PATENT COOPERATION TREATY
`
`PCT
`
`INTERNATIONAL PRELIMINARY REPORT ON PATENTABILITY
`
`(Chapter I of the Patent Cooperation Treaty)
`
`(PCT Rule 44bis)
`
`Applicant’s or agent’s file reference
`44854-730601
`
`FOR FURTHER ACTION
`
`See item 4 below
`
`International filing date (day/month/year)
`International application No.
`17 November 2017 (17.11.2017)
`PCT/U82017/062391
`International Patent Classification (8th edition unless older edition indicated)
`See relevant information in Form PCT/ISA/237
`
`Priority date (day/month/year)
`18 November 2016 (18.11.2016)
`
`Applicant
`TWIST BIOSCIENCE CORPORATION
`
`This international preliminary report on patentability (Chapter I) is issued by the International Bureau on behalf of the
`International Searching Authority under Rule 44 bis.1(a).
`
`This REPORT consists of a total of 18 sheets, including this cover sheet.
`
`In the attached sheets, any reference to the written opinion of the International Searching Authority should be read as a
`reference to the international preliminary report on patentability (Chapter I) instead.
`
`This report contains indications relating to the following items:
`
`Box \0. I
`
`Basis of the report
`
`Box \0. II
`
`Box \0.
`
`
`
`Priority
`
`Non—establishment of opinion with regard to novelty, inventive step and industrial
`applicability
`
`Lack of unity of invention
`
`Reasoned statement under Article 35(2) with regard to novelty, inventive step or
`industrial applicability; citations and explanations supporting such statement
`
`Certain documents cited
`
`Certain defects in the international application
`
`Certain observations on the international application
`
`'lhe International Bureau will communicate this report to designated Offices in accordance with Rules 44bis.3(c) and 93bis.1
`but not, except where the applicant makes an express request under Article 23(2), before the expiration of 30 months from
`the priority date (Rule 44bis .2).
`
`The International Bureau of WIPO
`34, chemin des Colombettes
`1211 Geneva 20, Switzerland
`Facsimile NO. +41 22 338 82 70
`Form PCT/IB/373 (January 2004)
`
`Date of issuance of this report
`21 May 2019 (21.05.2019)
`
`AthHZEd officer
`
`Athlna NICKItaS-Etlenne
`e—mail; pct.[eam4@wip0.int
`
`
`
`
`
`Box \0.
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`Box \0.
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`Box \0.
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`Box \0.
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`Box \0.
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`E |
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`:|
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`& g |
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`|:|
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`

`

`PCT/USZO17I062391 28.03.2018
`
`From the
`INTERNATIONA L SEARCHING AUTHORITY
`
`PATENT COOPERATION TREATY
`
`To: DAVID HARBURGER
`WILSON SONSINI GOODRICH & ROSATI
`650 PAGE MILL ROAD
`PALO ALTO, CA 94304
`
`PCT
`
`WRITTEN OPINION OF THE
`INTERNATIONAL SEARCHING AUTHORITY
`
`(PCT Rule 43 bis. I)
`
`Applicant’s or agent’s file reference
`44854-730601
`
`See paragraph 2 below
`
`International application No.
`
`International filing date (day/monrh/year)
`
`Priority date (day/monIh/year)
`
`International Patent Classification (IPC) or both national classification and IPC
`
`IPC(8) _ C40B 50/14, C120 1/68, C12N 15/10 (2018.01)
`CPC _
`C4OB 50/14, C120 1/6869, C120 1/6876, C408 50/00, C403 40/08
`
` Bfi/EZZ‘fi/EZE» Z 8 MA R 2018
`Applicant TWIST BIOSCIENCE CORPORATION
`EDDKEDEE Box No. VIII Certain observations on the international application
`
`
`
`1. This opinion contains indications relating to the following items:
`
`Box No.
`
`I
`
`Basis ofthe opinion
`
`Box No. II
`
`Priority
`
`Box No. III Non-establishment ofopinion with regard to novelty, inventive step and industrial applicability
`
`Box No. IV
`
`Lack ofunity ofinvention
`
`Box No. V
`
`Reasoned statement under Rule 4317/3. I(a)(i) with regard to novelty, inventive step and industrial applicability;
`citations and explanations supporting such statement
`
`Box No. VI
`
`Certain documents cited
`
`Box No. VII Certain defects in the international application
`
`2. FURTHER ACTION
`
`If a demand for international preliminary examination is made, this opinion will be considered to be a written opinion of the
`International Preliminary Examining Authority (“IPEA”) except that this does not apply where the applicant chooses an Authority
`other than this one to be the IPEA and the chosen IPEA has notified the International Bureau under Rule 66. lbis(b) that written
`opinions ofthis lntemational Searching Authority will not be so considered.
`lfthis opinion is, as provided above, considered to be a written opinion ofthe IPEA, the applicant is invited to submit to the IPEA
`a written reply together, where appropriate, with amendments, before the expiration of} months fiom the date ofmailing ofFonn
`PCT/ISA/220 or before the expiration 0f22 months from the priority date, whichever expires later.
`For further options, see Form PCT/ISA/ZZO.
`
`PCT osp: 571-272-7774
`
`Name and mailing address of the ISA/US Date ofcompletion ofthis opinion
`Mail Stop PCT, Attn: ISA/US
`Commissioner for Patents
`PO. Box 1450, Alexandria, Virginia 22313-1450
`Facsimile No. 571-273-8300
`
`15 March 2018
`
`Authorized officer
`Lee W. Young
`PCT Helpdesk: 5714724300
`
`Form PCT/ISA/237 (cover sheet) (January 20 I 5)
`
`

`

`PCTIUSZO17IO62391 28.03.2018
`
`Box No. 1
`
`Basis of this opinion
`
`I. With regard to the language, this opinion has been established on the basis of:
`
`the international application in the language in which it was filed.
`[X]
`I: a translation ofthe international application into
`
`
`
`which is the language ofa translation
`
` WRITTEN OPINION OF THE
`International application No.
`INTERNATIONA L SEARCHING A UTHORITY
`PCT/US 17/62391
`
`
`
`
`
`
`
`
`furnished for the purposes ofintemational search (Rules 12.3(a) and 21103)).
` 2. [:1 This opinion has been established taking into account the rectification of an obvious mistake authorized by or notified to
`this Authority under Rule 9| (Rule 43bis. l(a)).
` With regard to any nucleotide and/or amino acid sequence disclosed in the international application, this opinion has
`
`
`
`
`
`
`
`
`
`
`
`
`
`3-IZI
`
`been established on the basis ofa sequence listing:
`
`a.
`
`forming part ofthe international application as filed:
`
`E in the form of an Annex C/ST.25 text file.
`D on‘ paper or in the form ofan image file.
`
`b. [:1 furnished together with the international application under PCT Rule |31er.|(a) for the purposes ofintemational
`search only in the form ofan Annex C/ST.25 text file.
`
`e. I: furnished subsequent to the international filing date for the purposes ofintemational search only:
`I: in the form of an Annex C/ST.25 text file (Rule l3ter. 1(a)).
`[j on paper or in the form ofan image file (Rule 131err1(b) and Administrative instructions, Section 713).
`
` 4. [:1 In addition, in the case that more than one version or copy ofa sequence listing has been filed or fiJrnished, the required
`
`statements that the information in the subsequent or additional copies is identical to that forming part ofthe application as
`filed or does not go beyond the application as filed, as appropriate, were fiimished.
`
`
`
`5. Additional comments:
`
`
`
`Form FCT/lSA/237 (Box No. 1) (January 2015)
`
`

`

`PCT/U82017l062391 28.03.2018
`
`WRITTEN OPINION OF THE
`
`INTERNATIONA L SEA RCHING AUTHORITY
`
`Box No. IV
`
`Lack of unity ofinvention
`
`PCT/US17/62391
`
` lntemational application No.
`
`
`
`
`
`
`
`
`
`
`
`[2]
`not paid additional fees.
`
` 2. E] This Authority found that the requirement ofunity ofinvention is not complied with and chose not to invite the applicant to
`pay additional fees.
` 3. This Authority considers that the requirement ofunity ofinvention in accordance with Rule 13.], 13.2 and l3.3 is
`
`
`D complied with.
`v
`
`
`
`
`not complied with for the following reasons:
`This application contains the following inventions or groups of inventions which are not so linked as to form a single general inventive
`concept under PCT Rule 13.1. In order for all inventions to be examined, the appropriate additional examination fees must be paid.
`
`V
`
`1.
`
`In response to the invitation (Form PCT/lSA/206) to pay additional fees the applicant has, within the applicable time limit:
`
`paid additional fees.
`
`E]
`
`E]
`
`paid additional fees under protest and, where applicable, the protest fee.
`
`D paid additional fees under protest but the applicable protest fee was not paid.
`
`Group I: Claims 1-36, 55-78, drawn to compositions comprising a polynucleotide library and method for using said polynucleotide library
`to sequence genomic DNA
`
`Group II: Claims 37-45, drawn to a method for generating a polynucleotide library
`
` Group III: Claims 46-54, drawn to a method for amplifying a polynucleotide library having a preselected representation of a sequence
`
`
`feature
`
`The inventions listed as Groups I, II, ”I do not relate to a single general inventive concept under PCT Rule 13.1 because, under PCT
`
`
`Rule 13.2, they lack the same or corresponding special technical features for the following reasons:
`
`Special Technical Features
`
` Group I requires compositions of matter comprising a polynucleotide library, and method steps for using said polynucleotide library for
`
`
`sequencing genomic DNA, not required by Groups II, III.
`
`Group II requires method steps for generating a polynucleotide library by providing predetermined sequences encoding a set of
`
`
`polynucleotides, not required by Groups I, III.
`
`
`
`Group III requires method steps for amplifying a polynucleotide library having a preselected representation by obtaining an amplification
`distribution for a set of polynucleotides. clustering polynucleotides into two or more bins based on a sequence feature, and adjusting the
`
`
`ll. relative frequency of polynucleotides in at least one bin, not required by Groups I,
`
`Common Technical Features
`
`
`The feature shared by Groups I, II. III is a polynucleotide library comprising at least 5000 polynucleotides.
`
`The feature shared by Groups ll and III is synthesizing a polynucleotide library and amplifying the polynucleotide library.
`
` However, these shared technical features do not represent a contribution over prior art. because the shared technical features are taught
`
`
`by US 2015/0038373 A1 to Twist Bioscience Corp. (hereinafter 'Twist').
`
` Twist discloses a polynucleotide library comprising at least 5000 polynucleotides (para [0010] - "ln practicing any of the methods of
`
`
`constructing a gene library as provided herein, in some embodiments, the list of genes comprises at least 500 genes, In some
`embodiments, the list comprises at least 5000 genes“).
` ========= Please see continuation in supplemental box ---------
`
` Consequently, this opinion has been established in respect ofthe following parts ofthe international application:
`
`
`
`D all parts.
`
`
`the parts relating to claims Nos. 1-36l 55-78
`
`Form PCT/ISA/237 (Box No. lV) (January 2015)
`
`

`

`PCT/USZO17I062391 28.03.2018
`
`WRITTEN OPINION OF THE
`INTERNATIONAL SEARCHING AUTHORITY
`
`International application No.
`PCT/US17/62391
`
`Box No. V
`
`Reasoned statement under Rule 43bis.l(a)(i) with regard to novelty, inventive step or industrial applicability;
`citations and explanations supporting such statement
`
`Statement
`
`Novelty (N)
`
`Claims
`Claims
`
`1-36, 55-78
`None
`
`--------- Please see continuation in supplemental box —————————
`
`lnventivc step (IS)
`
`Claims
`Claims
`
`
`None
`1-35, 55-78
`
`Industrial applicability (IA)
`
`Claims
`Claims
`
`1-35. 55-78
`None
`
`Citations and explanations:
`2.
`Claims 1-7, 9-11, 13—26. 28-30, 32-36, (55-67, 69-70, 72-78)/(1-7, 9-11, 13-26, 28-30, 32-36) lack an inventive step under PCT Article
`33(3) as being obvious over US 2016/0032396 A1 to the Board of Trustees of the Leland Stanford Junior University (hereinafter
`'Stanford').
`
`Regarding claim 1, Standford discloses a polynucleotide library (para [0711] - "a next-generation sequencing (NGS) library"; para [0601] -
`"methods described herein for creating a selector set (e.g., library)"; para [0013] - "The selector population of DNA oligonucleotides, which
`may be referred to as a selector set, comprises probes for a plurality of genomic regions").
`Stanford does not teach that the polynucleotide library comprising at least 5000 polynucleotides, wherein each of the at least 5000
`polynucleotides is present in an amount such that, following hybridization with genomic fragments and sequencing of the hybridized
`genomic fragments, the polynucleotide library provides for at least 30 fold read depth of at least 90 percent of the bases of the genomic
`fragments under conditions for up to a 55 fold theoretical read depth for the bases of the genomic fragments. However, Stanford does
`teach that the polynucleotides of the library hybridize to a plurality of genomic regions and are used for sequencing (para [0293] - "The
`composition may comprise a set of oligonucleotides that selectively hybridize to a plurality of genomic regions"; para [0493] - "producing a
`library for sequencing, the library comprising the plurality of amplicons"). Stanford also teaches that the genomic regions can number over
`a thousand (para [0303] - "The genomic regions may comprise at least .
`.
`. 1500 regions"). Given that Stanford teaches that more than a
`thousand genomic regions can be targeted by the library, where the library hybridizes to said genomic regions and can be used for
`sequencing methods. one of ordinary skill in the art would have found it obvious that the polynucleotide library can comprise at least 5000
`polynucleotides, wherein each of the polynucleotides hybridize with genomic fragments and allow for sequencing of the hybridized
`genomic fragments. Furthermore, Stanford teach that a user-defined minimum read depth is used to select the genomic regions. where
`the read depth may be at least 10 times or more (para [0418] - "Producing the list of genomic regions may comprise selecting genomic
`regions with a minimum user-defined read depth"; para [0419] — "The minimum user-defined read depth may be at least 2.times., 3.times.,
`4.times., 5.times., 6.times., 7.times., 8.times., 9.times., 10.times. or more"). Given that effective parameters, such as the amount of
`polynucleotide needed for a pre-selected read depth is a parameter that a person of ordinary skill in the art would routinely optimize, it
`would have been customary for an artisan of ordinary skill to determine the optimal amount of polynucleotide needed to achieve the
`desired user—defined read depth. Thus, one of ordinary skill in the art would have found it obvious that the polynucleotides are present in
`an amount such that, the polynucleotide library provides for at least 30 fold read depth of at least 90 percent of the bases of the genomic
`fragments under conditions for up to a 55 fold theoretical read depth for the bases of the genomic fragments.
`
`Regarding claims 2 and 3, Stanford discloses the polynucleotide library of claim 1, but does not specifically teach wherein the
`polynucleotide library provides for at least 30 fold read depth of at least 95 or 98 percent of the bases of the genomic fragments under
`conditions for up to a 55 fold theoretical read depth for the bases of the genomic fragments. However. Stanford teach that a user-defined
`minimum read depth is used to select the genomic regions, where the read depth may be at least 10 times or more (para [0418] —
`"Producing the list of genomic regions may comprise selecting genomic regions with a minimum user-defined read depth"; para [0419] -
`"The minimum user-defined read depth may be at least 2.times., 3.times., 4.times., 5.times., 6.times., 7.times_, B.times., 9.times., 10.times.
`or more"). Given that effective parameters, such read depth is a parameter that a person of ordinary skill in the art would routinely
`optimize, it would have been customary for an artisan of ordinary skill to determine the optimal read depth needed to achieve the desired
`results. Thus, one of ordinary skill in the art would have found it obvious wherein the polynucleotide library provides for at least 30 fold
`read depth of at least 95 or 98 percent of the bases of the genomic fragments under conditions for up to a 55 fold theoretical read depth
`for the bases of the genomic fragments.
`
`Regarding claims 4 and 5, Stanford discloses the polynucleotide library of claim 1, but does not specifically teach wherein the
`polynucleotide library provides for at least 90 or 95 percent unique reads for the bases of the genomic fragments. However, Stanford does
`teach that the polynucleotide library is able to uniquely identify genomic regions and that the genomic regions are selected to minimize
`overlaps (para [0523] - "Oligonucleotides may have the general characteristic of sufficient length to uniquely identify the genomic region";
`para [0576] - "Producing the selector set may comprise selecting one or more genomic regions based on minimizing overlap of subjects
`already identified by the selector"). Thus, given the minimal overlap and ability to identify unique sequences, one of ordinary skill in the art
`would have found it obvious wherein the polynucleotide library provides for at least 90 or 95 percent unique reads for the bases of the
`genomic fragments.
`
`Form PCT/ISA/237 (Box No. V) (January 2015)
`
`

`

`PCT/USZO17I062391 28.03.2018
`
`WRITTEN OPINION OFTHE
`INTERNATIONAL SEARCHING AUTHORITY
`
`
`International application No.
`
`PCT/US 17/62391
`
`
`Supplemental Box
`
`
`
`In case the space in any of the preceding boxes is not sufficient.
`Continuation of:
`Box No. IV Lack of unity of invention
`
`Twist discloses a method comprising synthesizing a polynucleotide library and amplifying the polynucleotide library (para [0204] - "Various
`
`
`methods are contemplated for the de novo synthesis of gene libraries with low error rates. .
`.
`. For example, oligonucleotide synthesis
`typically in situ on a DNA synthesis wafer. may be followed by a gene assembly reaction, such as polymerase cycling assembly (PCA), of
`
`
`the synthesized oligonucleotides into longer sequences. The assembled sequences may be amplified. 9.9. through PCR").
`
`As the technical features were known in the art at the time of the invention, they cannot be considered special technical features that would
`
`
`
`otherwise unify the groups.
`
` Groups I, II, III therefore lack unity of invention under PCT Rule 13 because they do not share a same or corresponding special technical
`
`
`feature.
`
`
`
`
`
`Form PCT/lSA/237 (Supplemental Box) (January 2015)
`
`

`

`PCT/USZO17I062391 28.03.2018
`
`WRITTEN OPINION OF THE
`INTERNATIONAL SEARCHING AUTHORITY
`
` lntemational application No.
`
`PCT/US 17/62391
`
`
`
`in case the space in any of the preceding boxes is not sufficient.
`Continuation of:
`Box No. V2 Citations and Explanations
`
`
` Regarding claims 6 and 7, Stanford discloses the polynucleotide library of claim 1, but does not teach wherein the polynucleotide library
`
`
`provides for at least 90 or 95 percent of the bases of the genomic fragments having a read depth within about 1.5 times the mean read
`
`depth. However, Stanford teach that a user-defined minimum read depth is used to select the genomic regions, where the read depth may
`
`
`be at least 10 times or more (para [0418] — "Producing the list of genomic regions may comprise selecting genomic regions with a minimum
`user-defined read depth"; para [0419] - "The minimum user—defined read depth may be at least 2.times., 3.times., 4.times., 5.times.,
`
`
`6.times., 7.times., 8.times., 9.times., 10.times. or more"). Given that effective parameters. such read depth is a parameter that a person of
`
`
`ordinary skill in the art would routinely optimize, it would have been customary for an artisan of ordinary skill to determine the optimal read
`
`
`depth needed to achieve the desired results. Thus, one of ordinary skill in the art would have found it obvious wherein the polynucleotide
`
`
`library provides for at least 90 or 95 percent of the bases of the genomic fragments having a read depth within about 1.5 times the mean
`read depth.
`
`
`
`Regarding claim 9, Stanford discloses the polynucleotide library of claim 1, but does not teach wherein the polynucleotide library provides
`for at least about 80 percent of the genomic fragments having a repeating or secondary structure sequence percentage from 10 percent to
`
`
`
`30 percent or 70 percent to 90 percent having a read depth within about 1.5x of the mean read depth. However, Stanford does teach that
`
`
`the polyncleotides target genomic regions that comprise repeating structures (para [0140] - "Determining the quantities of ctDNA may
`comprise detecting one or more mutations. Determining the quantities of ctDNA may comprise detecting two or more different types of
`
`
`mutations. The types of mutations include, but are not limited to, SNVs, indels, fusions, breakpoints, structural variants, variable number of
`
`
`tandem repeats, hypervariable regions, minisatellites, dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats, simple sequence
`
`
`repeats, or a combination thereof in selected regions of the subject's genome"). Stanford further teaches that a user-defined minimum read
`
`
`depth is used to select the genomic regions (para [0418] - "Producing the list of genomic regions may comprise selecting genomic regions
`
`
`with a minimum user-defined read depth"; para [0419] - 'The minimum user-defined read depth may be at least 2.times.. 3.times., 4.times.,
`
`
`5.times., 6.times., 7.times., 6.times., 9.times., 10.times. or more"). Given that effective parameters. such as the read depth is a parameter
`
`
`that a person of ordinary skill in the art would routinely optimize, it would have been customary for an artisan of ordinary skill to determine
`
`
`the optimal read depth needed to achieve the desired results. Thus, one of ordinary skill in the art would have found it obvious wherein the
`
`
`polynucleotide library provides for at least about 80 percent of the genomic fragments having a repeating or secondary structure sequence
`
`
`percentage from 10 percent to 30 percent or 70 percent to 90 percent having a read depth within about 1.5x of the mean read depth.
`
`Regarding claim 10, Stanford discloses the polynucleotide library of claim 1, wherein each of the genomic fragments are about 100 bases
`to about 500 bases in length (para [0513] — "A genomic region may comprise a sequence of the human genome of sufficient size to capture
`
`one or more recurrent mutations. .
`.
`. The length of genomics region in a selector set may be on average around about 100 bp, about 125
`
`
`bp, about 150 bp, 175 bp, about 200 bp, about 225 bp, about 250 bp, about 275 bp, or around about 300 bp").
`
`Regarding claim 11, Stanford discloses the polynucleotide library of claim 1, but does not expressly teach wherein at least about 80
`percent of the at least 5000 polynucleotides are represented in an amount within at least about 1.5 times the mean representation for the
`
`polynucleotide library. However, Stanford does teach that the method for selecting the polynucleotides for the library uses a recurrence
`
`
`index to maximize coverage of the library and to maximize a median number of mutations per subject in the population of subjects (para
`[0590] - "A method of producing a selector set may comprise (a) calculating a recurrence index for a plurality of genomic regions from a
`
`
`population of subjects suffering from a cancer by dividing a number of subjects containing one or more mutations in a genomic region of
`
`
`the plurality of genomic regions by a size of the genomic region; and (b) producing a selector set comprising two or more genomic regions
`
`
`of the plurality of genomic regions by (i) using the recurrence index to maximize coverage of the selector set for the population of subjects;
`
`
`and/or (ii) using the recurrence index to maximize a median number of mutations per subject in the population of subjects"). Given that the
`
`
`polynucleotide library is reflective of the representation of genomic regions of 100 percent of the population of subjects of the subject
`
`
`patient genome whereby the polynucleotide library is used in an amount that is able to hybridize to the represented genomic regions
`
`
`present in the patient population genome, one of ordinary skill in the art would have found it obvious wherein at least about 80 percent of
`
`
`the at least 5000 polynucleotides are represented in an amount within at least about 1.5 times the mean representation for the
`
`
`polynucleotide library.
`
`Regarding claim 13, Stanford discloses the polynucleotide library of claim 1, but does not specifically teach wherein at least about 15
`percent of the at least 5000 polynucleotides comprise polynucleotides having a repeating or secondary structure sequence percentage
`
`from 10 percent to 30 percent or 70 percent to 90 percent. However, Stanford does teach that the polyncleotides target genomic regions
`
`
`that comprise repeating structures (para [0140] - "Determining the quantities of ctDNA may comprise detecting one or more mutations.
`Determining the quantities of ctDNA may comprise detecting two or more different types of mutations. The types of mutations include, but
`
`
`are not limited to, SNVs, indels, fusions, breakpoints, structural variants, variable number of tandem repeats, hypervariable regions,
`
`
`minisatellites, dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats, simple sequence repeats, or a combination thereof in
`
`
`selected regions of the subject's genome"). Given that the polynucleotide library hybridizes to genomic region targets that comprise
`
`
`repeating structures, one of ordinary skill in the art would have found it obvious wherein at least about 15 percent of the at least 5000
`
`
`polynucleotides comprise polynucleotides having a repeating or secondary structure sequence percentage from 10 percent to 30 percent
`
`
`or 70 percent to 90 percent.
`
` Regarding claim 14, Stanford discloses the polynucleotide library of claim 1, wherein the at least 5000 polynucleotides as discussed
`
`
`above, and Stanford further teaches that the polynucleotides encode for at least 1000 genes (para [0293] - "The composition may
`
`comprise a set of oligonucleotides that selectively hybridize to a plurality of genomic regions"; para [0303] — "The genomic regions may
`
`
`comprise at least 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, or 1500
`
`
`regions”; para [0126] - "The genomic regions may comprise genes").
`
` --------- Please see continuation in next supplemental box ---------
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Form PCT/lSA/237 (Supplemental Box) (January 20l5)
`
`

`

`WRITTEN OPINION OF THE
`INTERNATIONAL SEARCHING AUTHORITY
`
`Supplemental Box
`
`
`
`
`
`
`
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`
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`
`
`
`PCT/U82017l062391 28.03.2018
`
` International application No.
`
`PCT/US 17/62391
`
`
`
`
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`
`
`
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`
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`
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`In case the space in any ofthe preceding boxes is not sufficient.
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`Continuation of:
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`Box No. V2 Citations and Explanations
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`Regarding claims 15 and 16, Stanford discloses the polynucleotide library of claim 1, but does not specifically teach wherein the
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`polynucleotide library comprises at least 100, 000 polynucleotides or at least 700,000 polynucleotides. However, Stanford does teach that
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`the polynucleotides of the library hybridize to a plurality of genomic regions and are used for sequencing (para [0293] - "The composition
`may comprise a set of oligonucleotides that selectively hybridize to a plurality of genomic regions"; para [0493] - "producing a library for
`sequencing, the library comprising the plurality of amplicons"). Stanford also teaches that the genomic regions can number over a
`thousand (para [0303] — "The genomic regions may comprise at least .
`.
`. 1500 regions"). Given that Stanford teaches that more than a
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`thousand genomic regions can be targeted by the library, where the library hybridizes to said genomic regions, one of ordinary skill in the
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`art would have found it obvious that the method of producing a polynucleotide library as taught by Stanford can be used to generate a
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`large library of for example at least 100, 000 or at least 700, 000 polynucleotides.
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`Regarding claim 17, Stanford discloses the polynucleotide library of claim 1, comprising at least 5000 polynucleotides, as discussed
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`above, and Stanford further teaches wherein the polynucleotides comprise at least one exon sequence (para [0325] - "The plurality of
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`genomic regions may comprise .
`. 100 or more different protein-coding regions. The protein-coding regions may comprise an exon").
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`Regarding claim 18, Stanford discloses the polynucleotide library of claim 16, comprising at least 700,000 polynucleotides as discussed
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`above. Stanford does not expressly teach that the polynucleotides comprise at least one set of polynucleotides collectively comprising a
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`single exon sequence. However, Stanford does teach that the polynucleotides comprise at least one exon sequence (para [0325] - "The
`plurality of genomic regions may comprise .
`. 100 or more different protein-coding regions. The protein—coding regions may comprise an
`exon") and that the genome may comprise up to 1.5 megabases wherein at least 95% of the genimic regions may comprise exonic regions
`(para [0138] - “The subset of the genome may comprise less than 1.5 megabases (Mb)"; para [0127] - "At least about 10%, 15%, 20%,
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`25%. 30%, 35%, 40%, 45%. 50%, 55%. 60%, 65%, 70%, 75%. 80%, 85%. 90%, or 95% of the genomic regions may comprise exonic
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`regions"). Given that the polynucleotide library hybridizes to the genomic regions and that the genome may be large and comprise a large
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`exonic region, one of ordinary skill in the art would have found it obvious wherein the polynucleotides comprise at least one set of
`polynucleotides collectively comprising a single exon sequence.
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` Regarding claim 19, Stanford discloses the polynucleotide library of claim 18, comprising at least 700,000 polynucleotides as discussed
`
`
`above. Stanford does not expressly teach that the polynucleotides comprises at least 150,000 sets. However, Stanford does teach that
`sequencing can be performed using massively parallel sequencing on a subset of a genome (para [0137] - "Obtaining sequence
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`information may comprise performing massively parallel sequencing. Massively parallel sequencing may be performed on a subset of a
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`genome of the cell-free nucleic acids'from the sample"). Stanford also teaches that the genomic regions can number over a thousand
`(para [0303] - "The genomic regions may comprise at least .
`.
`. 1500 regions") and that the polynucleotides of the library hybridize to a
`plurality of genomic regions and are used for sequencing (para [0293] - "The composition may comprise a set of oligonucleotides that
`selectively hybridize to a plurality of genomic regions"; para [0493] - "producing a library for sequencing, the library comprising the plurality
`of amplicons"). Given that Stanford teaches that more than a thousand genomic regions can be targeted by the library, where the library
`hybridizes to said genomic regions, one of ordinary skill in the art would have found it obvious that the method of producing a
`polynucleotide library as taught by Stanford can be used to generate a large library that can be subdivided into subsets that are used for
`sequencing subsets of a genome. Thus, one of ordinary skill in the an would have found it obvious that the library can comprise a large
`number of sets, such as at least 150,000 sets.
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`Regarding claim 20, Stanford discloses a polynucleotide library (para [0711] - "a next—generation sequencing (NGS) library"; para [0601] -
`"methods described herein for creating a selector set (e.g., library)"; para [0013] - "The selector population of DNA oligonucleotides, which
`may be referred to as a selector set, comprises probes for a plurality of genomic regions"),
`wherein each of the polynucleotides is about 20 to 200 bases in length (para [0268] - "An oligonucleotide of the plurality of oligonucleotides
`may be between about 20 to 200 nucleotides in length").
`wherein the plurality of polynucleotides encode sequences from each exon for at least 1000 preselected genes (para [0293] - "The
`
`composition may comprise a set of oligonucleotides that selectively hybridize to a plurality of genomic regions"; para [0303] - "The genomic
`
`regions may comprise at least 250, 300. 350. 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400,
`or 1500 regions"; para [0126] - "The genomic regions may comprise genes"; para [0325] — "The plurality of genomic regions may comprise
`. 100 or more different protein-coding regions. The protein-coding regions may comprise an exon"),
`wherein each polynucleotide comprises a molecular tag (para [0294] - "An oligonucleotide of the set of oligonucleotides may comprise a
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`tag. The tag may be biotin. The tag may be a label. The label may be a fluorescent label or dye. The tag may be an adaptor"; para [273] -
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`"The adaptor may comprise a molecular barcode").
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`Stanford does not teach that the p