PCTIUSZO19I032992 06.09.2019
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`PATENT COOPERATION TREATY
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`From the INTERNATIONAL SEARCHING AUTHORITY
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`PCT
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`INVITATION TO PAY ADDITIONAL FEES
`AND, WHERE APPLICABLE, PROTEST FEE.
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`(PCT Article l7(3)(a) and Rules 40.1 and 40.2(e))
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`To: SEAN A. REED
`WILSON SONSIN] GOODRICH & ROSATI
`650 PAGE MILL ROAD
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`PALO ALTO. CA 94304
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`Date ofmailing
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`within ONE MONTH from
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`Applicant 5 or agent 5 file reference
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`the above date of mailing
`44354376601
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`international application No.
`International filing date
`
`
`PCTi‘US 19132992
`17 May 2019 (17.05.2019)
`(“y/””“Wm”
`
`
`Applicant TWIST BIOSCIENCE CORPORATION
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`
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`1. This International Searching Authority
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`(numberof) inventions claimed in the international application covered
`3
`(i) considers that there are
`by the claims indicated belowlon an extra sheet:
`—————see extra sheet—u—
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`(ii)
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`therefore considers that the international application does not comply with the requirement of unity of invention
`(Rules 13.1, 13.2 and [3.3) for the reasons indicated belowi’on an extra sheet:
`muses extra sheet—u-
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`(iii) [:1 has carried out a partial international search (see Annex) El will establish the international search repon
`1-4 on those parts ofthc international application which relate to the invention first mentioned in claims Nos:
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`(iv) will establish the international search report on the other parts of'the international application only if, and to the extent
`to which, additional fees are paid.
`
`Consequently, the applicant is hereby invited to pay, within the time limit indicated above, additional fees in the amount
`indicated below:
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`$2080
`Fee per additional invention
`
`x
`
`2
`number ofadditional inventions
`
`(See Item 2 of annex)
`= 54150
`total amount of additional feesfcurrency
`
`
`
`The applicant is informed that, according to Rule 40.2(c), the payment ol'any additional fees may be made under protest,
`that is, a reasoned statement to the effect that the international application complies with the requirement ofunity ofinvention
`or that the amount ofthe required additional fees is excessive, where applicable, subject to the payment ofa protest fee.
`
`Where the applicant pays additional fees under protest, the applicant is hereby invited, within the time limit indicated above,
`to pay a protest fee (Rule 40.2(e)) in the amount of
`{amortize/currency)
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`Where the applicant has not, within the lime limit indicated above, paid the required protest fee, the protestwill be considered
`not to have been made and the International Searching Authority will so declare.
`
`4.
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`have been found to be unsearchable under
`5-23 ZIP-37. 41-58. 63. 65-?5. 81-93. 97.10?
`Claim(s) Nos.
`" Article l?(2)(b) because ofdefects under Article l'r'(2}(a} and therefore have not been included with any invention.
`
`Name and mailing address of the ISAIUS
`Mail Stop PCT, Attn: ISNUS
`Commissioner for Patents
`
`PO. Box 1450. Alexandria. Virginia 22313-1450
`Facsimile No. 571-273-3300
`Form pcmsmzua (April 2005)
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`Authorized officer:
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`pm Halwask. 5?1,272_43m
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`Lee W. Young
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`

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`PCTIU8201 91032992 06.09.2019
`
`INVITATION TO PAY ADDITIONAL FEES
`AND, WHERE APPLICABLE, PROTEST FEE
`
`Perms 19.92992
`
`international application N0-
`
`Item 2 (continued). For International Applications filed on or after 01 January 2014.
`Applicant is reminded that the search fee per additional invention indicated in item 2 is the
`undiscounted fee per additional invention. An Applicant may pay the search fee per
`additional invention fee reduced by 50% (small entity assertion) or 75% (micro entity
`certification). as appropriate. See 37 CFR 1.27 and 1.29.
`
`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 searched. the appropriate additional search fees must be paid.
`
`Groups I: Claims 1-4. drawn to a method for sequencing genomic DNA comprising a polynucleotide library which provides a specific ratio
`of the read depth of the bases of the genomic fragments.
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`Groups II: Claims 24-26. 38-40. drawn to a composition comprising a polynucleotide library.
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`Grciups Ill: Claims 59-62. 64. ?6-80. 94-96. drawn to a composition for nucleic acid hybridization. and a method of use for nucleic acid
`hybridization or genomic sequencing.
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`The inventions listed as Groups 1 through lll 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:
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`Special Technical Features
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`Group | includes the special technical feature of adding a second polynucleotide library comprising at least one polynucleotide that binds
`to genomic fragments comprising the one or more Positions having less than average read depth. not required by Groups II and III.
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`Group It includes the special technical feature of a composition comprising a polynucleotide library. wherein less than all polynucleotides
`comprises a molecular tag. an index sequence. an adapter or a blocker. not required by Groups | and III.
`
`Group III includes the special technical feature of a composition for nucleic acid hybridization or genomic sequencing comprising an
`additive. wherein the additive reduces oft—target hybridization of the at least one polynucleotide or reduce the local concentration of
`nucleic acid at the air-liquid interface. not required by Groups | and II.
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`Common Technical Features
`
`The inventions of Groups l-III share the technical feature of a polynucleotide library. nucleic acid hybridization and genomic sequencing.
`
`The inventions of Groups I and II share the technical feature ofa polynucleotide library that provides a read depth of at least 30 percent of
`the bases of the genomic fragments corresponding to the polynucleotides; and a total number of sequencing reeds. wherein the total
`number of sequencing reads are capable of covering 100 percent of each of the bases of the genomic fragments corresponding to the
`polynucleotides at a theoretical read depth. wherein the ratio of the read depth of at least 30 percent of the bases of the genomic
`fragments corresponding to the polynucleotides to the theoretical read depth is at least 0.5 with a plurality of genomic fragments.
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`However. these shared technical features do not represent a contribution over prior art in view of US 2017i0355984 A1 to Counsyl. Inc.
`(hereinafter "Counsyi") and US 2016f0019341 A1 to Perscmalis. Inc. (hereinafter "Personalls").
`
`Counsyl teaches (instant claim 1) a method for sequencing genomic DNA(pare[011?]. methods of sequencing a nucleic acid molecule.
`methods of sequencing a nucleic acid library), comprising:
`(a) contacting a composition comprising a first polynucleotide library comprising at least 30.000 polynucleotides. wherein each of the at
`least 30.000 polynucleotides (para [018?]. A set of capture probes (also referred to as a capture probe library) can be used to enrich a
`plurality of nucleic acid molecules (for example. in a sequencing library which may or may not be amplified. for example using PCR). The
`capture probes comprise a nucleic acid sequence complementary to various portions within the region of interest. In some embodiments.
`a set of capture probes includes. for example. between about 2 and about 20.000 capture probe... A sequencing library can include
`nucleic acid molecules comprising a portion of the region of interest (for example. genomic DNA containing the region of interest can be
`fragmented into a plurality of nucleic acid molecules. with each nucleic acid molecule originating from the region of interest comprising a
`portion of the region of interest) is present in an amount such that. following hybridization with genomic fragments and sequencing of the
`hybridized genomic fragments (para [0190]. An equal number of each capture probe in the set of capture probes can result in sequencing
`depth variation because some capture probes may more efficiently bind a particular fragment of a sequence of interest than another
`capture probe. This may be due. for example. to a variance in the concentration of the capture probe.....Large variance in sequencing
`depth can decrease overall sequence quality by limiting sensitivity and specificity in low sequencing depth sub-regions of a region of
`interest). the polynucleotide library provides:
`a read depth of of the genomic fragments corresponding to the polynucleotides (para [0235]. Sequencing depth venetian (for example.
`after sequencing a nucleic acid sequencing library described herein) can be improve by balancing the different capture probes in the set
`of capture probes. Balanced capture probes are a set of capture probes for a sequence of interest. wherein the amount of each capture
`probe in the set is predetermined to account for varying efficiency of capture for each probe.)
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`———oontinued on next sheet———
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`Form PCTIISN'ZOS (extra shcct) (April 2005)
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`PCTIUSZO19I032992 06.09.2019
`
`TNVITATION To PAY ADDITIONAL FEES
`AND, WHERE APPLICABLE, PROTEST FEE
`
`inte’"a“°"a' “Pp"ca‘i‘m N0-
`PcTius 19:32992
`
`Continuation from prior sheet
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`Counsyl does not specifically teach wherein said read depth covers at lea st 80 percent of the bases of the genomic fragment and a total
`number of sequencing reads, wherein the total number of sequencing reads are capable of covering 100 percent of each of the bases of
`the genomic fragments corresponding to the polynucleotides at a theoretical read depth‘ wherein the ratio of the read depth of at least 80
`percent of the bases of the genomic fragments corresponding to the polynucleotides to the theoretical read depth is at least 0.5 with a
`plurality of genomic fragments. However, Counsyl teaches that a balanced capture probes provides improved sequencing efficiency by
`providing the ratio of a predicted sequencing depth profile and a desired sequencing depth profile as an objective function of the balanced
`capture probes (para [0236]. a balanced set of capture probes is constructed to enrich a nucleic acid library (or nucleic acid sequencing
`library) to minimize the difference between a predicted sequencing depth profile and a desired sequencing depth profile. The desired
`sequencing depth profile can be uniform or non-uniform. and the desired sequencing depth profile can be implied by an objective function
`used to minimize the difference between the predicted and desired sequencing depth profiles. In some embodiments, construction of the
`balanced set of capture probes is constrained by a minimum amount or a maximum amount of the capture probes in the balanced set of
`capture probes. The desired sequencing depth profile is made in reference to a reference sequencing library. which is a nucleic acid
`library used to prepare the balanced set of capture probes. Thus. when a set of capture probes is balanced using a reference sequencing
`library, the sequencing depth profile obtained after sequencing a test sequencing library may be different from the desired sequencing
`depth profile due to differences between the reference sequencing library and the test sequencing library.). Thus. it would have been
`obvious to one of ordinary skill in the art to have provided a balanced capture probes to achieve the claimed limitations: "wherein the ratio
`of the read depth of at least 80 percent of the bases of the genomic fragments corresponding to the polynucleotides to the theoretical
`read depth is at least 0.5 with a plurality of genomic fragments.".
`
`Counsyl further teaches:
`(b) enriching at least one genomic fragment that binds to the first polynucleotide library to generate at least one enriched target
`polynucleotide [para [02321 hybrid capture methods are used to enrich the region of interest by combining capture probes that are
`substantially complementary to a portion of the region of interest with the nucleic acid library {or nucleic acid sequencing library), thereby
`hybridizing the capture probes to nucleic acid molecules comprising the portion of the region of interest):
`(:3) sequencing the at least one enriched target polynucleotide (para [0232], the enriched nucleic acid molecules from the nucleic acid
`library (or nucleic acid sequencing library) can be sequenced):
`(e) repeating steps a—c. wherein a second polynucleotide library comprising capture probes is added to the composition, wherein the
`second polynucleotide library comprises at least one polynucleotide that binds to genomic fragments (para [0231]. the enriched nucleic
`acid molecules are re—enriched in a second (or more) round of hybridization to the capture probes).
`
`Counsyl does not specifically teach (d) identifying one or more positions of the at least one enriched polynucleotide having less than
`average reed depth: adding at least 1500 polynucleotides to the composition: wherein the presence of the second polynucleotide library
`increases the read depth at the one or more positions having less than average read depth. Personalis teaches (b) enriching at least one
`genomic fragment that binds to the first polynucleotide library to generate at least one enriched target polynucleotide (Claim 1. A method
`for genetic analysis of a subject. comprising: b. subjecting a second nucleic acid sample of said subject to target—specific sequencing to
`generate target—specific sequencing data)‘ and (cl) identifying one or more positions of the at least one enriched polynucleotide having
`less than average read depth (para [0025]. FIG. 3 illustrates a statistical model on sensitivity vs. coverage of genomic regions with single
`run whole genome sequencing data). It would have been obvious to one of ordinary skill in the art to have adding a second balanced
`caplure probes. such as at least 1 500 polynucleotides, to the genomic region having less than average read, to improve the read depth of
`said region.
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`As said technical features were known in the art at the time of the invention, these cannot be considered special technical features that
`would otherwise unity the groups.
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`Groups i through Ill therefore lack unity under PCT Rule 13 because they do not share a same or corresponding special technical
`feature.
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`accordance with the second and third sentences of Rule 6.4(e).
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`Item 4 (continued):
`Claims 5-23. ZT-Si" 41-56, 63, 65-?5, 81-93, Sit-10? are held unsearchable because they are dependent claims and are not drafted in
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`Form PCTIISAIZOG (cxtra sheet) (April 2005)
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