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. IPR2022-01152
`
`U.S. Patent No. 11,118,221
`___________________
`
`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 11,118,221
`Declaration of Paul T. Spellman, Ph.D.
`
`
`TABLE OF CONTENTS
`
`- 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 ........ 19
`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 ................... 41
`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 11,118,221
`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 .............. 53
`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 ’221 PATENT SPECIFICATION AND CLAIMS ............................. 61
`VIII. PROSECUTION HISTORY ........................................................................ 67
`IX. THE MEANING OF CLAIM TERMS ........................................................ 73
`X.
`LEGAL BASIS FOR MY ANALYSIS ........................................................ 74
`XI. KEY PRIOR ART ........................................................................................ 76
`A. Narayan (EX1082) .............................................................................. 76
`B.
`Schmitt (EX1009), Schmitt-623 (EX1083) ......................................... 76
`C. Meyer (EX1005) .................................................................................. 82
`D. Kivioja (EX1006) ................................................................................ 83
`E.
`Craig (EX1007) ................................................................................... 84
`XII. GROUND 1: CLAIMS 1-4, 6-7, 9-15, 18-22, AND 24-28 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 of the
`population with a set of duplex tags comprising
`molecular barcodes from a set of molecular barcodes to
`
`1.
`
`2.
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`- ii -
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`

`

`3.
`
`4.
`
`5.
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`Inter Partes Review of US Patent 11,118,221
`Declaration of Paul T. Spellman, Ph.D.
`produce tagged parent polynucleotides, wherein duplex
`tags from the set of duplex tags are attached at both ends
`of a molecule of the plurality of the cfDNA molecules ........... 86
`(c) amplifying a plurality of the tagged parent
`polynucleotides to produce amplified progeny
`polynucleotides ........................................................................ 88
`(d) sequencing at least a subset of the amplified progeny
`polynucleotides to produce a set of sequence reads; and ........ 89
`(f) reducing or tracking redundancy in the set of sequence
`reads using at least sequence information from the
`molecular barcodes to generate a plurality of consensus
`sequences representative of original cfDNA molecules
`from among the tagged parent polynucleotides, wherein
`the plurality of consensus sequences is generated from (i)
`paired reads corresponding to sequence reads generated
`from a first tagged strand and a second tagged
`complementary strand derived from a cfDNA molecule
`from among the tagged parent polynucleotides, and (ii)
`unpaired reads corresponding to sequence reads
`generated from a first tagged strand having no second
`tagged complementary strand derived from a cfDNA
`molecule from among the tagged parent polynucleotides ....... 90
`B. Motivation to combine ........................................................................ 93
`1.
`A POSA would have been motivated to use Schmitt’s 3-
`mer hybrid tagging approach to tag cfDNA molecules. .......... 96
`A POSA would have been motivated to amplify,
`sequence, and reduce or track redundancy through paired
`and unpaired reads. .................................................................. 98
`Reasonable expectation of success ....................................................100
`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. ................................................................................... 102
`A POSA would have reasonably expected to successfully
`tag cfDNA molecules. ............................................................ 104
`
`C.
`
`2.
`
`2.
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`- iii -
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`

`

`D.
`
`3.
`
`3.
`
`4.
`
`2.
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`Inter Partes Review of US Patent 11,118,221
`Declaration of Paul T. Spellman, Ph.D.
`A POSA would have reasonably expected to successfully
`amplify, sequence, and reduce or track redundancy to
`generate a plurality of consensus sequences as recited in
`claim 1. ................................................................................... 105
`Claim 18 ............................................................................................105
`1.
`A method, comprising: (a) providing a population of
`double-stranded cell-free deoxyribonucleic acid (cfDNA)
`molecules having first and second complementary
`strands .................................................................................... 105
`(b) non-uniquely tagging a plurality of the double-
`stranded cfDNA molecules of the population with a set of
`duplex tags comprising molecular barcodes from a set of
`molecular barcodes to produce non-uniquely tagged
`parent polynucleotides; .......................................................... 106
`wherein the double-stranded cfDNA molecules that map
`to a mappable base position of a reference sequence are
`tagged with a number of different molecular barcodes
`ranging from at least 2 to fewer than a number of the
`double-stranded cfDNA molecules that map to the
`mappable base position; ......................................................... 108
`(c) amplifying a plurality of the non-uniquely tagged
`parent polynucleotides to produce amplified progeny
`polynucleotides; ..................................................................... 110
`(d) sequencing at least a subset of the amplified progeny
`polynucleotides to produce a set of sequence reads .............. 110
`(e) reducing or tracking redundancy in the set of
`sequence reads using at least sequence information from
`the molecular barcodes; ......................................................... 111
`(f) sorting the set of sequence reads into paired reads and
`unpaired reads, wherein (i) a paired read corresponds to
`sequence reads generated from a first tagged strand and a
`second tagged complementary strand derived from a
`double-stranded cfDNA molecule from among the non-
`uniquely tagged parent polynucleotides, and (ii) an
`unpaired read corresponds to sequence reads generated
`from a first tagged strand having no second tagged
`complementary strand derived from a double-stranded
`
`5.
`
`6.
`
`7.
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`- iv -
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`

`

`8.
`
`I.
`
`J.
`
`E.
`F.
`G.
`H.
`
`Inter Partes Review of US Patent 11,118,221
`Declaration of Paul T. Spellman, Ph.D.
`cfDNA molecule from among the non-uniquely tagged
`parent polynucleotides; and ................................................... 111
`(g) determining, at one or more loci of a reference
`sequence, quantitative measures of at least two of (i) the
`paired reads, (ii) the unpaired reads, (iii) read depth of
`the paired reads, and (iv) read depth of the unpaired reads ... 114
`Claims 2 and 19 .................................................................................121
`Claims 3 and 20 .................................................................................123
`Claims 4 and 21 .................................................................................125
`Claim 6: The method of claim 1, wherein the tagging comprises
`non-uniquely tagging the plurality of the cfDNA molecules
`with the set of duplex tags comprising molecular barcodes from
`the set of molecular barcodes, wherein the cfDNA molecules
`that map to a mappable base position of a reference sequence
`are tagged with a number of different molecular barcodes
`ranging from at least 2 to fewer than a number of the cfDNA
`molecules that map to the mappable base position. ..........................130
`Claim 7: The method of claim 1, wherein the molecular
`barcodes of the set of molecular barcodes have pre-determined
`sequences. ..........................................................................................131
`Claim 22: The method of claim 18, wherein the molecular
`barcodes of the set of molecular barcodes have between 2 and
`10,000 different molecular barcode sequences. ................................132
`Claims 9-10 and 24-25 ......................................................................134
`K.
`Claims 11 and 26 ...............................................................................138
`L.
`M. Claims 12 and 13 ...............................................................................140
`N.
`Claim 14: The method of claim 1, wherein the reducing or
`tracking redundancy in the set of sequence reads comprises
`mapping a plurality of the set of sequence reads to a reference
`sequence. ...........................................................................................143
`Claim 15: The method of claim 1, further comprising: (f)
`determining quantitative measures of at least two of (i) the
`paired reads, (ii) the unpaired reads, (iii) read depth of the
`paired reads, and (iv) read depth of the unpaired reads at one or
`more loci of a reference sequence. ....................................................147
`
`O.
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`- v -
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`

`

`P.
`
`Q.
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`Inter Partes Review of US Patent 11,118,221
`Declaration of Paul T. Spellman, Ph.D.
`Claim 27: The method of claim 18, wherein reducing or
`tracking the redundancy in the set of sequence reads comprises
`collapsing a plurality of the set of sequence reads to generate
`consensus sequences representative of original double-stranded
`cfDNA molecules from among the non-uniquely tagged parent
`polynucleotides. .................................................................................147
`Claim 28: The method of claim 27, further comprising mapping
`a plurality of the set of sequence reads or the consensus
`sequences to a reference sequence. ...................................................149
`XIII. GROUND 2: CLAIM 5 WOULD HAVE BEEN OBVIOUS IN VIEW
`OF NARAYAN, SCHMITT, AND MEYER ............................................. 150
`XIV. GROUND 3: CLAIMS 8 AND 23 WOULD HAVE BEEN
`OBVIOUS IN VIEW OF NARAYAN, SCHMITT, AND CRAIG ........... 154
`A. A POSA would have been motivated to incorporate Craig’s pre-
`determined barcode sequences into Schmitt’s Duplex
`Sequencing method. ..........................................................................156
`Expectation of Success ......................................................................158
`B.
`XV. GROUND 4: CLAIMS 16-17 AND 29-30 WOULD HAVE BEEN
`OBVIOUS OVER NARAYAN, SCHMITT, AND KIVIOJA .................. 159
`1.
`Claims 16-17 and 29-30 ......................................................... 159
`XVI. OBJECTIVE EVIDENCE OF NONOBVIOUSNESS DO NOT
`SUPPORT PATENTABILITY .................................................................. 166
`XVII. CONCLUSION ........................................................................................... 167
`
`
`
`- vi -
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`

`

`Inter Partes Review of US Patent 11,118,221
`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. 11,118,221 ("the ’221 patent") (EX1001), which resulted
`
`from U.S. Patent Application No. 16/672,267 ("the ’267 application"), filed on
`
`January 7, 2020. I understand that the ’221 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 ’221 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 ’221 is currently assigned to
`
`- 1 -
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`

`

`Inter Partes Review of US Patent 11,118,221
`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
`
`- 2 -
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`

`

`Inter Partes Review of US Patent 11,118,221
`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,
`
`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 11,118,221
`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 ’221 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 11,118,221
`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 ’221 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 ’221 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 11,118,221
`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-
`
`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
`
`the number of molecules generated without actually observing all of them.
`
`
`declaration.
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`Inter Partes Review of US Patent 11,118,221
`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 1-4, 6-7, 9-15, 18-22, 24-28 of
`
`the ’221 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 11,118,221
`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. Claim 5 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, claims 8 and 23 of the ’221 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
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`Inter Partes Review of US Patent 11,118,221
`Declaration of Paul T. Spellman, Ph.D.
`at greater 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 16-17, 29-30 of the ’221 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], p. 48;
`
`EX1083, ¶[0048], p. 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
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`Inter Partes Review of US Patent 11,118,221
`Declaration of Paul T. Spellman, Ph.D.
`
`screening. EX1032; EX1051.
`
`25. Furthermore, a POSA would have reasonably expected success in
`
`calculating these quantitative measures of claims 16-17, 29-30 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 1-30, as I explain in Section XV.
`
`1001
`
`1004
`
`IV. LIST OF DOCUMENTS CONSIDERED
`Ex. No.
`Description
`Eltoukhy, H., et al., “Methods And Systems For Detecting Genetic
`Variants,” U.S. Patent No. 11,118,221 (filed January 7, 2020; issued
`September 14, 2021)
`Murtaza, M., et al., “Non-invasive analysis of acquired resistance to
`cancer therapy by sequencing of plasma DNA,” Nature 497: 108-112
`(2013)
`1005 Meyer, M., et al., “Parallel tagged sequencing on the 454 platform,”
`Nature Protocols 3(2): 267-278 (2008)
`1006 Kivioja, T., et al., “Counting absolute numbers of molecules using
`unique molecular identifiers,” Nature Methods 9(1): 72-76 (2012)
`Craig, D.W., et al., “Identification Of Genetic Variants Using
`Barcoded Multiplexed Sequencing”, Nature Methods 5:887–893
`(2008)
`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)
`
`1007
`
`1008
`
`- 10 -
`
`

`

`Ex. No.
`
`1009
`
`1010
`
`1013
`
`1014
`
`1016
`
`1019
`
`Inter Partes Review of US Patent 11,118,221
`Declaration of Paul T. Spellman, Ph.D.
`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)
`1011 Metzker, M.L., “Sequencing technologies — the next generation,”
`Nature Reviews 11:31-46 (2010)
`1012 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)
`1015 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)
`1017 Zheng, Z., “Titration-free 454 sequencing using Y adapters,” Nature
`Protocols 6(9): 1367-1376 (2011)
`1018 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)
`
`1020
`
`1021
`
`- 11 -
`
`

`

`Ex. No.
`
`1022
`
`1023
`
`1024
`
`Inter Partes Review of US Patent 11,118,221
`Declaration of Paul T. Spellman, Ph.D.
`Description
`van Nieuwerburgh, F., et al., “Quantitative Bias in Illumina TruSeq
`and a Novel Post Amplification Barcoding Strategy for Multiplexed
`DNA and Small RNA Deep Sequencing,” PLoS ONE 6(10): e2