`
`UNITED STATES PATENT AND TRADEMARK OFFICE
`
`____________________________________________
`
`BEFORE THE PATENT TRIAL AND APPEAL BOARD
`
`____________________________________________
`
`FOUNDATION MEDICINE, INC.,
`Petitioner,
`v.
`
`GUARDANT HEALTH, INC.,
`Patent Owner.
`
`_____________________
`
`Case No. IPR2019-00637
`U.S. Patent No. 9,902,992
`_____________________
`
`PETITION FOR INTER PARTES REVIEW
`OF U.S. PATENT NO. 9,902,992
`
`
`
`
`
`TABLE OF CONTENTS
`
`
`I.
`INTRODUCTION ............................................................................................... 1
`II. OVERVIEW ..................................................................................................... 1
`III. TECHNOLOGY BACKGROUND ................................................................. 1
`A. DNA and Cell-Free DNA .............................................................................. 2
`B. Genetic Mutations ......................................................................................... 3
`C.
`Sequencing .................................................................................................... 4
`D. Base Calling................................................................................................... 6
`E. Alignment/Mapping ...................................................................................... 6
`F.
`Error Correction and Molecular Barcodes .................................................... 8
`G.
`Identifying Errors ........................................................................................ 11
`H. Analysis of Sequence Reads ....................................................................... 13
`IV. THE ’992 PATENT AND THE SKILL IN THE ART .................................. 13
`A. Brief Description of ’992 Patent ................................................................. 13
`B.
`Prosecution History ..................................................................................... 16
`C.
`Person of Ordinary Skill in the Art ............................................................. 17
`V. CLAIM CONSTRUCTION ........................................................................... 18
`VI. PRIOR ART RELIED ON ............................................................................. 20
`A.
`Schmitt (EX1011) ........................................................................................ 20
`B.
`Fan (EX1048) .............................................................................................. 25
`C.
`Forshew (EX1004) ...................................................................................... 26
`D. Kucera (EX1071) ........................................................................................ 27
`E.
`Schwarzenbach (EX1054) ........................................................................... 28
`VII.
`STATEMENT OF THE PRECISE RELIEF REQUESTED FOR EACH
`CLAIM CHALLENGED ......................................................................................... 29
`
`i
`
`
`
`VIII. DETAILED EXPLANATION OF GROUNDS FOR
`UNPATENTABILITY............................................................................................. 30
`A. Ground 1: Claims 27-33 Are Unpatentable under 35 U.S.C. § 103 over
`Schmitt and Fan or Forshew ................................................................................. 31
`1. Motivation to Combine Schmitt with Fan or Forshew ............................... 31
`2. Reasonable Expectation of Success ............................................................ 36
`3. Claim 1 ........................................................................................................ 39
`4. Claim 27: The method of claim 21, wherein the single base substitution is
`detected with a sensitivity of at least 1%. .......................................................... 53
`Claim 28: The method of claim 21, wherein the single base substitution is
`detected with a sensitivity of at least 0.1%. ....................................................... 53
`5. Claim 29: The method of claim 1, wherein the plurality of genetic
`aberrations comprises three or more different members selected from the group
`of members consisting of a single base substitution, a copy number variation
`(CNV), an insertion or deletion (indel), and a gene fusion. .............................. 54
`Claim 30: The method of claim 1, wherein the plurality of genetic aberrations
`comprises a single base substitution, a copy number variation (CNV), an
`insertion or deletion (indel), and a gene fusion. ................................................ 54
`Claim 31: The method of claim 1, wherein the plurality of genetic aberrations
`comprises a plurality of each of two or more different members selected from
`the group of members consisting of a single base substitution, a copy number
`variation (CNV), an insertion or deletion (indel), and a gene fusion. ............... 55
`6. Claim 32: The method of claim 1, wherein each of the tagged parent
`polynucleotides is uniquely tagged.................................................................... 56
`7. Claim 33: The method of claim 1, wherein each of the tagged parent
`polynucleotides is non-uniquely tagged. ........................................................... 56
`B. Ground 2: Claims 11 and 12 are Unpatentable under 35 U.S.C. § 103 over
`Schmitt in view of Fan or Forshew, further with Kucera ..................................... 57
`1. Motivation to combine ................................................................................ 57
`2. Reasonable expectation of success .............................................................. 58
`
`ii
`
`
`
`3. Claim 11: The method of claim 1, wherein at least 50% of the cfDNA
`molecules are tagged by the attaching. .............................................................. 59
`Claim 12: The method of claim 1, wherein at least 80% of the cfDNA
`molecules are tagged by the attaching. .............................................................. 59
`C. Ground 3: Claim 14 is Unpatentable under 35 U.S.C. § 103 over Schmitt in
`view of Fan or Forshew, further in view of Schwarzenbach ................................ 60
`4. Motivation to combine ................................................................................ 60
`5. Reasonable expectation of success .............................................................. 61
`6. Claim 14: The method of claim 13, wherein the selectively enriching
`comprises enriching for polynucleotides mapping to the following genes: V-
`Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS), adenomatous
`polyposis coli (APC), and tumor protein 53 (ΤΡ53). ........................................ 62
`D. No Objective Indicia of Nonobviousness.................................................... 65
`IX. THE BOARD SHOULD NOT DENY INSTITUTION UNDER § 325(d) ... 66
`X. CONCLUSION .............................................................................................. 68
`XI. MANDATORY NOTICES ............................................................................ 68
`A. Real Party-in-Interest (37 C.F.R § 42.8(b)(1)) ............................................ 68
`B. Related Matters (37 C.F.R. § 42.8(b)(2)) .................................................... 69
`C. Counsel (37 C.F.R. §§ 42.8(b)(3) and 42.10(a)) ......................................... 70
`D.
`Service Information (37 C.F.R. § 42.8(b)(4)) ............................................. 71
`E.
`Standing (37 C.F.R. § 42.104(a)) ................................................................ 71
`
`
`
`
`
`
`
`iii
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`
`
`TABLE OF AUTHORITIES
`
`Page(s)
`
`Federal Cases
`
`Dynamic Drinkware, LLC v. Nat’l Graphics, Inc., 800 F.3d 1375
`(Fed. Cir. 2015)…………………………………………………………………...21
`
`In re Huai-Hung Kao, 639 F.3d 1057, 1068 (Fed. Cir. 2011……………………..65
`
`Leapfrog Enters., Inc. v. Fisher-Price Inc., 485 F.3d 1157 (Fed. Cir. 2007)……..65
`
`MCM Portfolio LLC v. Hewlett-Packard Co., 812 F.3d 1284 (Fed. Cir. 2015)…..68
`
`Phillips v. AWH Corp., 415 F.3d 1303 (Fed. Cir. 2005)………………………….18
`
`Patent Trial and Appeal Board Cases
`
`Donghee America, Inc. v. Plastic Omnium Advanced Innovation and Research,
`IPR2017-01654, Slip. Op (PTAB Jan. 19, 2018)…………………………………66
`
`Eli Lilly & Co. v. Trustees of U. Penn., IPR2016-00458, Slip. Op (PTAB July 14,
`2016)………………………………………………………………………………65
`
`Medtronic, Inc. v. Niazi Licensing Corp., IPR2018-00609, Slip. Op (PTAB Aug.
`20, 2018)…………………………………………………………………………..20
`
`Umicore AG & Co. KG v. BASF Corp., IPR2015-01124, Slip. Op (PTAB Nov. 2,
`2015)………………………………………………………………………………65
`
`Statutes
`
`35 U.S.C. § 102(a)(1)……………………………………………………25, 26, 28
`
`35 U.S.C. § 102(a)(2) ………………………………………………………21, 27
`
`iv
`
`
`
`35 U.S.C. § 103………………………………………………………………..27,29
`35 U.S.C. § 103 ..........................................................................27,29
`
`35 U.S.C. § 119 …………………………………………………………………..20
`35 U.S.C. § 119 ............................................................................. 20
`
`35 U.S.C. § 282(b) ……..…………………………………………………………18
`35 U.S.C. §282(b) .......................................................................... 18
`
`35 U.S.C. § 311………………………………………………………………...1, 29
`35 U.S.C. § 311 ........................................................................... 1, 29
`
`35 U.S.C. § 325(d) ………………………………………………………………..66
`35 U.S.C. § 325(d) .......................................................................... 66
`
`Regulations
`Regulations
`
`37 C.F.R § 42.8………………………………………………..………68, 69, 70, 71
`37 CPR § 42.8 ................................................................. 68, 69, 70, 71
`
`37 C.F.R. § 42.6(e) ……………………………………………………………….73
`37 C.F.R. § 42.6(e) ......................................................................... 73
`
`37 C.F.R. § 42.10(a) ………………………………………………………….…..70
`37 C.F.R. § 42.10(a) ........................................................................ 70
`
`37 C.F.R. § 42.24(a) ………………………………………………………….…..74
`37 C.F.R. § 42.24(a) ........................................................................ 74
`
`37 C.F.R. § 42.100(b) …………………………………………………………….18
`37 C.F.R. §42.100(b) ...................................................................... 18
`
`37 C.F.R. § 42.104(a) …………………………………………………………….71
`37 C.F.R. §42.104(a) ...................................................................... 71
`
`37 C.F.R. § 42.105(a) …………………………………………………………….73
`37 C.F.R. §42.105(a) ...................................................................... 73
`
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`
`
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`
`
`
`
`
`
`
`v
`
`
`
`
`
`Exhibit
`No.
`1001
`
`1002
`
`1003
`
`1004
`
`EXHIBIT LIST
`
`Description
`
`U.S. Patent No. 9,902,992 (“the '992 patent”)
`
`Declaration of Dr. Stacey Gabriel (“Gabriel Declaration”)
`
`Curriculum Vitae of Dr. Stacey Gabriel
`
`Forshew et al., “Noninvasive Identification and Monitoring of Cancer
`Mutations by Targeted Deep Sequencing of Plasma DNA,” Science
`Translational Medicine, 2012, 4(136) (“Forshew”)
`
`1005 Meyerson et al., “Advances in understanding cancer genomes through
`second-generation sequencing,” Nature Review Genetics, Vol. 11
`(2010), 685-696 (“Meyerson”)
`
`1006 Williford and Betrán, “Gene Fusion,” eLS 2013, 1-8 (“Williford”)
`
`1007
`
`1008
`
`1009
`
`1010
`
`1011
`
`1012
`
`1013
`
`Ding et al., “Analysis of next-generation genomic data in cancer:
`accomplishments and challenges,” Hum. Mol. Genet. 2010 19(R2),
`R188-R196 (“Ding”)
`
`Sehnert et al., “Optimal Detection of Fetal Chromosomal
`Abnormalities by Massively Parallel DNA Sequencing of Cell-free
`Fetal DNA from Maternal Blood,” Clinical Chemistry 2011, 57:7,
`1042-1049 (“Sehnert”)
`
`Redon et al., “Global variation in copy number in the human
`genome,” Nature 2006 444(7118), 444-454 (“Redon”)
`
`Gordon et al., “Causes and consequences of aneuploidy in cancer,”
`Nat. Rev. Genet. 2012 13(3), 189-203 (“Gordon”)
`
`U.S. Patent No. 9,752,188 (“Schmitt”)
`
`U.S. Provisional Application 61/613,413 (“Schmitt '413 provisional”)
`
`Duncan and Patel, Diagnostic Molecular Pathology: A Guide to
`Applied Molecular Testing 25-33 (Coleman and Tsongalis eds., 1st
`ed. 2016)
`
`vi
`
`
`
`1014
`
`1015
`
`1016
`
`1017
`
`1018
`
`1019
`
`1020
`
`1021
`
`1022
`
`Nielsen et al., “Genotype and SNP calling from next-generation
`sequencing data,” Nat. Rev. Genet. 2011 12(6), 443-451 (“Nielsen”)
`
`Shendure and Ji, “Next-generation DNA sequencing,” Nat.
`Biotechnol. 2008 26(10), 1135-1145 (“Shendure”)
`
`Kao et al., “BayesCall: A model-based base-calling algorithm for
`high-throughput short-read sequencing,” Genome Res. 2009 19(10),
`1884-1895 (“Kao”)
`
`Quinlan et al., “Pyrobayes: an improved base caller for SNP discovery
`in pyrosequences,” Nat. Methods 2008 5(2), 179-181 (“Quinlan”)
`
`Liang et al., “Bayesian basecalling for DNA sequence analysis using
`hidden Markov models,” IEE/ACM Trans. Comput. Biol. Bioinform.
`2007 4(3), 430-440 (“Liang”)
`
`Ledergerber and Dessimoz, “Base-calling for next-generation
`sequencing platforms,” Brief Bioinform. 2011 12(5), 489-497
`(“Ledergerber”)
`
`Kircher et al., “Improved base calling for the Illumina Genome
`Analyzer using machine learning strategies,” Genome Biol. 2009,
`10(8), R83 (“Kircher”)
`
`U.S. Patent No. 6,582,908 (“Fodor”)
`
`U.S. Patent No. 9,476,095 (“Kinde”)
`
`1023 Metzker, “Sequencing technologies - the next generation,” 2010
`11(1), 31-46 (“Metzker”)
`
`1024
`
`1025
`
`Lo et al., “Quantitative analysis of fetal DNA in maternal plasma and
`serum: implications for noninvasive prenatal diagnosis,” Am. J. Hum.
`Genet. 1998, 62(4), 768-775 (“Lo”)
`
`Narayan et al., “Ultrasensitive Measurement of Hotspot Mutations in
`Tumor DNA in Blood Using Error-Suppressed Multiplexed Deep
`Sequencing,” Cancer Res. 2012 72(14), 3492-3498 (published July
`15, 2012) (“Narayan”)
`
`1026
`
`International Publication No. WO 2012/042374 A2 (“Taipale”)
`
`vii
`
`
`
`1027
`
`1028
`
`1029
`
`1030
`
`1031
`
`1032
`
`1033
`
`1034
`
`1035
`
`1036
`
`1037
`
`1038
`
`1039
`
`1040
`
`1041
`
`1042
`
`1043
`
`U.S. Patent Application No. 15/076,565 (the “'565 application”)
`
`U.S. Patent Application No. 15/076,565 March 21, 2016 TrackOne
`Request
`
`U.S. Patent Application No. 15/076,565 April 29, 2016 Application
`Data Sheet
`
`U.S. Patent Application No. 15/076,565 June 10, 2016 TrackOne
`Request Granted
`
`U.S. Patent Application No. 15/076,565 June 15, 2017 Notice of
`Allowance
`
`U.S. Patent Application No. 15/076,565 February 9, 2017 Non-Final
`Rejection
`
`U.S. Patent Application No. 15/076,565 February 9, 2017 List of
`References Cited by Examiner
`
`U.S. Patent Application No. 15/076,565 February 9, 2017 List of
`References Cited by Applicant and Considered by Examiner
`
`U.S. Patent Application No. 15/076,565 May 9, 2017 Amendment and
`Response to Non-Final Office Action
`
`U.S. Provisional 61/696,734
`
`U.S. Provisional 61/704,400
`
`U.S. Provisional 61/793,997
`
`U.S. Provisional 61/845,987
`
`PCT/US2013/058061
`
`U.S. Patent Application No. 13/969,260
`
`U.S. Provisional 61/948,530
`
`Sparks et al., “Selective analysis of cell-free DNA in maternal blood
`for evaluation of fetal trisomy,” Prenat. Diagn. 2012, 32(1), 3-9
`(“Sparks”)
`
`viii
`
`
`
`1044
`
`1045
`
`U.S. Patent No. 8,209,130 (“Kennedy”)
`
`April 5, 2018 Memorandum from Deputy Commissioner for Patent
`Examination Policy to Patent Examining Corps
`
`1046 Mertes et al., “Targeted enrichment of genomic DNA regions for
`next-generation sequencing,” Brief Funct. Genomics 2011, 10(6),
`374-386 (“Mertes”)
`
`1047
`
`1048
`
`1049
`
`1050
`
`1051
`
`1052
`
`1053
`
`1054
`
`1055
`
`Schmitt et al., “Detection of ultra-rare mutations by next-generation
`sequencing,” Proc. Natl. Acad. Sci. USA 2012, 109(36), 14508-14513
`(“Schmitt 2012”)
`
`Fan et al., “Noninvasive diagnosis of fetal aneuploidy by shotgun
`sequencing DNA from maternal blood,” Proc. Natl. Acad. Sci. USA
`2008, 105(42), 16266-16271 (“Fan”)
`
`Kinde et al., “Detection and quantification of rare mutations with
`massively parallel sequencing,” Proc. Natl. Acad. Sci. USA 2011,
`108(23), 9530-9535 (“Kinde 2011”)
`
`Chiu et al., “Non-invasive prenatal assessment of trisomy 21 by
`multiplexed maternal plasma DNA sequencing: large scale validity
`study”, BMJ 2011;342, c7401 (“Chiu”)
`
`Diehl et al., “Detection and quantification of mutations in the plasma
`of patients with colorectal tumors,” Proc. Natl. Acad. Sci. USA 2005,
`102(45), 16368-16373 (“Diehl”)
`
`Liao et al., “Targeted massively parallel sequencing of maternal
`plasma DNA permits efficient and unbiased detection of fetal alleles,”
`Clin. Chem. 2011, 57(1), 92-101 (“Liao”)
`
`Vasyukhin et al., Challenges of Modern Medicine, 141-150 (Verna
`and Shamoo eds, 1994) (“Vasyukhin”)
`
`Schwarzenbach et al., “Cell-free nucleic acids as biomarkers in cancer
`patients,” Nat. Rev. Cancer 2011, 11(6), 426-437 (“Schwarzenbach”)
`
`Koboldt et al., “The next-generation sequencing revolution and its
`impact on genomics,” Cell 2013, 155(1), 27-38 (“Koboldt”)
`
`ix
`
`
`
`1056
`
`1057
`
`1058
`
`1059
`
`1060
`
`Kirsch and Klein, “Sequence error storms and the landscape of
`mutations in cancer,” Proc. Natl. Acad. Sci. USA 2012, 109(36),
`14289-14290 (“Kirsch and Klein”)
`
`Kennedy et al., “Detecting ultralow-frequency mutations by Duplex
`Sequencing,” Nat. Protoc. 2014, 9(11), 2586-2606 (“Kennedy 2014”)
`
`Kivioja et al., “Counting absolute numbers of molecules using unique
`molecular identifiers,” Nat. Methods 2011, 9(1), 72-74 (“Kivioja”)
`
`Kinde et al., “FAST-SeqS: a simple and efficient method for the
`detection of aneuploidy by massively parallel sequencing,” PLoS One
`2012, 7(7), e41162 (“Kinde 2012”)
`
`Krimmel et al., “Ultra-deep sequencing detects ovarian cancer cells in
`peritoneal fluid and reveals somatic TP53 mutations in noncancerous
`tissues,” Proc. Natl. Acad. Sci. USA 2016, 113(21), 6005-6010
`(“Krimmel”)
`
`1061 Meyer and Kircher, “Illumina sequencing library preparation for
`highly multiplexed target capture and sequencing,” Cold Spring Harb.
`Protoc. 2010, (6), prot5448 (“Meyer and Kircher”)
`
`1062
`
`1063
`
`1064
`
`1065
`
`1066
`
`1067
`
`Quail et al., “A large genome center's improvements to the Illumina
`sequencing system,” Nat. Methods 2008, 5(12), 1005-1010 (“Quail”)
`
`Lohman, Efficient Adaptor Ligation for the Preparation of dsDNA
`Libraries using the Blunt/TA Ligase Master Mix (“Lohman”)
`
`U.S. Patent No. 8,865,410
`
`Ng et al., “Targeted capture and massively parallel sequencing of 12
`human exomes,” Nature 2009, 461(7261), 272-276 (“Ng”)
`
`Albert et al., “Direct selection of human genomic loci by microarray
`hybridization,” Nat. Methods 2007, 4(11), 903-905 (“Albert”)
`
`Schweiger et al., “Genome-wide massively parallel sequencing of
`formaldehyde fixed-paraffin embedded (FFPE) tumor tissues for
`copy-number- and mutation-analysis,” PLoS One 2009, 4(5), e5548
`(“Schweiger”)
`
`x
`
`
`
`1068 McKernan et al., “Sequence and structural variation in a human
`genome uncovered by short-read, massively parallel ligation
`sequencing using two-base encoding,” Genome Res. 2009, 19(9),
`1527-1541 (“McKernan”)
`
`1069
`
`1070
`
`1071
`
`1072
`
`1073
`
`Ajay et al., “Accurate and comprehensive sequencing of personal
`genomes,” Genome Res. 2011, 21(9), 1498-1505 (“Ajay”)
`
`U.S. Patent Application No. 14/712,754 December 4, 2015 Office
`Action
`
`U.S. Patent No. 8,697,408 (“Kucera”)
`
`Nawroz et al., “Microsatellite alterations in serum DNA of head and
`neck cancer patients,” Nat. Med. 1996, 2(9), 1035-1037 (“Nawroz”)
`
`Korbel et al., “Paired-end mapping reveals extensive structural
`variation in the human genome,” Science 2007, 318(5849), 420-426
`(“Korbel”)
`
`1074 Wheeler et al., “The complete genome of an individual by massively
`parallel DNA sequencing,” Nature 2008, 452(7189), 872-876
`(“Wheeler”)
`
`1075 Mamanova et al., “Target-enrichment strategies for next-generation
`sequencing,” Nat. Methods 2010, 7(2), 111-118 (“Mamanova”)
`
`1076
`
`1077
`
`1078
`
`1079
`
`1080
`
`Parkinson et al., “Preparation of high-quality next-generation
`sequencing libraries from picogram quantities of target DNA,”
`Genome Res. 2012, 22(1), 125-133 (“Parkinson”)
`
`Li and Durbin, “Fast and accurate short read alignment with
`Burrows–Wheeler transform,” Bioinformatics 2009, 25(14), 1754-
`1760
`
`Arneson et al., “Whole-Genome Amplification by Adaptor-Ligation
`PCR of Randomly Sheared Genomic DNA (PRSG),” CSH Protocols,
`2008, 3(1) (“Arneson”)
`
`Intentionally Left Blank
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`Intentionally Left Blank
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`xi
`
`
`
`1081
`
`1082
`
`1083
`
`1084
`
`1085
`
`1086
`
`1087
`
`1088
`
`1089
`
`1090
`
`1091
`
`1092
`
`1093
`
`1094
`
`Intentionally Left Blank
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`Intentionally Left Blank
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`Intentionally Left Blank
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`Intentionally Left Blank
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`Intentionally Left Blank
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`Intentionally Left Blank
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`Intentionally Left Blank
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`Intentionally Left Blank
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`U.S. Provisional Application 61/625,319 (“Schmitt '319 provisional”)
`
`Intentionally Left Blank
`
`Bryzgunova et al., “A reliable method to concentrate circulating
`DNA,” Analytical Biochem., 2010, 408:354-356 (“Bryzgunova”)
`
`xii
`
`
`
`I.
`
`INTRODUCTION
`
`Pursuant to 35 U.S.C. § 311 and 37 C.F.R. § 42.100 et seq., Foundation
`
`Medicine, Inc. (“Petitioner”) hereby respectfully requests inter partes review of
`
`claims 11-12, 14, 27-33 of United States Patent No. 9,902,992 (“the ’992 patent”)
`
`(EX1001). The ’992 patent issued on February 27, 2018 and is assigned to
`
`Guardant Health, Inc. (“Patent Owner”). The ’992 patent is subject to the first
`
`inventor to file provisions of the AIA. EX1029, at 5. This Petition demonstrates
`
`by a preponderance of the evidence that it is more likely than not that claims 11-
`
`12, 14, and 27-33 of the ’992 patent are unpatentable over the prior art.
`
`II. OVERVIEW
`The claims of the ’992 patent are directed to methods for detecting genetic
`
`aberrations (i.e., genetic variations) in cell-free DNA, but merely recite producing
`
`sequence reads using ordinary sequencing techniques and then sorting the
`
`sequence reads into groups, to be compared to one another and to a known
`
`reference to identify mutations. As described further below, a person of ordinary
`
`skill in the art (“POSA”) would have found claims 11-12, 14, and 27-33 to be
`
`obvious in view of the prior art as of September 4, 2012, the earliest claimed
`
`priority date.
`
`III. TECHNOLOGY BACKGROUND
`The claims of the ’992 patent are directed to methods for detecting genetic
`
`aberrations in cell-free DNA. EX1001.
`
`1
`
`
`
`A. DNA and Cell-Free DNA
`DNA molecules consist of two strands, each of which is a linear
`
`arrangement of repeating units called “nucleotides.” EX1002, ¶18. DNA is also
`
`therefore referred to as a “polynucleotide.” Id. As illustrated below, four chemical
`
`bases comprise the nucleotides of DNA: adenine (A), guanine (G), cytosine (C),
`
`and thymine (T). Id., ¶19. The order of these bases—referred to as the
`
`“sequence”—determines the information available for building and maintaining an
`
`organism:
`
`Id.
`
`
`
`DNA is typically present in cells, but in an organism, DNA may be released
`
`from cells into the bloodstream. EX1002, ¶21. Once released, this DNA is called
`
`“circulating cell-free DNA”—or sometimes simply “cell-free DNA” or
`
`“circulating DNA.” Id. Tumor cell DNA that has been released into the
`
`bloodstream is sometimes called “circulating tumor DNA” or “ctDNA.” Id. DNA
`
`that has been released from circulating fetal cells into the bloodstream of pregnant
`
`2
`
`
`
`women is sometimes called “circulating fetal DNA” (“cfDNA”) or “cell-free fetal
`
`DNA” (“cffDNA”). Id.; EX1004, 1 (citing publications).
`
`B. Genetic Mutations
`DNA sometimes undergoes changes to its structure referred to as genetic
`
`variations or mutations. EX1002, ¶22. Mutations can range in size, affecting
`
`anywhere from a single base pair to a large segment of a chromosome. Id., ¶23.
`
`Substitutions involving a single base pair are called “single nucleotide variants,”
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`and are the most common type of tumor-related genetic mutation. Id., ¶24;
`
`EX1005, 690.
`
`Another type of variation is a “gene fusion,” occurring when partial or
`
`complete sequences of two or more distinct genes join together to form a chimeric
`
`gene or “fusion gene.” EX1006, Abstract; EX1002, ¶25. Fusion genes may be
`
`found in several types of cancer, including leukemia. EX1002, ¶25; EX1007,
`
`R190.
`
`Another common type of variation is known as “copy number variation,” or
`
`CNV, occurring when copies of some section of the genome are either amplified or
`
`lost. EX1002, ¶26. Such sections of the genome may include sections of genes,
`
`entire genes, large portions of a chromosome, or entire chromosomes. Id.;
`
`EX1008, 1048; EX1009, 444; EX1010, 189-191. For example, aneuploidy is a
`
`type of CNV associated with the presence of an abnormal number of
`
`3
`
`
`
`chromosomes, and includes “trisomy” in which there are three copies of a
`
`particular chromosome instead of the normal two. EX1002, ¶26. Trisomy 21
`
`(“T21”), referring to the presence of all or part of a third copy of chromosome 21,
`
`is also known as Down syndrome. Id.
`
`Sequencing
`
`C.
`One way to identify genetic variations in an individual’s DNA is through a
`
`process called “sequencing,” which determines the order of nucleotides in a DNA
`
`fragment. EX1002, ¶29. DNA fragments are read by a sequencer to produce
`
`sequence reads, as illustrated below:
`
`
`
`Id., ¶30.
`
`The sequencing technology employed today for mutation detection is
`
`referred to as Next Generation Sequencing, or NGS, is sometimes also called
`
`massively parallel sequencing or high-throughput sequencing. Id., ¶32. Next
`
`Generation Sequencing technologies began to emerge in the mid-2000s and were
`
`in widespread use by no later than September 2012. Id.; EX1011, 1:28-37;
`
`EX1012, [0002]. The specification of the ’992 patent acknowledges that the
`
`4
`
`
`
`alleged invention includes “sequencing of cell free polynucleotides by techniques
`
`known in the art,” EX1001, 30:21-22, and identifies numerous methods of
`
`sequencing known in the art. Id., 37:66-38:11.
`
`Identifying genomic variations through NGS can include making copies of
`
`the DNA fragments of interest through a process called amplification. EX1002,
`
`¶33. The ’992 patent explains that amplification can be performed using
`
`polymerase chain reaction (“PCR”, developed in the 1980s). Id.; EX1001, 38:52-
`
`64. PCR, creates copies of DNA fragments exponentially as illustrated below:
`
`
`
`EX1002 ¶33. As described further below, the ’992 patent refers to the original
`
`DNA fragments as “parent polynucleotides” and to the amplified copies as
`
`“progeny polynucleotides.” EX1001, Figure 9.
`
`5
`
`
`
`Following amplification, each amplified copy is sequenced to produce a
`
`“sequence read,” indicating the order of nucleotide bases in each amplified DNA
`
`molecule. EX1002, ¶34; EX1011; 3:10-20; EX1012, [0009].
`
`D. Base Calling
`The process by which the sequence read is converted into nucleotide bases is
`
`called “base calling.” EX1013, 29. This process utilizes base calling algorithms,
`
`which also assign a measure of the uncertainty (called a “quality score”) to each
`
`base call. EX1014, 444. Base-calling procedures vary according to the sequencing
`
`platform used and are prone to different types of errors. Id. The typical error rate
`
`for different NGS platforms ranges from a few tenths of a percent to several
`
`percent. Id. Reducing the error rate of base calls and improving the accuracy of
`
`the per-base quality score is important for reliable post-sequencing analysis.
`
`EX1002, ¶35. As of September 2012, several base-calling algorithms had been
`
`developed that provided ~5–30% improvement in error rates. EX1002, ¶36;
`
`EX1015; EX1016; EX1017; EX1018; EX1019. As the ’992 patent acknowledges,
`
`such methods were known in the art. EX1001, 47:42-50.
`
`E. Alignment/Mapping
`After sequence reads are generated, they typically are compared to a
`
`reference sequence, in a process called “alignment” or “mapping.” EX1002, ¶37.
`
`The reference sequence may consist of a sequence from the genome of an
`
`6
`
`
`
`individual known to be healthy. Id. As illustrated below, if a sequence read aligns
`
`perfectly with the reference sequence, it does not contain a mutation, whereas if it
`
`differs at one or more nucleotide bases, the sequence read is identified as
`
`containing a mutation:
`
`
`
`Id.
`
`Mapping a sequence read to a reference sequence is a necessary step in
`
`identifying genetic mutations because a genetic variant can be identified only when
`
`the sequence differs from the reference at one or more nucleotide positions.
`
`EX1002, ¶37. Mapping sequence reads enables one to detect any number of
`
`genetic mutations, including but not limited to, deletions, insertions, gene fusions,
`
`and copy number variation. EX1002, ¶¶38-39, ¶38 (figure).
`
`
`
`Accordingly, as of September 2012, a POSA would have understood any
`
`method of identifying rare mutations to include mapping sequence reads to a
`
`reference genome. EX1002, ¶40. The ’992 patent explains that reference
`
`sequences were readily available: “Human genome sequences useful as references
`
`can include the hg19 assembly or any previous or available hg assembly. Such
`
`7
`
`
`
`sequences can be
`
`interrogated using
`
`the genome browser available at
`
`genome.ucsc.edu/index.html.” EX1001, 35:35-40.
`
`F. Error Correction and Molecular Barcodes
`Amplification and sequencing can produce errors. EX1002, ¶41. For
`
`example, if the sequence of the original fragment is A-G-C-T-A-G, one of the
`
`nucleotide bases may be miscopied during the amplification process, resulting in
`
`an amplified copy with the sequence A-G-C-T-G-G. Id. The sequencer may also
`
`misread one or more nucleotide bases. Id. For example, even if the original
`
`fragments are correctly copied, the sequencer could erroneously produce the
`
`sequence read A-G-C-T-A-A for one of the amplified copies. Id.
`
`Amplification and sequencing errors are problematic because they may be
`
`mistaken for true genetic variations. Id., ¶¶42-43; EX1011, 16:37-57; EX1012,
`
`[0043].
`
`One common approach to correcting for errors is to assign molecular
`
`identifiers known as “barcodes” to DNA fragments. EX1002, ¶44. A barcode is a
`
`nucleotide or sequence of nucleotides attached to each original DNA fragment to
`
`serve as an identifier. Id.; EX1021, 16:55-64. For example, the DNA fragments in
`
`the initial genetic material may be attached to barcodes as illustrated below:
`
`8
`
`
`
`
`
`EX1002, ¶44. The ’992 patent acknowledges that methods for attaching barcodes
`
`to DNA had been described in the art as early as 2000. EX1001, 38:31-36;
`
`EX1021; EX1002, ¶45.
`
`After a barcode is attached to a DNA fragment they can then be amplified
`
`together. EX1002, ¶46; EX1011 3:10-20; EX1012, [0009]. The amplified copies
`
`can then be sequenced, such that the resulting sequence reads include sequences
`
`for both the barcode and the DNA fragment. EX1002, ¶46; EX1011 3:10-20;
`
`EX1012, [0009]. The sequence reads can then be placed in groups (or families)
`
`based on their barcodes, as illustrated below:
`
`9
`
`
`
`
`
`The object of the grouping is to ensure that each sequence read in a family is
`
`an amplified copy of the same unique DNA fragment. EX1002, ¶46; EX1011,
`
`21:55-22:15; EX1012, [0063]. In some cases, the families may be grouped based
`
`on the barcodes alone. For example, if there are 1000 original DNA fragments and
`
`at least 1000 different barcodes, each fragment will have its own unique barcode
`
`(i.e., be “uniquely tagged”). EX1002, ¶47. The resulting sequence reads can be
`
`placed into 1000 families—one for each barcode—wherein each read in each
`
`family is a copy of a unique original fragment. Id.
`
`However, if the number of original DNA fragments is large, using a unique
`
`barcode for each original fragment may be impractical. Id., ¶48. An alternative
`
`10
`
`
`
`approach is to use fewer barcodes than original DNA fragments, such that not
`
`every fragment has a unique barcode (i.e., is “non-uniquely” tagged). Id. Under
`
`this approach, looking at the barcode alone will not allow one to accurately group
`
`sequence reads into families, and it is necessary to look at additional information,
`
`including optionally a portion of the sequence read corresponding to the original
`
`DNA fragment itself. Id.; EX1011, 9:1-13; EX1012, [0030]; EX1049, SI1. For
`
`example, suppose a fragment with the sequence A-A-C-C-G-G and another with
`
`the sequence C-A-G-G-T-T are both assigned the same barcode. If the grouping is
`
`based on barcodes alone, the sequence reads corresponding to the two separate
`
`fra