SHUSAKU•YAMAMOTO 
` 
`Japanese Patent No. 6275145
`Opposition no: 2018-700659 WSGR Ref: 42534-704.761
`(Translation)
`
`
`
`Decision
`
`
`
`Opposition 2018-700659
`
`505 Penobscot Drive, Redwood City, CA 94063, USA
`Patent right holder
`
`
`Guardant Health, Incorporated
`
`SHUSAKU YAMAMOTO, Grand Front Osaka Tower C, 3-1 Ofuka-cho,
`Kita-ku, Osaka-shi, Osaka
`Shusaku YAMAMOTO, Patent Attorney
`
`SHUSAKU YAMAMOTO, Grand Front Osaka Tower C, 3-1 Ofuka-cho,
`Kita-ku, Osaka-shi, Osaka
`Natsuki MORISHITA, Patent Attorney
`
`SHUSAKU YAMAMOTO, Grand Front Osaka Tower C, 3-1 Ofuka-cho,
`Kita-ku, Osaka-shi, Osaka
`Takatoshi IIDA, Patent Attorney
`
`SHUSAKU YAMAMOTO, Grand Front Osaka Tower C, 3-1 Ofuka-cho,
`Kita-ku, Osaka-shi, Osaka
`Daisuke ISHIKAWA, Patent Attorney
`
`SHUSAKU YAMAMOTO, Grand Front Osaka Tower C, 3-1 Ofuka-cho,
`Kita-ku, Osaka-shi, Osaka
`Kensaku YAMAMOTO, Attorney at law
`
`10-2-202 Tsukuda-cho, Nishikyogoku, Sakyo-ku, Kyoto-shi,
`Kyoto
`Opponent
`
`The following decision has been rendered in connection
`
`with the Opposition filed against the inventions of Japanese
`
`Sumiko NODA
`
`
`
`
`
`
`
`- 1/24 -
`
`0001
`
`GUARDANT - EXHIBIT 2005
`Foundation Medicine, Inc. v. Guardant Health, Inc.
`IPR2019-00637
`
`

`

`SHUSAKU•YAMAMOTO 
` 
`Japanese Patent No. 6275145
`Opposition no: 2018-700659 WSGR Ref: 42534-704.761
`
`Patent No. 6275145 entitled "SYSTEMS AND METHODS TO DETECT RARE
`MUTATIONS AND COPY NUMBER VARIATIONS".
`
`Conclusion
`
`The patent for claims 1-29 in Japanese Patent No. 6275145
`shall be maintained.
`
`Reasons
`I
`Timeline of proceeding
`
`The application for the inventions defined by claims 1-29
`in Japanese Patent No. 6275145 is an application with an
`international filing date of September 4, 2013 (priority claims
`under the Paris convention: September 4, 2012, US; September
`21, 2012, US; March 15, 2013, US; July 13, 2013, US). A patent
`was registered for the inventions on January 19, 2018.
`
`Opposition was filed thereafter against the patent on
`August 7, 2018 by the Opponent, Sumiko NODA.
`
`Instant inventions
`II
`The inventions defined by claims 1-29 of Japanese Patent
`
`No. 6275145 are specified by the recitations of the respective
`claims 1-29 (hereinafter, referred to as "instant inventions
`1", "instant invention 2", and the like, respectively). Claim
`1 thereof is the following.
`
`[Claim 1]
`A method for detecting copy number variation comprising:
`
`a. non-uniquely tagging extracellular polynucleotides or
`fragments thereof from a bodily sample from a subject, thereby
`producing a population of non-uniquely tagged extracellular
`polynucleotides;
`
`b. sequencing said non-uniquely tagged extracellular
`polynucleotides, wherein each of the extracellular
`polynucleotide generates a plurality of sequencing reads;
`
`c. filtering out reads that fail to meet a set threshold;
`
`
`
`- 2/24 -
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`0002
`
`

`

`SHUSAKU•YAMAMOTO 
` 
`Japanese Patent No. 6275145
`Opposition no: 2018-700659 WSGR Ref: 42534-704.761
`
`
`d. mapping the sequencing reads obtained from step (b),
`after reads are filtered out, to a reference sequence;
`
`e. quantifying or enumerating mapped reads or unique
`sequencing reads in a plurality of predefined regions of the
`reference sequence; and
`
`f. determining copy number variation in one or more of the
`plurality of predefined regions by:
`
` i. normalizing a number of sequencing reads in each of
`the plurality of predefined regions to each other and/or a
`number of unique sequencing reads in the plurality of predefined
`regions to each other; and/or
`
` ii. comparing a number of sequencing reads in each of the
`plurality of predefined regions and/or a number of unique
`sequencing reads in the plurality of predefined regions to
`normalized numbers obtained from a control sample.
`
`III Summary of Reasons for Opposition
`
`The summary of reasons for opposition that have been set
`forth by the Opponent and the methods of proof are the following.
`1
`Instant inventions should be revoked because instant
`inventions 1-10, 13, 14, 15, 20, 21, 23, and 29 should be rejected
`under Sec. 29(1)(iii) of the Japanese Patent Law as being
`anticipated by the subject matter described in Exhibit Ko No.
`1, and instant inventions 1-29 should be rejected under Sec.
`29(2) of the Japanese Patent Law as being obvious to those
`skilled in the art over the subject matter described in Exhibit
`Ko No. 1 and the subject matter described in Exhibit Ko Nos.
`2-9.
`Instant inventions 1-29 should be revoked because said
`2
`inventions should be rejected under Sec. 29(2) of the Japanese
`Patent Law as being obvious to those skilled in the art over
`the subject matter described in Exhibit Ko No. 5, as well as
`Exhibit Ko Nos. 3,4, and 7-9.
`
`[Method of proof]
`
`
`
`- 3/24 -
`
`0003
`
`

`

`
`
`SHUSAKU•YAMAMOTO 
` 
`Japanese Patent No. 6275145
`Opposition no: 2018-700659 WSGR Ref: 42534-704.761
`Exhibit Ko No. 1:
`Chiu, et al., "Non-invasive prenatal assessment of trisomy 21
`by multiplexed maternal plasma DNA sequencing: large scale
`validity study", BMJ (2011) p. 1/9-9/9 and Web extra appendices
`(p. 1-8), <URL> https://doi.org/10.1136/bmj.c7401
`Exhibit Ko No. 2:
`Chiu, et al., "Noninvasive prenatal diagnosis of fetal
`chromosomal aneuploidy by massively parallel genomic
`sequencing of DNA in maternal plasma", Proc. Natl. Acad. Sci.
`USA (2008) vol. 105, no. 51, p. 20458-20463 and Supporting
`Information (p. 1/17-17/17)
`Exhibit Ko No. 3:
`"Multiplexed Sequencing with the Illumina Genome Analyzer
`System" (2008) p. 1-4 <URL> https://www.illumina.com/
`Documents/products/datasheets/datasheet_sequencing_multiple
`x.pdf
`Exhibit Ko No. 4:
`Kinde, et al., "Detection and quantification of rare mutations
`with massively parallel sequencing", Proc. Natl. Acad. Sci. USA
`(2011) vol. 108, no. 23, p. 9530-9535 and Supporting Information
`(p. 1/10-10/10)
`Exhibit Ko No. 5:
`US Patent No. 8195415
`Exhibit Ko No. 6:
`Wang, et al., "Digital karyotyping", Pro. Natl. Acad. Sci. USA
`(2002) vol. 99, no. 25, p. 16156-16161
`Exhibit Ko No. 7:
`<URL>
`(2011),
`Guide
`User
`CASAVA
`v1.8.2
`http://gensoft.pasteur.fr/docs/casava/1.8.2/CASAVA_1_8_2_UG
`_15011196C.pdf
`Exhibit Ko No. 8:
`Shaw, et al., "Genomic analysis of circulating cell-free DNA
`infers breast cancer dormancy", Genome Res. (published online
`OCT 2011) vol. 22, p. 220-231
`Exhibit Ko No. 9:
`
`
`
`- 4/24 -
`
`0004
`
`

`

`SHUSAKU•YAMAMOTO 
` 
`Japanese Patent No. 6275145
`Opposition no: 2018-700659 WSGR Ref: 42534-704.761
`
`Nord et al., "Accurate and exact CNV identification from
`targeted high-throughput sequence data," BMC Genomics (2011)
`p.
`1/10-10/10,
`<URL>
`http://www.biomedcentral.com/1471-2164/12/184
`
`Findings of facts
`IV
`The descriptions in Exhibit Ko Nos. 1-9 are summarized
`
`as follows (The Appeal Bench prepared the translation from
`English to Japanese)
`
` 1
`
`Exhibit Ko No. 1
`
`(1) Descriptions of Exhibit Ko No. 1
`
`Exhibit Ko No. 1, which is a document made available to
`the public prior to the priority dates, describes that: to rule
`out fetal trisomy 21 among high risk pregnancies, multiplexed
`maternal plasma DNA sequencing analysis was used to measure the
`proportions of DNA molecules that originated from chromosome
`21, and a fetus was diagnosed as a trisomy 21 fetus when the
`z score for the chromosome 21 DNA molecules was > 3 (page 1,
`left column, lines 19-25; and page 1, right column, lines 1-3).
`
`Exhibit Ko No. 1 also describes that maternal plasma DNA
`molecules were extracted and sequenced using the same protocol
`described in Reference 24 (Note by the Appeal Bench:
`corresponding to Exhibit Ko No. 2) other than the introduction
`of multiplexing (page 2, right column, lines 21-24). As the
`summary of the protocol, Exhibit Ko No. 1 further describes
`that: a unique synthetic DNA "barcode" of six base pairs, which
`served as a signature for a sample, with one index used per
`maternal plasma sample, was introduced onto one end of each
`plasma DNA molecule, and multiplexed sample mixtures consisting
`of multiple maternal plasma DNA preparations were co-sequenced
`(page 2, right column, lines 41-51); and the sequencing was
`performed on the Genome Analyzer II (Illumina) or Genome
`Analyzer IIx (Illumina), and after the sequencing, the actual
`DNA molecules that belonged to a specific sample were
`
`
`
`- 5/24 -
`
`0005
`
`

`

`SHUSAKU•YAMAMOTO 
` 
`Japanese Patent No. 6275145
`Opposition no: 2018-700659 WSGR Ref: 42534-704.761
`
`distinguished from those belonging to other samples by sorting
`the index sequence attached to the DNA molecules (page 2, right
`column, lines 52-59).
`
`Furthermore, Exhibit Ko No. 1 describes that: a
`sequencing result was considered as valid only if the analysis
`of the sample passed a set of quality control measures (page
`2, right column, line 59 to page 3, left column, line 2); the
`chromosomal origin of sequenced "read" was identified by
`comparing with the reference human genome available from the
`Ensembl website using a software package named ELAND program
`provided by Illumina (page 3, left column, lines 7-11 and
`Appendix 2: lines 3-9); the percentage chromosome 21 within each
`maternal plasma sample was then calculated (page 3, left column,
`lines 11-12); a z score for chromosome 21 in a test sample is
`obtained by subtracting the mean percentage chromosome 21 of
`a reference set of euploid pregnancies (controls) from the
`percentage chromosome 21 of the test case and divided by the
`standard deviation of the value for percentage chromosome 21
`among the reference sample set, and a z score of > 3 was used
`as the cut-off value to determine if the percentage chromosome
`21 was increased and fetal trisomy 21 was present (page 3, left
`column, line 12 to right column, line 11); and with a chromosome
`21 z score of 3 as the diagnostic cut-off point, trisomy 21
`fetuses were detected at 100% sensitivity and 97.9% specificity
`(page 4, right column, lines 22-27).
`
`(2) Subject matter of Ko 1
`
`In view of the disclosures in (1), Exhibit Ko No. 1
`describes the following subject matter (hereinafter, referred
`to as the "subject matter of Ko 1").
`
`"A method for detecting fetal trisomy 21, comprising:
`a
`extracting a maternal plasma DNA molecule;
`b
`introducing a unique synthetic DNA of six base pairs,
`which serves as a signature for each sample per maternal plasma
`sample, onto one end of each plasma DNA molecule, and using the
`
`
`
`- 6/24 -
`
`0006
`
`

`

`
`
`SHUSAKU•YAMAMOTO 
` 
`Japanese Patent No. 6275145
`Opposition no: 2018-700659 WSGR Ref: 42534-704.761
`synthetic DNA as an index for each sample;
`c
`co-sequencing multiplexed sample mixtures consisting of
`multiple maternal plasma DNA preparations;
`d
`distinguishing, after the sequencing, DNA molecules that
`belong to a specific sample from those belonging to other
`samples with the index attached to the DNA molecules;
`e
`considering a sequencing result as valid only if analysis
`of the sample passed a set of quality control measures;
`f
`identifying a chromosomal origin of a sequenced read by
`comparing with a reference human genome;
`g
`obtaining a z score for chromosome 21 in each maternal
`plasma sample by subtracting mean percentage chromosome 21 of
`a reference set of euploid pregnancies (controls) from
`percentage chromosome 21 of a test case and divided by a standard
`deviation of a value for percentage chromosome 21 among a
`reference sample set; and
`h
`determining that fetal trisomy 21 is present if the z score
`is greater than 3."
`
`Exhibit Ko No. 2
`2
`Exhibit Ko No. 2, which is a document made available to
`
`the public prior to the priority dates, is an academic article
`entitled "Noninvasive prenatal diagnosis of fetal chromosomal
`aneuploidy by massively parallel genomic sequencing of DNA in
`maternal plasma". Exhibit Ko No. 2 was cited by Exhibit Ko No.
`1 as Reference 24 as discussed in 1. Exhibit Ko No. 2 describes
`that the presence of fetal trisomy 21 was determined by
`analyzing the sequence of maternal plasma DNA.
`
` 3
`
`
`
`Exhibit Ko No. 3
`Exhibit Ko No. 3, which is a document made available to
`
`the public prior to the priority dates, describes a summary of
`multiplexed sequence analysis system of Illumina.
`
`
`
`Exhibit Ko No. 4
`
` 4
`
`
`
`- 7/24 -
`
`0007
`
`

`

`SHUSAKU•YAMAMOTO 
` 
`Japanese Patent No. 6275145
`Opposition no: 2018-700659 WSGR Ref: 42534-704.761
`
`
`Exhibit Ko No. 4, which is a document made available to
`the public prior to the priority dates, is an academic article
`entitled "Detection and quantification of rare mutations with
`massively parallel sequencing". Exhibit Ko No. 4 describes a
`method called Safe-Sequencing System (Safe-SeqS) that
`substantially increases the sensitivity of a massively parallel
`sequencing instrument for ensuring identification of mutations
`that are present in a small fraction of DNA templates with the
`massively parallel sequencing instrument (page 9530,
`Abstract).
`
`Exhibit Ko No. 4 also describes that: Safe-SeqS performs
`step 1 for assigning a unique identifier (UID) to each DNA
`template molecule to be analyzed and step 2 for amplifying each
`uniquely tagged template so that many daughter molecules with
`the identical sequence are generated (UID family); and if a
`mutation preexisted in the template molecule used for
`amplification, the mutation should be present in every daughter
`molecule containing that UID, barring any subsequent
`replication or sequencing errors (page 9530, right column, line
`32 to page 9531, left column, line 2).
`
`Exhibit Ko No. 4 further describes that: endogenous UIDs
`(page 9531, left column, lines 3-14) and exogenous UIDS (page
`9532, left column, lines 1-17) are used as UIDs; and Safe-SeqS
`decreased the presumptive sequencing error by at least 70-fold
`(page 9531, left column, lines 31-38).
`
`Furthermore, Exhibit Ko No. 4 describes that a
`conventional method or Safe-SeqS was used to analyze single base
`substitutions of the CTNNB1 gene of three normal, unrelated
`individuals (Figs. 3 and 4).
`
`In addition, Exhibit Ko No. 4 describes that one of the
`advantages of the strategy is that it yields the number of
`templates analyzed as well as the fraction of templates
`containing variant bases (page 9534, left column, lines 2-4).
`
`
`
`Exhibit Ko No. 5
`
` 5
`
`
`
`- 8/24 -
`
`0008
`
`

`

`
`
`SHUSAKU•YAMAMOTO 
` 
`Japanese Patent No. 6275145
`Opposition no: 2018-700659 WSGR Ref: 42534-704.761
`(1) Descriptions of Exhibit Ko No. 5
`
`Exhibit Ko No. 5, which is a document made available to
`the public prior to the priority dates, describes about
`noninvasive diagnosis of fetal aneuploidy by sequencing (title
`of the invention). Specifically, Exhibit Ko No. 5 describes that
`5 million sequence tags per patient sample were obtained on
`average by direct sequencing of cell-free DNA from plasma of
`pregnant women, and the sequences obtained were mapped to
`specific
`chromosomal
`locations,
`and
`over-
`or
`under-representation of chromosomes were measured from an
`aneuploidy fetus to successfully identify 9 cases of trisomy
`21 (Down syndrome), 2 cases of trisomy 18, and 1 case of trisomy
`13 in a cohort of 18 normal and aneuploid pregnancies (column
`20, lines 30-46).
`
`Exhibit Ko No. 5 also describes that: fetal DNA which is
`present in the maternal plasma of peripheral blood is subjected
`to analysis (column 3, lines 35-39); fetal and maternal DNA
`contained in peripheral blood is sequenced in a method which
`gives a large number of short reads (column 3,lines 43-47);
`short reads act as sequence tags, and a significant fraction
`of the reads are sufficiently unique to be mapped to specific
`chromosomes or chromosomal locations known to exist in the human
`genome (column 3, lines 47-50); by counting the number of
`sequence tags mapped to each chromosomes (1-22, X and Y), the
`over- or under-representation of any chromosome or chromosome
`portion in the mixed DNA contributed by an aneuploidy fetus can
`be detected (column 3, lines 50-56); and the median count of
`the number of sequences per autosome is used as a normalization
`constant to account for differences in the total number of
`sequence tags and used for comparison between samples and
`between chromosomes (column 4, lines 1-5).
`
`Exhibit Ko No. 5 further describes that: DNA fragments
`from multiple chromosome portions of a mixed sample are
`sequenced to obtain a number of sequence tags of sufficient
`length of determined sequence to be assigned to a chromosome
`
`
`
`- 9/24 -
`
`0009
`
`

`

`SHUSAKU•YAMAMOTO 
` 
`Japanese Patent No. 6275145
`Opposition no: 2018-700659 WSGR Ref: 42534-704.761
`
`location within a genome and of sufficient number to reflect
`abnormal distribution (column 4, lines 34-40); specifically,
`on average about 10 million 25 bp sequence tags were obtained
`per sample (column 14, lines 60-64); a sequence tag is a DNA
`sequence of sufficient length that it may be assigned
`specifically to one of chromosomes 1-22, X or Y (column 8, lines
`50-61); and the sequencing is performed with an Illumina/Solexa
`1G Genome Analyzer provided by Illumina (column 9, lines 25-29).
`
`Furthermore, Exhibit Ko No. 5 describes that: sequence
`tags are assigned to their corresponding chromosomes by
`comparing the sequence to reference genomic sequence (column
`4, lines 40-46); in doing so, sequence tags that mapped uniquely
`to the human genome with at most 1 mismatch are selected (column
`23, lines 11-13); the value of the sequence tag density is
`normalized within a sample by counting the number of sequence
`tags falling within a defined window of a sequence on a
`chromosome (in a preferred embodiment, the window is about 50
`kb), obtaining a median value of the total sequence tag count
`for each chromosome, obtaining a median value of all of the
`autosomal values, and using this value as a normalization
`constant to account for the difference in total number of
`sequence tags obtained for different samples; and a sequence
`tag density calculated in this way would ideally be about 1 for
`a disomic chromosome (column 8, line 50 to column 9, line 4).
`
`(2) Subject matter of Ko 5
`
`In view of the disclosures in (1), Exhibit Ko No. 5
`describes the following subject matter (hereinafter, referred
`to as the "subject matter of Ko 5").
`
`"A method for noninvasively diagnosing fetal aneuploidy
`by sequencing, comprising:
`a
`sequencing fetal and maternal DNA contained in peripheral
`blood in a method which gives about 10 million 25 bp short reads
`per sample on average, with fetal DNA which is present in
`maternal plasma of peripheral blood as a subject of analysis;
`
`
`
`- 10/24 -
`
`0010
`
`

`

`SHUSAKU•YAMAMOTO 
` 
`Japanese Patent No. 6275145
`Opposition no: 2018-700659 WSGR Ref: 42534-704.761
`
`b
`utilizing the short reads as sequence tags to select a
`sequence tag that is mapped uniquely to the human genome with
`at most 1 mismatch and to map the sequence tag uniquely to each
`chromosome (1-22, X and Y) of the human genome;
`c
`counting a number of sequence tags falling within a window
`defined by dividing a sequence on a chromosome into about 50
`kb;
`normalizing a value of a sequence tag density within a
`d
`sample by counting a number of tags within each window on each
`chromosome, obtaining a median value of the total sequence tag
`count for each chromosome, obtaining a median value of all of
`the autosomal values, and using this value as a normalization
`constant to account for a difference in total number of sequence
`tags obtained for different samples; and
`e
`detecting over- or under-representation of any
`chromosome or chromosome portion in mixed DNA contributed by
`an aneuploidy fetus."
`
` 6
`
`
`
`Exhibit Ko No. 6
`Exhibit Ko No. 6, which is a document made available to
`
`the public prior to the priority dates, is an academic article
`entitled "Digital karyotyping". Exhibit Ko No. 6 describes a
`method for digital karyotyping consisting of: step 1 for
`obtaining a fragment with a genomic tag by isolating a genomic
`DNA and cleaving with a mapping enzyme (SacI); step 2 for
`ligating a biotinylated linker to both ends of the fragment;
`step 3 for cleaving the fragment with a fragmenting enzyme
`(NlaIII) and then isolating the fragment with streptavidin
`magnetic beads; step 4 for ligating the fragment to a linker
`containing a tagging enzyme site (MmeI); step 5 for releasing
`genomic tags using a tagging enzyme (MmeI); step 6 for
`performing ligation to form ditags, PCR amplification,
`concatenation, and sequencing; and step 7 for mapping the tags
`to each chromosome to evaluate the tag density (Fig. 1).
`
`
`
`
`- 11/24 -
`
`0011
`
`

`

`SHUSAKU•YAMAMOTO 
` 
`Japanese Patent No. 6275145
`Opposition no: 2018-700659 WSGR Ref: 42534-704.761
`7
`Exhibit Ko No. 7
`
`Exhibit Ko No. 7, which is a document made available to
`the public prior to the priority dates, is a user guide for the
`sequence analysis software "CASAVA v1.8.2" of Illumina.
`
`
`
` 9
`
`
`
`Exhibit Ko No. 9
`Exhibit Ko No. 9, which is a document made available to
`
`the public prior to the priority dates, is an academic article
`entitled "Accurate and exact CNV identification from targeted
`high-throughput sequence data". Exhibit Ko No. 9 describes
`that: CNV calls are confirmed by testing for a signature of
`sequences that span the CNV breakpoint (page 1/10: Abstract);
`and this method can detect a 200 bp CNV with 87% sensitivity
`and a 100 bp CNV with 80% sensitivity (column 2, right column,
`lines 30-49).
`
`Judgment of the Appeal Bench
`V
`Instant invention 1
`1
`The novelty and inventive step of instant invention 1 are
`
`first examined when the subject matter of Ko 1 is the primary
`sited subject matter, and then inventive step of instant
`invention 1 is examined when the subject matter of Ko 5 is the
`primary cited subject matter.
`
`(1) When the subject matter of Ko 1 is the primary cited
`
`
`
`- 12/24 -
`
` 8
`
`
`
`Exhibit Ko No. 8
`Exhibit Ko No. 8, which is a document made available to
`
`the public prior to the priority dates, is an academic article
`entitled "Genomic analysis of circulating cell-free DNA infers
`breast cancer dormancy". Exhibit Ko No. 8 describes that
`specific CNVs were detected, mirroring the primary tumor, up
`to 12 years after diagnosis in patients diagnosed as having
`breast cancer by genomic analysis of cfDNA isolated from plasma
`(page 220, Abstract).
`
`0012
`
`

`

`
`
`SHUSAKU•YAMAMOTO 
` 
`Japanese Patent No. 6275145
`Opposition no: 2018-700659 WSGR Ref: 42534-704.761
`subject matter
`A
`Comparison
`
`Instant invention 1 is compared to the subject matter of
`Ko 1.
`
`The "maternal plasma DNA molecule" in the subject matter
`of Ko 1 corresponds to the "extracellular polynucleotides … from
`a bodily sample from a subject" in instant invention 1. In
`addition, "introducing a unique synthetic DNA of six base pairs,
`which serves as a signature for each sample per maternal plasma
`sample, onto one end of each plasma DNA molecule, and using the
`synthetic DNA as an index for each sample" in the subject matter
`of Ko 1 enables "distinguishing … DNA molecules that belong to
`a specific sample from those belonging to other samples" with
`the index in the subject matter of Ko 1, and a single index,
`i.e., single synthetic DNA, is used for a single maternal plasma
`DNA molecule. Thus, the same synthetic DNA is introduced into
`a single maternal plasma DNA molecule. Therefore, "introducing
`a unique synthetic DNA of six base pairs, which serves as a
`signature for each sample per maternal plasma sample, onto one
`end of each plasma DNA molecule, and using the synthetic DNA
`as an index for each sample" in the subject matter of Ko 1
`corresponds to the "non-uniquely tagging extracellular
`polynucleotides or fragments thereof from a bodily sample from
`a subject, thereby producing a population of non-uniquely
`tagged extracellular polynucleotides" in instant invention 1.
`
`It is also apparent that multiple sequencing reads are
`produced by "co-sequencing multiplexed sample mixtures
`consisting of multiple maternal plasma DNA preparations" in the
`subject matter of Ko 1. Therefore, the above step corresponds
`to "sequencing said non-uniquely tagged extracellular
`polynucleotides, wherein each of the extracellular
`polynucleotide generate a plurality of sequencing reads" in
`instant invention 1.
`
`Further, "considering a sequencing result as valid only
`if analysis of the sample passed a set of quality control
`
`
`
`- 13/24 -
`
`0013
`
`

`

`SHUSAKU•YAMAMOTO 
` 
`Japanese Patent No. 6275145
`Opposition no: 2018-700659 WSGR Ref: 42534-704.761
`
`measures" in the subject matter of Ko 1 can be rephrased as
`considering a sequencing result, i.e., sequencing read, that
`does not pass a certain quality control measure is not valid,
`i.e., invalid. Thus, said step corresponds to "filtering out
`reads that fail to meet a set threshold" in instant invention
`1.
`Furthermore, "identifying a chromosomal origin of a
`
`sequenced read by comparing with a reference human genome" in
`the subject matter of Ko 1 is performed after considering a
`sequencing read that does not pass a certain quality control
`measure as invalid in the previous step, thus corresponding to
`"mapping the sequencing reads obtained from step (b)", "after
`reads are filtered out", "to a reference sequence" in instant
`invention 1.
`
`In addition, the step of calculating "percentage
`chromosome 21 of a test case" in "obtaining a z score for
`chromosome 21 in each maternal plasma sample by subtracting mean
`percentage chromosome 21 of a reference set of euploid
`pregnancies (controls) from percentage chromosome 21 of a test
`case and divided by a standard deviation of a value for
`percentage chromosome 21 among a reference sample set" in the
`subject matter of Ko 1 corresponds to "quantifying … mapped
`reads or unique sequencing reads in a plurality of predefined
`regions of the reference sequence" in instant invention 1, and
`the "reference set of euploid pregnancies (controls)"
`corresponds to the "control sample" in instant invention 1.
`Moreover, mean percentage chromosome 21 and the standard
`deviation of a value for percentage chromosome 21 are calculated
`for the "reference set of euploid pregnancies (controls)". Thus,
`the reference set is normalized. In view of the above, the step
`of obtaining a "z score for chromosome 21 in each maternal plasma
`sample" by "subtracting mean percentage chromosome 21 of a
`reference set of euploid pregnancies (controls) from percentage
`chromosome 21 of a test case and divided by a standard deviation
`of a value for percentage chromosome 21 among a reference sample
`
`
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`0014
`
`

`

`SHUSAKU•YAMAMOTO 
` 
`Japanese Patent No. 6275145
`Opposition no: 2018-700659 WSGR Ref: 42534-704.761
`
`set" corresponds to "comparing … to normalized numbers obtained
`from a control sample" in instant invention 1.
`
`Meanwhile, the "fetal trisomy 21" in the subject matter
`of Ko 1 is a genome level abnormality, but should be considered
`in view of common general knowledge as of the priority dates
`as "aneuploidy", which does not correspond to "copy number
`variation". This is also consistent with the instant
`specification describing "copy number variation" separately
`from "aneuploidy", as described in [0013] in the instant
`specification (translator's note: corresponding to [0013] of
`WO 2014/039556) "In some embodiments, the subject may be a
`pregnant female in which the abnormal condition may be a fetal
`abnormality selected from the group consisting of … copy number
`variations … aneuploidy …" and [0095] in the instant
`specification (translator's note: corresponding to [0095] of
`WO 2014/039556) "In some embodiments, the subject is a pregnant
`female. In some embodiments, the copy number variation or rare
`mutation or genetic variant is indicative of a fetal abnormality.
`In some embodiments, the fetal abnormality is selected from the
`group consisting of … copy number variations … aneuploidy …"
`
`Therefore, the point of agreement and difference between
`instant invention 1 and the subject matter of Ko 1 are the
`following.
`
`(Point of agreement)
`
`"A method for detecting a genome level abnormality
`comprising:
`
`a. non-uniquely tagging extracellular polynucleotides or
`fragments thereof from a bodily sample from a subject, thereby
`producing a population of non-uniquely tagged extracellular
`polynucleotides;
`
`b. sequencing said non-uniquely tagged extracellular
`polynucleotides, wherein each of the extracellular
`polynucleotide generates a plurality of sequencing reads;
`
`c. filtering out reads that fail to meet a set threshold;
`
`
`
`- 15/24 -
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`0015
`
`

`

`SHUSAKU•YAMAMOTO 
` 
`Japanese Patent No. 6275145
`Opposition no: 2018-700659 WSGR Ref: 42534-704.761
`
`
`d. mapping the sequencing reads obtained from step (b),
`after reads are filtered out, to a reference sequence;
`
`e. quantifying or enumerating mapped reads or unique
`sequencing reads in a plurality of predefined regions of the
`reference sequence; and
`
`f. determining a genome level abnormality in one or more
`of the plurality of predefined regions by:
`
` i. normalizing a number of sequencing reads in each of
`the plurality of predefined regions to each other and/or a
`number of unique sequencing reads in the plurality of predefined
`regions to each other; and/or
`
` ii. comparing a number of sequencing reads in each of the
`plurality of predefined regions and/or a number of unique
`sequencing reads in the plurality of predefined regions to
`normalized numbers obtained from a control sample."
`
`(Difference)
`
`The genome level abnormality subjected to detection is
`"copy number variation" in instant invention 1, whereas it is
`"fetal trisomy 21" in the subject matter of Ko 1.
`
` B
`
`Judgement on the difference
`
`(A) The difference will be examined. As discussed in IV-2 and
`5, Exhibit Ko Nos. 2 and 5 have a description on detection of
`"aneuploidy", but no description related to the detection of
`"copy number variation". As discussed in IV-3 and 7, Exhibit
`Ko Nos. 3 and 7 also do not have a description related to the
`detection of "copy number variation".
`
`As discussed in IV-4, Exhibit Ko No. 4 describes a method
`called Safe-SeqS, which can detect and quantify a rare mutation
`by massively parallel sequencing. Exhibit Ko No. 4 also
`describes that endogenous UIDs and exogenous UIDs, which are
`identifiers unique to each DNA template molecule to be analyzed,
`are used to decrease the presumptive sequencing error by at
`least 70-fold. However, Exhibit Ko No. 4 specifically describes
`
`
`
`- 16/24 -
`
`0016
`
`

`

`SHUSAKU•YAMAMOTO 
` 
`Japanese Patent No. 6275145
`Opposition no: 2018-700659 WSGR Ref: 42534-704.761
`
`that a single base variation of a specific gene, i.e., CTNNB1
`gene from three individuals, was analyzed with Safe-SeqS. In
`view of the above, Safe-SeqS is for accurate detection of a
`mutation of a base sequence in a gene such as a single base
`variation, but not for detecting "copy number variation".
`Exhibit Ko No. 4 describes that the number of templates analyzed
`is obtained by Safe-SeqS. However, in view of the entire
`description in Exhibit Ko No. 4, it can be understood that
`information is obtained as to whether the number of genes with
`a base sequence mutation is 1 or 2, i.e., whether the genetic
`mutation is hetero or homo, from the description of "yields the
`number of templates analyzed", but the description cannot be
`considered as suggesting that copy number variation can be
`detected.
`
`Furthermore, as discussed in IV-6, the method described
`in Exhibit Ko No. 6 is "karyotyping", i.e., a method of studying
`the overall number and structure of chromosomes, but is not a
`method intended to detect "copy number variation". Thus, it is
`not suggested that copy number variation can be detected by the
`method described in Exhibit Ko No. 6.
`
`In view of the above, Exhibit Ko Nos. 2-7 fail to describe
`or suggest the detection of "copy number variation".
`(B) The descriptions in Exhibit Ko Nos. 8-9 are now examined.
`While both have a description on copy number variations, as
`discussed in IV-8, Exhibit Ko No. 8 merely describes that copy
`number variations from tumor were detectable over a long period
`of time in plasma. As discussed in IV-9, Exhibit Ko No. 9 merely
`describes a method of determining copy number variation by
`testing for a signature of sequences that span the CNV
`breakpoint. From the descriptions in Exhibit Ko Nos. 8 and 9,
`it can be understood that copy number variations were commonly
`detected prior to the priority dates of the present application.
`Meanwhile, it is not possible to understand that detection of
`"copy numb