 11,149,306
`Declaration of Paul T. Spellman, Ph.D.
`ranges from at least 2 to no more than 100,000*z
`(100,000 (cid:117) the number of duplicate molecules that map
`to identical start and stop positions). .....................................105
`A POSA would have been motivated to amplify,
`sequence, map, and reduce or track redundancy through
`paired and unpaired reads......................................................106
`Reasonable expectation of success..................................................108
`1.
`A POSA would have reasonably expected to successfully
`use Schmitt’s Duplex Sequencing method on Narayan’s
`cfDNA because of knowledge in the art that a routine
`blood draw contains more than sufficient quantities of
`cfDNA. .................................................................................110
`A POSA would have reasonably expected to successfully
`tag cfDNA molecules............................................................112
`A POSA would have reasonably expected to successfully
`tag, amplify, sequence, map, and reduce or track
`redundancy to generate a plurality of consensus
`sequences as recited in claim 1..............................................113
`Claim 17 .........................................................................................113
`1.
`A method, comprising: (a) tagging a population of
`double-stranded cell-free deoxyribonucleic acid (cfDNA)
`molecules obtained or derived from a sample of a subject
`with a set of tags comprising molecular barcodes to
`produce tagged parent polynucleotides .................................113
`(b) amplifying a plurality of the tagged parent
`polynucleotides to produce amplified progeny
`polynucleotides;....................................................................115
`(c) sequencing at least a subset of the amplified progeny
`polynucleotides to produce a set of sequence reads...............116
`(d) sorting a plurality of sequence reads from the set of
`sequence reads into (i) families comprising paired reads
`corresponding to sequence reads generated from a first
`tagged strand and a second tagged complementary strand
`derived from double-stranded cfDNA molecules from
`among the tagged parent polynucleotides, and (ii)
`families comprising unpaired reads corresponding to
`
`2.
`
`3.
`
`4.
`
`- iv -
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`

`

`J.
`K.
`
`L.
`
`E.
`F.
`G.
`H.
`I.
`
`Inter Partes Review of US Patent 11,149,306
`Declaration of Paul T. Spellman, Ph.D.
`sequence reads generated from a first tagged strand
`having no second tagged complementary strand derived
`from double-stranded cfDNA molecules from among the
`tagged parent polynucleotides...............................................116
`Claims 2 and 18 ..............................................................................119
`Claims 3 and 19 ..............................................................................122
`Claims 5 and 20 ..............................................................................124
`Claim 7: The method of claim 1, wherein z is between 2 and 8.......129
`Claim 21: The method of claim 17, wherein the set of tags
`comprises 2 to 10,000 different molecular barcode sequences. .......130
`Claims 9-10 and 22-23....................................................................132
`Claim 11: The method of claim 10, further comprising, prior to
`the sequencing, amplifying a plurality of the enriched progeny
`polynucleotides...............................................................................136
`Claim 12: The method of claim 1, wherein the duplex tags are
`part of sequencing adapters.............................................................138
`M. Claim 13: The method of claim 1, wherein reducing or tracking
`the redundancy of the plurality of sequence reads comprises
`grouping the paired reads or the unpaired reads into families
`based at least in part on (i) the molecular barcodes associated
`with te paired reads or the unpaired reads and (ii) sequence
`information at the start and stop positions of the paired reads or
`the unpaired reads. ..........................................................................139
`Claims 14 and 27 ............................................................................141
`Claim 24: The method of claim 17, wherein (d) further
`comprises reducing or tracking redundancy of a plurality of
`sequence reads from the set of sequencing reads, wherein the
`reducing or tracking comprises mapping at least a subset of the
`plurality of sequence reads to a reference sequence, and the
`reducing or tracking is based on (i) the molecular barcodes
`associated the paired reads or the unpaired reads and (ii)
`sequence information at the start and stop positions of the
`paired reads or the unpaired reads. ..................................................145
`Claim 25: The method of claim 24, wherein reducing or
`tracking the redundancy of the plurality of sequence reads
`
`N.
`O.
`
`P.
`
`- v -
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`

`

`R.
`
`Q.
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`Inter Partes Review of US Patent 11,149,306
`Declaration of Paul T. Spellman, Ph.D.
`comprises determining a base call at one or more genomic loci
`for a plurality of sequence reads that map to the one or more
`genomic loci on the reference sequence. .........................................146
`Claim 26: The method of claim 24, wherein reducing or
`tracking the redundancy of the plurality of sequence reads
`comprises collapsing the plurality of sequence reads to produce
`consensus sequences representative of a sequence of the
`original double-stranded cfDNA molecules from among the
`tagged parent polynucleotides.........................................................149
`Claim 29: The method of claim 1, wherein the distinct cfDNA
`molecules in (e) are determined based on (i) the paired reads
`and (ii) the unpaired reads...............................................................150
`XIII. GROUND 2: CLAIMS 4 AND 6 WOULD HAVE BEEN OBVIOUS
`IN VIEW OF NARAYAN, SCHMITT, AND MEYER ............................151
`XIV. GROUND 3: CLAIM 8 WOULD HAVE BEEN OBVIOUS IN VIEW
`OF NARAYAN, SCHMITT, AND CRAIG..............................................158
`A. A POSA would have been motivated to incorporate Craig’s pre-
`determined barcode sequences into Schmitt’s Duplex
`Sequencing method.........................................................................159
`Expectation of Success ...................................................................162
`B.
`XV. GROUND 4: CLAIMS 15-16 AND 28 WOULD HAVE BEEN
`OBVIOUS OVER NARAYAN, SCHMITT, AND KIVIOJA...................162
`1.
`Claims 15 and 28 ..................................................................163
`2.
`Claim 16: The method of claim 15, further comprising:
`(h) detecting copy number variation in the population of
`cfDNA molecules by determining a normalized
`quantitative measure determined in (g) at each of one or
`more genomic loci. ...............................................................169
`XVI. OBJECTIVE EVIDENCE OF NONOBVIOUSNESS DO NOT
`SUPPORT PATENTABILITY .................................................................171
`XVII. CONCLUSION ........................................................................................172
`
`- vi -
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`

`

`Inter Partes Review of US Patent 11,149,306
`Declaration of Paul T. Spellman, Ph.D.
`I, Paul T. Spellman, hereby declare as follows.
`
`I.
`
`INTRODUCTION
`1.
`I have been retained as an expert witness on behalf of TwinStrand
`
`Biosciences, Inc. for the above-captioned inter partes review (IPR). I am being
`
`compensated for my time in connection with this IPR at my standard consulting
`
`rate, which is $400 per hour.
`
`2.
`
`I understand that this Declaration accompanies a petition for IPR
`
`involving U.S. Patent No. 11,149,306 ("the ’306 patent") (EX1001), which resulted
`
`from U.S. Patent Application No. 16/945,124 ("the ’124 application"), filed on July
`
`31, 2020. I understand that the ’306 patent alleges a priority date of December 28,
`
`2013. I refer to this date throughout this declaration.
`
`3.
`
`In preparing this Declaration, I have reviewed the ’306 patent and
`
`each of the documents cited herein, in light of general knowledge in the art before
`
`December 28, 2013. In formulating my opinions, I have relied upon my
`
`experience, education, and knowledge in the relevant art. In formulating my
`
`opinions, I have also considered the viewpoint of a person of ordinary skill in the
`
`art ("POSA") (i.e., a person of ordinary skill in the field of molecular biology, as
`
`defined further below in §V) prior to December 28, 2013.
`
`4.
`
`I further understand that, according to the United States Patent and
`
`Trademark Office (“USPTO”) assignment records, the ’306 is currently assigned to
`
`- 1 -
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`

`

`Inter Partes Review of US Patent 11,149,306
`Declaration of Paul T. Spellman, Ph.D.
`
`Guardant Health, Inc.
`
`II. MY BACKGROUND AND QUALIFICATIONS
`5.
`I am a Full Professor with Tenure in the Department of Molecular and
`
`Medical Genetics at Oregon Health & Science University School of Medicine
`
`(“OHSU”). I have held this position since 2013. I am also co-leader of the
`
`Quantitative Oncology Program in the OHSU Knight Cancer Institute, a position I
`
`have held since 2017. I am also Co-Director of the Cancer Early Detection
`
`Advanced Research (“CEDAR”) Center. From 2018-2020, I was Interim Director
`
`for the Program of Computational Biology at OHSU School of Medicine. From
`
`2011-2013, I served as an Associate Professor at OHSU School of Medicine. From
`
`2003-2011, I was a Staff Scientist at the Life Science Division of Lawrence
`
`Berkeley Lab.
`
`6.
`
`I received a Bachelor’s degree in Biology in 1995 from the
`
`Massachusetts Institute of Technology. In 2000, I received my Ph.D. in Genetics
`
`from Stanford University Medical School. My thesis topic was “Generating and
`
`Analyzing Genome Scale Data.” From 2000-2003, I was a Post-Doctoral Fellow in
`
`the Department of Molecular and Cellular Biology at University of California,
`
`Berkeley.
`
`7.
`
`My current research focuses on computational biology, cancer
`
`biology, and cancer genomics. My lab develops technologies and approaches to
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`- 2 -
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`

`

`Inter Partes Review of US Patent 11,149,306
`Declaration of Paul T. Spellman, Ph.D.
`understand the processes by which cancer develops, monitor disease, and to
`
`identify therapeutic strategies. Within this broad area of research, I have specific
`
`experience in using population genetics to help determine who is at risk for cancer,
`
`how to computationally analyze genomic data to identify early changes in cancers,
`
`and how to accurately screen different populations for the disease. My team
`
`develops new methodologies for identifying changes in the cancer genome,
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`systematic integration of multiple genomic data types, including copy number,
`
`expression and mutation, to better understand the process by which cancer
`
`develops.
`
`8.
`
`CEDAR is a $314 million effort funded by a gift from Phil and Penny
`
`Knight to detect and treat early cancers. Among other activities, my work as Co-
`
`Director of CEDAR includes identifying populations at risk for developing cancer
`
`and understanding the early biology of breast cancers.
`
`9.
`
`I have over 20 years of experience in the field of genomics and 15
`
`years of experience in cancer genomics. I have taught undergraduate and graduate
`
`level courses at OHSU in the area of sequencing technologies, computational
`
`analysis, and genetics. I have also supervised three completed Ph.D. theses since I
`
`joined the OHSU faculty in 2011.
`
`10.
`
`I have authored or co-authored more than 115 peer-reviewed
`
`publications that discuss methods of DNA manipulation and analysis, including
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`Inter Partes Review of US Patent 11,149,306
`Declaration of Paul T. Spellman, Ph.D.
`DNA sample preparation for sequencing, amplification (e.g. PCR), methods of
`
`DNA sequencing (including NGS and related sequencing methods), and
`
`bioinformatics methods for raw data analysis. In addition, I have presented over
`
`100 invited lectures or conference presentations regarding these topics. My
`
`curriculum vitae includes a sample list of these publications and presentations.
`
`11.
`
`I have received over $16 million in federal grants for my research in
`
`the area of cancer genomic and cancer precision medicine. I am currently one of 30
`
`principal investigators for the Genome Data Analysis Network, sponsored by the
`
`National Cancer Institute. The Genome Data Analysis Network is a consortium of
`
`computational researchers from across the United States focused on developing the
`
`framework for relating the genomics and outcomes of patients in cancer clinical
`
`trials.
`
`12. As a postdoctoral scholar, I was selected for the prestigious National
`
`Science Foundation Biocomputing Fellowship, which covered both my stipend and
`
`significant research costs. In 2017, I was named the inaugural holder of the Penny
`
`and Phil Knight Endowed Professorship for Cancer Research Innovation.
`
`III.
`
`SUMMARY OF OPINIONS
`13. Generally speaking, the ’306 patent is directed to methods of
`
`sequencing cfDNA molecules using commonly known, routinely performed
`
`sequencing methods that were taught in the art. In particular, the claims recite
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`Inter Partes Review of US Patent 11,149,306
`Declaration of Paul T. Spellman, Ph.D.
`methods comprising the following general steps: (1) tagging with adapters; (2)
`
`amplifying; (3) sequencing; and (4) reducing or tracking redundancy in the set of
`
`sequence reads based on paired and unpaired reads. There is nothing inventive in
`
`the steps of the ’306 patent claims.
`
`14.
`
`The claimed methods would have been obvious to a POSA in view of
`
`disclosures in the art cited in the Grounds below.
`
`15. Narayan teaches sequencing of circulating cell-free tumor DNA in
`
`plasma samples to track treatment-associated changes in circulating tumor DNA
`
`levels in patients with non-small cell lung cancer. EX1082, Abstract, 3492. To do
`
`so, Narayan discloses performing ultradeep sequencing on plasma samples of 30
`
`patients with stage 1-IV non-small cell lung cancer. EX1082, 3493.
`
`16.
`
`Schmitt teaches all the steps of Guardant’s claimed methods,
`
`including tagging DNA fragments (EX1009, ¶¶[0022], [0026], [0032]-[0034];
`
`Figs. 1-2; EX10831, ¶¶[0015], [0016], [0024]-[0026], Figs. 1-2), amplifying the
`
`1 As discussed below, Schmitt (EX1009) claims priority to provisional
`
`application no. 61/625,623 (“Schmitt-623,” EX1083). Since Schmitt-623 provides
`
`support for at least one claim in Schmitt, Schmitt is prior art to the ’306 patent at
`
`least as of Schmitt-623’s filing date of April 17, 2012. As such, I have provided
`
`citations to both Schmitt and Schmitt-623 in support of my opinions in this
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`Inter Partes Review of US Patent 11,149,306
`Declaration of Paul T. Spellman, Ph.D.
`tagged fragments (EX1009, ¶¶[0006], [0010]; EX1083, ¶¶[0005], [0009]),
`
`sequencing the DNA (EX1009, ¶¶[0074], [0095]; EX1083, ¶¶[0042], [0059]), and
`
`reducing or tracking redundancy in sequencing reads (EX1009, ¶¶[0096], [0099],
`
`[00105]; EX1083, ¶¶[0060], [0063], [0068]) in a method called Duplex
`
`Sequencing.
`
`17. Meyer discloses parallel tagged sequencing as a molecular barcoding
`
`method designed to adapt the 454 parallel sequencing technology for use with
`
`multiple samples. EX1005, Abstract. Meyer discloses an overview of the tagging
`
`protocol and teaches that after each DNA sample is blunt-end repaired and sample-
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`specific barcoding adapters are ligated to both ends of the molecules, the barcoded
`
`samples are pooled in equimolar ratios and unligated molecule ends are excluded
`
`from sequencing. EX1005, Fig. 1, 272-274. Meyer discloses that “the expected
`
`overall recovery” after quantifying tagged samples “is between 40 and 60%.”
`
`EX1005, 274.
`
`18. Kivioja teaches methods of quantitating unseen DNA molecules using
`
`count statistics by adding random DNA sequence labels (unique molecular
`
`identifiers). EX1006, 1. Specifically, Kivioja teaches how to accurately estimate
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`the number of molecules generated without actually observing all of them.
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`declaration.
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`Inter Partes Review of US Patent 11,149,306
`Declaration of Paul T. Spellman, Ph.D.
`
`EX1006, 74, 16-18.
`
`19.
`
`Craig discloses “[a] total of 48 different 6-mer index sequences” that
`
`were “appended to adapter sequence[s]” and used for sequencing on the Illumina
`
`platform. EX1007, Suppl. Table 4, Suppl. Methods. Craig teaches that the
`
`disclosed 6-mer barcode design can “control, tolerate, and measure error during
`
`base-calling.” EX1007, 888. Craig also discloses that the 48 6-mer barcodes that
`
`were disclosed that indexes were designed “so that one, and in some cases two,
`
`sequencing errors could be tolerated without [a barcode] being incorrectly
`
`identified as being a different valid [barcode].” EX1007, 888.
`
`20. As discussed more fully below, claims 1-3, 5, 7, 9-14, 17-27, 29 of the
`
`’306 patent would have been obvious to a POSA over the combination of Narayan
`
`and Schmitt. A POSA would have had a reason to combine the teachings of
`
`Narayan and Schmitt to arrive at the claimed methods. This is because (1) a POSA
`
`would have been motivated to apply Schmitt’s Duplex Sequencing error correction
`
`and improved accuracy to Narayan’s explicit teachings of cfDNA and specific
`
`cancer genes; and (2) a POSA would have been motivated to tag cfDNA molecules
`
`using Schmitt’s hybrid tagging method.
`
`21. A POSA also would have reasonably expected to use Schmitt’s
`
`Duplex Sequencing method on cfDNA because the prior art taught that Duplex
`
`Sequencing could be used to enhance accuracy when sequencing cfDNA from
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`Inter Partes Review of US Patent 11,149,306
`Declaration of Paul T. Spellman, Ph.D.
`cancer patients. EX1008, 7. Furthermore, the art taught that sufficient quantities of
`
`cfDNA could be extracted from a routine blood draw and sequenced using
`
`Schmitt’s Duplex Sequencing methods. Thus, a POSA would have reasonably
`
`expected to be able to perform the claimed sequencing methods based on the
`
`disclosures in Narayan and Schmitt.
`
`22.
`
`Claims 4 and 6 would have been obvious to a POSA over the
`
`combination of Narayan, Schmitt, and Meyer. A POSA would have been
`
`motivated to combine Narayan and Schmitt as discussed above. In addition, a
`
`POSA would have been motivated to optimize the adapter-DNA ligation efficiency
`
`to maximize the percentage of adapter-ligated DNA fragments after ligation based
`
`on the disclosures in Meyer. Moreover, a POSA would have reasonably expected
`
`to successfully tag cfDNA molecules to achieve a high adapter-DNA ligation
`
`efficiency because Schmitt and Meyer disclose that improving ligation efficiency
`
`increases the sensitivity of sequence detection. EX1009, ¶[0006], EX1083,
`
`¶[0005], EX1005, 274.
`
`23.
`
`In addition, claim 8 of the ’306 patent would have been obvious to a
`
`POSA over the combination of Narayan, Schmitt, and Craig. A POSA would have
`
`been motivated to combine Narayan and Schmitt as discussed above. In addition, a
`
`POSA would have been motivated to use Craig’s 6-mer barcodes in combination
`
`with Schmitt because Schmitt teaches performing Duplex Sequencing at greater
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`Inter Partes Review of US Patent 11,149,306
`Declaration of Paul T. Spellman, Ph.D.
`depths for greater sensitivity and Craig discloses barcodes that are able to mitigate
`
`errors in the barcode sequence itself, which is especially important when
`
`sequencing at greater depths. Moreover, a POSA would have reasonably expected
`
`to successfully use Craig’s barcodes in Schmitt’s Duplex Sequencing method
`
`because Schmitt discloses that a 6-mer barcode can be used in the hybrid tag
`
`embodiment, and that the molecular barcodes can be pre-determined sequences,
`
`just like Craig’s.
`
`24. As discussed below, claims 15-16, 28 of the ’306 patent would have
`
`been obvious to a POSA over the combination of Narayan, Schmitt, and Kivioja. A
`
`POSA would have had a reason to combine the teachings of Narayan and Schmitt,
`
`as already discussed above. A POSA would further have been motivated to
`
`combine Narayan and Schmitt with Kivioja because Schmitt expressly cites and
`
`incorporates Kivioja in its disclosure. EX1009, ¶[0084], 48; EX1083, ¶[0048], 41.
`
`In addition, Kivioja teaches methods of quantitating total and unseen DNA
`
`molecules, which would be beneficial because calculating the absolute numbers of
`
`molecules, including unseen cfDNA molecules, can “improve accuracy of almost
`
`any next-generation sequencing method.” EX1006, Abstract, EX1009, ¶[0084];
`
`EX1083, ¶[0048]. A POSA would have also been motivated to quantitate the total
`
`and unseen cfDNA molecules because such data are useful in determining gene
`
`copy number variations, such as in cancer diagnostics and screening. EX1032;
`
`- 9 -
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`

`

`Inter Partes Review of US Patent 11,149,306
`Declaration of Paul T. Spellman, Ph.D.
`
`EX1051.
`
`25.
`
`Furthermore, a POSA would have reasonably expected success in
`
`calculating these quantitative measures of claims 15-16, 28 because doing so
`
`requires nothing more than mathematical calculations after determining the
`
`quantities of paired reads, unpaired reads, and absolute molecules, as disclosed in
`
`Kivioja and Schmitt. As such, a POSA would have arrived at the claimed methods
`
`based on the disclosures in Narayan, Schmitt, and Kivioja.
`
`26.
`
`In addition, I am aware of no objective evidence that would support
`
`patentability of claims 1-29, as I explain in Section XV.
`
`1001
`
`1004
`
`1005
`
`1006
`
`1007
`
`IV. LIST OF DOCUMENTS CONSIDERED
`Ex. No. Description
`Eltoukhy, H., et al., “Methods And Systems For Detecting Genetic
`Variants,” U.S. Patent No. 11,149,306 (filed July 31, 2020; issued
`October 19, 2021)
`Murtaza, M., et al., “Non-invasive analysis of acquired resistance to
`cancer therapy by sequencing of plasma DNA,” Nature 497: 108-112
`(2013)
`Meyer, M., et al., “Parallel tagged sequencing on the 454 platform,”
`Nature Protocols 3(2): 267-278 (2008)
`Kivioja, T., et al., “Counting absolute numbers of molecules using
`(cid:88)(cid:81)(cid:76)(cid:84)(cid:88)(cid:72)(cid:3)(cid:80)(cid:82)(cid:79)(cid:72)(cid:70)(cid:88)(cid:79)(cid:68)(cid:85)(cid:3)(cid:76)(cid:71)(cid:72)(cid:81)(cid:87)(cid:76)(cid:191)(cid:72)(cid:85)(cid:86)(cid:15)(cid:180)(cid:3)Nature Methods 9(1): 72-76 (2012)
`Craig, D.W., et al., “Identification Of Genetic Variants Using
`Barcoded Multiplexed Sequencing”, Nature Methods 5:887–893
`(2008)
`Kukita, Y., et al., “Quantitative Identification of Mutant Alleles
`Derived from Lung Cancer in Plasma Cell-Free DNA via Anomaly
`Detection Using Deep Sequencing Data,” PLOS One 8(11): 1-31
`(2013)
`
`1008
`
`- 10 -
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`

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`Inter Partes Review of US Patent 11,149,306
`Declaration of Paul T. Spellman, Ph.D.
`
`1009
`
`Ex. No. Description
`Schmitt, M., et al., “Method of Lowering the Error Rate of Massively
`Parallel DNA Sequencing Using Duplex Consensus Sequencing,”
`International Publication Number WO2013/142389 (filed on March
`15, 2003; published on September 26, 2013)
`Alberts, B., et al., Eds., “Chapter 4: DNA and Chromosomes,” and
`“Chapter 8: Manipulating Protein, DNA and RNA”, Molecular
`Biology of the Cell, pp. 191-234 and pp. 469-546, Fourth Edition,
`Garland Science, United States (2002)
`Metzker, M.L., “Sequencing technologies — the next generation,”
`Nature Reviews 11:31-46 (2010)
`Mardis, E.R., “Next-Generation Sequencing Platforms,” Annu. Rev.
`Anal. Chem. 6:287–303 (2013)
`Franca, L.T.C., et al., “A Review of DNA Sequencing techniques,”
`Quarterly Reviews of Biophysics 35(2): 169-200 (2002)
`Ong, J., et al., “Overview of the Agilent Technologies SureSelectTM
`Target Enrichment System,” Journal of Biomolecular Techniques,
`22(Suppl.): S30 (2011)
`Technical Data Sheet, KAPA HTP Library Preparation Kit Illumina®
`platforms, KAPA Biosystems (July 2013)
`Rohland, N. and Reich, D., “Cost-effective, high-throughput DNA
`sequencing libraries for multiplexed target capture,” Genome Research
`22:939–946 (2012)
`Zheng, Z., “Titration-free 454 sequencing using Y adapters,” Nature
`Protocols 6(9): 1367-1376 (2011)
`Glenn, T.C., “Field guide to next-generation DNA sequencers,”
`Molecular Ecology Resources 11: 759–769 (2011)
`Neiman, M., et al., “Library Preparation and Multiplex Capture for
`Massive Parallel Sequencing Applications Made Efficient and Easy,”
`PLOS ONE 7(11): e48616 (2012)
`Blumenstiel, B., et al., “Targeted Exon Sequencing by In-Solution
`Hybrid Selection,” Current Protocols in Human Genetics 18.4.1-
`18.4.24 (2010)
`So, A.P., et al., “Increasing the efficiency of SAGE adaptor ligation
`by directed ligation chemistry,” Nucleic Acids Research 32(12): e96
`(2004)
`
`1019
`
`1010
`
`1011
`
`1012
`
`1013
`
`1014
`
`1015
`
`1016
`
`1017
`
`1018
`
`1020
`
`1021
`
`- 11 -
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`

`

`Inter Partes Review of US Patent 11,149,306
`Declaration of Paul T. Spellman, Ph.D.
`
`1022
`
`1023
`
`1024
`
`1025
`
`1026
`
`1027
`
`Ex. No. Description
`van Nieuwerburgh, F., et al