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`EXHIBIT 91
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`Case 1:20-cv-01580-LPS Document 41-1 Filed 03/05/21 Page 2 of 528 PageID #: 8902
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`WSGR Reference No.: 42534~710.301
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`.........uww———.m.m._m.
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`Inventor: ArnirAli TALASAZ
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`Group Art Unit: 1639
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`Serial Number: 14/855,301
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`Examiner: KAUP, SAHANA S
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`Filing Date: September 15, 2015
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`CONFIRMATION NO.: 9854
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`SYSTEMS AND METHODS TO
`Title:
`DETECT RARE M’UTATIONS AND COPY
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`NUMBER VARIATIONuW
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`fl
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`CUSTOMER NO: 115823
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`Mail Stop RCE
`Commissioner for Patents
`PO. Box 1450
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`Alexandria, VA 22313-1450
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`DECLARATION OF STEFANIE .MORTIMER PURSUANT TO 37 CFR §l.132
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`I, Dr. Stefanie Mortimer, residing in Morgan Hill, California, hereby declare as follows:
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`1.
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`I earned a BS. in Chemistry and Biochemistry from McMaster University in 2004. I earned
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`a PhD. in Biological Chemistry from University of North Carolina at Chapel Hill in 2008.
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`2.
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`I have been employed by Guardant Health, Inc. since 2013 and currently hold the position of
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`Associate Director of Technology Development. My responsibilities include developing processes
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`and products related to liquid biopsy, including sequencing assays, sample processing and
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`bioinformatics techniques. Previously I held a position as a Leukemia and Lymphoma Society
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`Fellow at University ofCalifornia at Berkeley, where I investigated the biomolecular mechanisms
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`of miRNAs implicated in B cell Lymphomas.
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`3.
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`I am familiar with the disclosure and the pending claims of US. Patent Application Serial
`
`No. 14/855,301 (“ '301 application”), assigned to Guardant Health, and I am an inventor of the
`same.
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`4.
`
`I have reviewed US. Patent Application Pro-Grant Publication No. U820030165978 to Firth
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`et a1. (“Firth”), and I believe that my experience in sequencing assays and sample processing
`
`techniques qualifies me to interpret the disclosure of Firth at an expert level of analysis.
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`WSGR Reference No.: 42534—710301
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`Application No.: 14/855,301
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`5.
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`I have reviewed US. Patent No. 8,209,130 to Kennedy et at]. (“Kennedy”), and I believe that
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`my experience in sequencing assays and bioinformatics qualifies me to interpret the disclosure of
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`Kennedy at an expert level of analysis.
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`6.
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`I have read the Final Rejection of November 21, 2016 issued in the '301 application, and I
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`understand the Examiner’s comments on the art cited in the pending claims.
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`7.
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`I have analyzed Firth and conclude that Firth does not disclose examples of b1unt~end
`
`ligation using a molar excess of adapters.
`8. Firth only describes examples in which a molar excess of adapters are used in the context of
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`sticky—end ligation. Some of these examples are provided in paragraphs [0069], [0082], and [0140]
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`of Firth. Firth does not describe any particular use of a molar excess of adapters in combination
`
`with blunt—end ligation.
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`9. For example, in paragraph [0069] ofFirth, Firth describes a 50X molar excess of sticky-end
`
`adapters that was ligated to Fstl—digested DNA. Pstl digestion generates 4 nucleotide single—
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`stranded overhangs in DNA molecules. The sticky—end adapters were combinations of oligos A and
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`B or oligos A and D, as described in paragraph [0066] of Firth. Oligos A, B, and D have 33, 29,
`
`and 27 nucleotides, respectively. Thus, combining these oligos as described in Firth results in sticky
`
`end adapters used for sticky end ligation.
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`10. Further, in Example 2, Firth. describes using a 20-fold molar excess of sticky-end adapters
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`KL and KM, described in paragraph [0076] of Firth. It is noted in Firth that these adapters have a 1
`
`base—pair overhang and are thus sticky—end adapters. In Example 4, Firth describes ligating a 100—
`
`fold excess of adapters comprising oligonucleotides E and H, described in paragraph [0098]. Again,
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`these adapters are sticky-end. These are the only instances in which Firth describes using a molar
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`excess of adapters. Nowhere does Firth describe an example ofblunt—end ligation in combination
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`with using a molar excess of adapters.
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`11. Sticky-end ligation is fundamentally different than blunt end ligation. Sticky end ligation
`
`involves double stranded nucleic acid molecules that have a single stranded overhang. For ligation
`
`of two molecules to occur, the single stranded overhangs of the nucleic acid molecules must be
`
`complementary. Accordingly, adapters having the same sticky ends can be ligated to molecules
`
`having complementary sticky ends. However, such adapters cannot be ligated to each other because
`
`they have the same, not complementary, o‘verhangs.
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`In contrast, blunt end ligation involves
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`ligation of double-stranded nucleic acid molecules that do not have single-stranded overhangs. For
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`WSGR Reference No.: 42534—710301
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`Application No.: 14/855,301
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`blunt end ligation, there is no specificity in terms of which molecules can be ligated to one another.
`
`Thus, sticky—end ligation is different from blunt end ligation.
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`12. It is my opinion that, based on the teachings in Firth, one would not use polynucleotide
`
`adapters for blunt-end ligation in a molar excess sufficient to tag at least 20% of DNA molecules
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`because it contaminates the sequencing output for adapter-tagged molecules. To be more specific,
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`is well-established that an excess of blunt-ended adapters results in adapter dimerization (i.e.,
`
`adapters ligated to each other). Adapter dimerization makes detection of variants more difficult at
`
`least because the adapter dimers contaminate sequencing output. For instance, adapter dimers can
`
`bind to a flow cell and undergo sequencing, but provide no data other than the sequence of the
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`adapter present. Such data is not useable for cancer diagnostics, for instance, because sample DNA
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`is not sequenced. In addition, the presence of adapter dimers in a sample to be sequenced may take
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`away sequencing reads from nucleic acid molecules that may have potential aberrations indicative
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`of cancer such that they may not be detectable.
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`13. In contrast, using a molar excess of sticky end adapters to tag molecules does not produce
`
`adapter dimers (or does so in miniscule amounts) since the adapters do not have complementary
`
`ends with respect to each other. In many instances, sticky~end adapters are used in sequencing
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`library preparation as a strategy to mitigate adapter dimerization. For example, it is standard
`
`practice in next—generation sequencing library preparation to ligate T-tailed (i.e., sticky—end)
`
`adapters to DNA fi‘agments with single A—base overhangs.
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`14. Therefore, based on the teachings of Firth, which describes ligating a molar excess of sticky»
`
`end adapters, it would not be obvious to use a molar excess of blunt ended adapters. Rather, due to
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`the problem of adapter dimerization, using a molar excess of adapters in blunt—end ligation would
`
`worsen sequencing output because adapter dimers provide no useful sequence information. As
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`such, it would not be obvious to use a molar excess of blunt-end adapters when preparing nucleic
`
`acids for sequencing particularly for chNA.
`
`15. The claimed methods address the problem of adapter dimerization by enriching tagged DNA
`
`molecules by selective sequence capture of at least a subset of the DNA molecules. Selective
`
`sequence capture enriches tagged DNA molecules from adapter dimers in a sample.
`
`16. I note that Firth does refer to the use of blunt end adapters in paragraph [0015]. However, it
`
`is my interpretation that this refers to the use of blunt end adapters in the general context of
`
`providing mis-matched adapters to a molecule, not the more specific application of providing a
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`WSGR Reference No.: 42534—710301
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`Application No.: 14/855,301
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`molar excess of adapter in a process of ligation. As I mentioned, all examples in Firth that use a
`
`molar excess of adapter do so sticky-end adapters.
`
`17. Accordingly, it is my belief that Firth does not teach a method capable of using a molar
`
`excess of adapters for blunt end ligation.
`
`18. l have also analyzed Kennedy and conclude that Kennedy does not disclose ligating a molar
`
`excess of blunt-end adapters to DNA molecules such that at least 20% of the DNA molecules are
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`tagged with one or more adapters.
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`19. I declare that all statements made herein of my own knowledge are true and that all
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`statements made on information and belief are believed to be true; and further that these statements
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`were made with the knowledge that willful false statements and the like so made are punishable by
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`fine or imprisonment, or both, under 18 U.S.C. §1001 and that such willful false statements may
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`jeopardize the validity and enforceability of any United States Patent that would issue from US.
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`Patent Application Serial No. 14/855,301.
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`Date: April 19, 2017
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`Signature:
`
`
`
`Stefanie Mortimer, Phi).
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`EXHIBIT 92
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`Case 1:20-cv-01580-LPS Document 41-1 Filed 03/05/21 Page 7 of 528 PageID #: 8907
`Case 1:20-cv-01580-LPS Document 41-1 Filed 03/05/21 Page 7 of 528 PageID #: 8907
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`Electronically filed on: May 9, 2017
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`PATENT
`Attorney Docket No.: 42534-710302
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`Inventor: AmirAli TALASAZ
`
`Serial Number: 15/076,565
`
`Filing Date: March 21, 2016
`
`Title: SYSTEMS AND METHODS TO DETECT
`RARE MUTATIONS AND COPY NUMBER
`VARIATION
`
`Group Art Unit: 1637
`
`Examiner: WILDER, CYNTHIA
`
`CONFIRMATION NO.: 3340
`
`CUSTOMER NO.: 115823
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`
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`AMENDMENT AND RESPONSE TO NON-FINAL OFFICE ACTION
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`Mail Stop Amendment
`Commissioner for Patents
`
`PO. Box 1450
`
`Alexandria, Virginia 22313
`
`Dear Commissioner:
`
`This paper is filed in response to the Non—Final Office Action mailed February 9, 2017.
`
`Reconsideration of the above—referenced application is respectfully requested in View of the
`
`following remarks.
`
`Amendments to the Specification begin on page 2 of this paper.
`
`Amendments to the Claims begin on page 3 of this paper.
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`A Summary of the Interview appears on page 9 of this paper.
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`Remarks begin on page 10 of this paper.
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`9143702_1.docx
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`Case 1:20-cv-01580-LPS Document 41-1 Filed 03/05/21 Page 8 of 528 PageID #: 8908
`US. Application No: 15/076,565
`Non-Final Office Action Mailed on February 9, 2017
`Amendment and Response Filed on May 9, 2017
`
`AMENDMENTS TO THE SPECIFICATION
`
`Please amend paragraph [0227] the Specification of the published application, US. Patent
`
`Publication No. 2016/0251704, as follows:
`
`[0227] After collection of bodily fluid, cell free polynucleotides may be isolated and extracted using
`
`a variety of techniques known in the art. In some cases, cell free DNA may be isolated, extracted and
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`prepared using commercially available kits such as theQiage1+QiampTRiPM.—a_Circulating Nucleic
`
`Acid Kit protocol by Qiagen. In other examples, Qiagen—Qa-bitéllMTadsDNA HS Assay kit protocol
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`by Qiagen, AgilentTT—MTQDNA 1000 kit by Agilent, er—TruSeqéllMTaSequencing Library
`
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`Preparation protocol,[[;]] or a Low—Throughput (LT) protocol may be used.
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`9143702_1.docx
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`—2—
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`Case 1:20-cv-01580-LPS Document 41-1 Filed 03/05/21 Page 9 of 528 PageID #: 8909
`Case 1:20-cv-01580-LPS Document 41-1 Filed 03/05/21 Page 9 of 528 PageID #: 8909
`US. Application No: 15/076,565
`Non-Final Office Action Mailed on February 9, 2017
`Amendment and Response Filed on May 9, 2017
`
`AMENDMENTS TO THE CLAIMS
`
`1.
`
`(Currently Amended) A method for detecting genetic aberrations in cell—free DNA
`
`flchNAfl (“chNA”) molecules from a subject, comprising:
`
`a) providing chNA molecules obtained from a bodily sample of the subject;
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`b) attaching tetheefDN—A—meleeulesrtags comprising barcodes having a plurality of different
`
`distinet—barcode sequences to the chNA molecules to tag generatetagged—parent—pel—yqeueleetides;
`
`
`wherein—at least [110% || 20% of the chNA molecules—is—tagged—by—theattaehing—step which
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`attaching comprises ligating adaptors comprising the barcodes to both ends of the chNA
`
`molecules2 wherein ligating comprises using more than 10X molar excess of the adaptors as
`
`compared to the chNA molecules2 thereby generating tagged parent polynucleotides;
`
`c) amplifying the tagged parent polynucleotides to produce amplified tagged progeny
`
`polynucleotides;
`
`d) sequencing the amplified tagged progeny polynucleotides to produce a plurality of
`
`
`sequence reads from each of the tagged parent polynucleotides, wherein each sequence read of the
`
`plurality of sequence reads comprises a barcode sequence and a sequence derived from a chNA
`
`molecule of the chNA molecules;
`
`e) mapping sequence reads of the plurality of sequence reads to one or more reference
`
`sequences from a human genome;
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`fle)]]_f) grouping the sequence reads mapped in e) into families based at least on thebarcode
`
`sequences of the sequence reads2 each of the families comprising sequence reads comprising the
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`same barcode sequence2 whereby each of the families comprises sequence reads amplified from the
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`same tagged parent polynucleotide;
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`Case 1:20-cv-01580-LPS Document 41-1 Filed 03/05/21 Page 10 of 528 PageID #: 8910
`Case 1:20-cv-01580-LPS Document 41-1 Filed 03/05/21 Page 10 of 528 PageID #: 8910
`U.S. Application No: 15/076,565
`Non-Final Office Action Mailed on February 9, 2017
`Amendment and Response Filed on May 9, 2017
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`Hf) H g) at each of a plurality of genetic loci in the one or more reference sequences,
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`eemparing—theseq-ueneereads—gretmed—withi—n—collapsing sequence reads in each family to
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`determineeensensus—seq-aenees—yield a base call for each family at the genetic locusrwherein—eaeh
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`
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`pel—ynueleetides; and
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`Hg) H h) detecting, at one or more genetic loci, a plurality of genetic aberrations, wherein the
`
`plurality of genetic aberrations comprises two or more different members ared—if—ferent—l—yLselected
`
`from the group of members consisting of[[:]] a single base substitution, a copy number variation
`
`1CNV), an insertion or deletion gindel), and a gene fusion.
`
`2.
`
`(Currently Amended) The method of claim 1, comprising providing less than 100
`
`nanograms gng) of the chNA molecules.
`
`3.
`
`(Currently Amended) The method of claim 1, comprising providing less than 10
`
`nanograms gng) of the chNA molecules.
`
`4.
`
`(Currently Amended) The method of claim 1, comprising providing between 100 and
`
`100,000 human haploid genome equivalents of th_echNA molecules, wherein the chNA molecules
`
`are tagged with between 2 and 1,000,000 unique identifiers.
`
`5.
`
`(Currently Amended) The method of claim 1, comprising providing between 1,000
`
`and 50,000 human haploid genome equivalents of th_echNA molecules, wherein the chNA
`
`molecules are tagged with between 2 and 1,000 unique identifiers.
`
`6.
`
`(Currently Amended) The method of claim 1, wherein each of the plurality of
`
`different barcode sequences is at least 5 nucleotides in length.
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`Case 1:20-cv-01580-LPS Document 41-1 Filed 03/05/21 Page 11 of 528 PageID #: 8911
`Case 1:20-cv-01580-LPS Document 41-1 Filed 03/05/21 Page 11 of 528 PageID #: 8911
`U.S. Application No: 15/076,565
`Non-Final Office Action Mailed on February 9, 2017
`Amendment and Response Filed on May 9, 2017
`
`7.
`
`(Currently Amended) The method of claim 1, wherein the attaching comprises non—
`
`uniquely tagging the chNA molecules with at least 10 and at most 1,000 different—distinet barcode
`
`sequences.
`
`8.
`
`(Withdrawn) The method of claim 1, wherein the attaching comprises uniquely
`
`tagging the chNA molecules.
`
`9.
`
`(Withdrawn) The method of claim 1, wherein the attaching comprises performing
`
`blunt—end ligation or sticky end ligation.
`
`10.
`
`(Canceled)
`
`1 1.
`
`(Canceled)
`
`12.
`
`(Currently Amended) The method of claim 1, wherein the attaching comprises non—
`
`uniquely tagging the chNA molecules such that no more than 5% of the tgggflparent
`
`polynucleotides are uniquely tagged.
`
`13.
`
`(Currently Amended) The method of claim 1, wherein at least L[30%]]M of the
`
`chNA molecules are tagged by the attaching flstepfl.
`
`14.
`
`(Currently Amended) The method of claim 1, wherein at least L[50%]]M of the
`
`chNA molecules are tagged by the attaching flstepfl.
`
`15.
`
`(Currently Amended) The method of claim 1, further comprising selectively
`
`enriching for polynucleotides mapping to one or more selected reference sequences ef—interest—prior
`
`to the sequencing, wherein the selectively enriching comprises 1i) subjecting the chNA molecules
`
`to selective amplification against the one or more selected reference sequences, (ii) subjecting the
`
`tagged parent polynucleotides to selective amplification against the one or more selected reference
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`sequences, (iii) subjecting the amplified progeny polynucleotides to selective sequence capture
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`9143702_1.docx
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`Case 1:20-cv-01580-LPS Document 41-1 Filed 03/05/21 Page 12 of 528 PageID #: 8912
`Case 1:20-cv-01580-LPS Document 41-1 Filed 03/05/21 Page 12 of 528 PageID #: 8912
`U.S. Application No: 15/076,565
`Non-Final Office Action Mailed on February 9, 2017
`Amendment and Response Filed on May 9, 2017
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`against the one or more selected reference sequences, or (iv) subjecting the chNA molecules to
`
`selective sequence capture against the one or more selected reference sequences.
`
`16.
`
`(Currently Amended) The method of claim 15, wherein the selectively enriching
`
`comprises enriching for polynucleotides mapping to the following genes: V—Ki—ras2 Kirsten rat
`
`sarcoma viral oncogene homolog gKRAS), adenomatous polyposis coli (AFC), and tumor protein 53
`
`(TP53).
`
`17.
`
`(Original) The method of claim 1, wherein sequencing comprises massively parallel
`
`sequencing.
`
`18.
`
`(Currently Amended) The method of claim 1, wherein the amplified taggflprogeny
`
`polynucleotides are sequenced to produce an average of 5 to 10 sequence reads for each family.
`
`19.
`
`(Currently Amended) The method of claim 1, wherein the eensensus—seq-ueneelfl
`
`call for each family possesses an error rate below 0.0001%.
`
`20.
`
`(Currently Amended) The method of claim 1, wherein each base of the tagged parent
`
`polynucleotides has at least 99% chance of being represented by at least one sequence readMg
`
`the sequence reads mapped in e).
`
`21.
`
`(Currently Amended) The method of claim 4, wherein grouping the sequence reads
`
`mapped in e) is further based on one or more of: sequence information at a beginning of the
`
`sequence derived from the chNA molecule, sequence information at an end of the sequence derived
`
`from the chNA molecule, and length of the sequence read.
`
`22.
`
`(Currently Amended) The method of claim 4, wherein grouping the sequence reads
`
`mapped in e) is further based on a plurality of: sequence information at a beginning of the sequence
`
`derived from the chNA molecule, sequence information at an end of the sequence derived from the
`
`chNA molecule, and length of the sequence read.
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`Case 1:20-cv-01580-LPS Document 41-1 Filed 03/05/21 Page 13 of 528 PageID #: 8913
`Case 1:20-cv-01580-LPS Document 41-1 Filed 03/05/21 Page 13 of 528 PageID #: 8913
`U.S. Application No: 15/076,565
`Non-Final Office Action Mailed on February 9, 2017
`Amendment and Response Filed on May 9, 2017
`
`23.
`
`(Withdrawn) The method of claim 1, wherein at least one single base substitution is
`
`detected.
`
`24.
`
`(Currently Amended) The method of claim 1, wherein at—least—ene the two or more
`
`members comprise a copy number variation (CNVg—is—det-eeted.
`
`25.
`
`(Withdrawn) The method of claim 1, wherein at least one indel is detected.
`
`26.
`
`(Withdrawn) The method of claim 1, wherein at least one gene fusion is detected.
`
`27.
`
`(Withdrawn) The method of claim 1, wherein at least one single base substitution
`
`and at least one copy number variation is detected.
`
`28.
`
`(Withdrawn) The method of claim 1, further comprising detecting, at one or more
`
`genetic loci, one or more genetic aberrations selected from: a transversion, a translocation, an
`
`inversion, a deletion, aneuploidy, partial aneuploidy, polyploidy, chromosomal instability,
`
`chromosomal structure alterations, chromosome fusions, a gene truncation, a gene amplification, a
`
`gene duplication, a chromosomal lesion, a DNA lesion, abnormal changes in nucleic acid chemical
`
`modifications, abnormal changes in epigenetic patterns and abnormal changes in nucleic acid
`
`methylation.
`
`29.
`
`(Withdrawn) The method of claim 23, wherein the single base substitution is
`
`detected with a sensitivity of at least 1%.
`
`30.
`
`(Withdrawn) The method of claim 23, wherein the single base substitution is
`
`detected with a sensitivity of at least 0.1%.
`
`31.
`
`(New) The method of claim 12 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 gCNV), an insertion or deletion (indel)= and a gene fusion.
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`Case 1:20-cv-01580-LPS Document 41-1 Filed 03/05/21 Page 14 of 528 PageID #: 8914
`Case 1:20-cv-01580-LPS Document 41-1 Filed 03/05/21 Page 14 of 528 PageID #: 8914
`U.S. Application No: 15/076,565
`Non-Final Office Action Mailed on February 9, 2017
`Amendment and Response Filed on May 9, 2017
`
`32.
`
`{New} The method of claim 12 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.
`
`33.
`
`{New} The method of claim 12 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.
`
`34.
`
`{New} The method of claim 12 wherein each of the tagged parent polynucleotides is
`
`uniguely tagged.
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`35.
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`{New} The method of claim 12 wherein each of the tagged parent polynucleotides is
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`non—uniguely tagged.
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`9143702_1.docx
`
`—S—
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`Attorney Docket No.: 42534—710302
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`A1410
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`A1410
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`

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`Case 1:20-cv-01580-LPS Document 41-1 Filed 03/05/21 Page 15 of 528 PageID #: 8915
`Case 1:20-cv-01580-LPS Document 41-1 Filed 03/05/21 Page 15 of 528 PageID #: 8915
`US. Application No: 15/076,565
`Non-Final Office Action Mailed on February 9, 2017
`Amendment and Response Filed on May 9, 2017
`
`SUMMARY OF THE INTERVIEW
`
`Applicant is appreciative of Examiner Wilder for extending the courtesy of a telephonic
`
`interview to Applicant’s representatives, Ali Alemozafar, Dawson Wong, and John Storella, on April
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`25, 2017. During the interview, amendments and arguments consistent with those presented herein
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`were discussed.
`
`9143702_1.docx
`
`—9—
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`Attorney Docket No.: 42534—710302
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`A1411
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`A1411
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`

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`Case 1:20-cv-01580-LPS Document 41-1 Filed 03/05/21 Page 16 of 528 PageID #: 8916
`Case 1:20-cv-01580-LPS Document 41-1 Filed 03/05/21 Page 16 of 528 PageID #: 8916
`U.S. Application No: 15/076,565
`Non-Final Office Action Mailed on February 9, 2017
`Amendment and Response Filed on May 9, 2017
`
`REMARKS
`
`Claims 1—30 were pending prior to entry of the abovementioned amendments. Claims 10—11
`
`have been canceled. Claims 1—7, 12—16, 18—22, and 24 have been amended. New claims 31—35 have
`
`been added. No new matter is added by any of these amendments. Accordingly, claims 1—9 and 12—
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`35 are now pending.
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`Reconsideration and allowance of this application is respectfully requested in light of the
`
`abovementioned amendments and the following remarks.
`
`Claims amendments
`
`Claims 1, 13—16, and 19 have been amended to clarify the claimed subject matter. This
`
`amendment is fully supported by the application as originally filed, U.S. Patent Application No.
`
`15/076,565, (“the Application”) at, for example, paragraphs [0055], [0064], [00237], [00254],
`
`[00265], and [00270], and original claims 10—11.
`
`Claims 2—7, 12, 15—16, 18, 20—22, and 24 have been amended for antecedent basis purposes.
`
`New claims 31—35 are fully supported by the Application at, for example, paragraphs [0012],
`
`[0024], [0057], [00116], and [00250]—[00251], and original claims 23—26.
`
`Objection to the Specification
`
`Applicant has amended the Specification to address the objection raised by the Office.
`
`35 U.S.C.
`
`112 b
`
`Claims 1—7, 11—22, and 24 stand rejected under 35 U.S.C. § 112(b) or 35 U.S.C. § 112 (pre—
`
`AIA), second paragraph.
`
`Without conceding in the basis of rejection, and solely to expedite the prosecution of this
`
`application, Applicant has amended Claims 1 and 15—16 to clarify the claimed subject matter.
`
`9143702_1.docx
`
`— 10—
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`Attorney Docket No.: 42534—710302
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`A1412
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`A1412
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`

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`Case 1:20-cv-01580-LPS Document 41-1 Filed 03/05/21 Page 17 of 528 PageID #: 8917
`Case 1:20-cv-01580-LPS Document 41-1 Filed 03/05/21 Page 17 of 528 PageID #: 8917
`US. Application No: 15/076,565
`Non-Final Office Action Mailed on February 9, 2017
`Amendment and Response Filed on May 9, 2017
`
`Accordingly, Applicant submits that Claim 1 and dependent claims 2—7, 12—22, and 24
`
`thereof are not indefinite. Applicant respectfully requests that the § 112(b) rejections of Claims 1—7,
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`12—22, and 24 be Withdrawn.
`
`Without conceding in the basis of rejection, Applicant submits that the § 112(b) rejections of
`
`claims 10—11 are moot in View of the cancelation of these claims.
`
`35 U.S.C.
`
`103 a
`
`Claims 1—3, 7, 15, 17, and 24 stand rejected under 35 U.S.C. § 103(a) over Sparks et al.,
`
`Prenatal Diagnosis, 32 (2012), 3—9 (“Sparks”), in View of Kennedy et al., US 8209130 (“Kennedy”).
`
`Without conceding in the basis of rejection, and solely to expedite the prosecution of this
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`application, Applicant has amended Claim 1 to clarify various differences between the claimed
`
`subject matter and the cited art. Applicant submits that Claim 1 is not obvious over the asserted
`
`combination of Sparks and Kennedy because these references, alone or in combination, do not meet
`
`all of the elements of this claim. More specifically, the asserted combination of Sparks and Kennedy
`
`fails to teach or disclose, or even suggest, the elements of (b), (f), (g), and (h) of Claim 1 as
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`amended.
`
`These references, alone or in combination, do not teach, among other things, (1) “attaching
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`tags comprising barcodes having a plurality of different barcode sequences to the chNA molecules
`
`to tag at least 20% of the chNA molecules” in a single bodily sample, (2) “grouping the sequence
`
`reads mapped in e) into families based at least on barcode sequences of the sequence reads, each of
`
`the families comprising sequence reads comprising the same barcode sequence, whereby each of the
`
`families comprises sequence reads amplified from the same tagged parent polynucleotide,” and (3)
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`“collapsing sequence reads in each family to yield a base call for each family” at each of a plurality
`
`of genetic loci.
`
`9143702_1.docx
`
`—1 1—
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`Attorney Docket No.: 42534—710302
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`A1413
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`A1413
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`Case 1:20-cv-01580-LPS Document 41-1 Filed 03/05/21 Page 18 of 528 PageID #: 8918
`Case 1:20-cv-01580-LPS Document 41-1 Filed 03/05/21 Page 18 of 528 PageID #: 8918
`US. Application No: 15/076,565
`Non-Final Office Action Mailed on February 9, 2017
`Amendment and Response Filed on May 9, 2017
`
`0
`
`S arks does not teach or disclose claim 1 b : “attachin ta s com risin barcodes
`
`having a plurality of different barcode seguences to” cell-free DNA molecules from a
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`bodily sample
`
`Claim 1(b) as amended recites:
`
`“b) attaching tags comprising barcodes having a plurality of different barcode
`sequences to the chNA molecules to tag at least 20% of the chNA
`molecules, which attaching comprises ligating adaptors comprising the
`barcodes to both ends of the chNA molecules, wherein ligating comprises
`using more than 10X molar excess of the adaptors as compared to the chNA
`molecules, thereby generating tagged parent polynucleotides.”
`
`The Office Action states that Sparks met the “limitation of tags comprising barcodes having
`
`from 5 to 1000 distinct barcode sequences to generate non—uniquely tagged parent polynucleotides”
`
`in the “use of 96 different patient samples that were tagged and pooled and sequenced together.”
`
`(Office action, page 17.) However, this statement from Sparks merely shows a sample tagging
`
`method in which each different sample is tagged with one of 96 different tags. Thus, all of the
`
`
`molecules in any one sample bear the same tag.
`
`In contrast, claim 1 recites that molecules in the same sample are tagged with barcodes
`
`having a plurality of different barcode sequences:
`
`“a) providing chNA molecules obtained from a bodily sample of th_e
`subject;
`b) attaching tags comprising barcodes having a plurality of different
`barcode sequences to th_e chNA molecules to tag at least 20% of the
`chNA molecules, which attaching comprises ligating adaptors
`comprising the barcodes to both ends of the chNA molecules, wherein
`ligating comprises using more than 10X molar excess of the adaptors as
`compared to the chNA molecules, thereby generating tagged parent
`polynucleotides.” (Emphasis added.)
`
`Accordingly, Sparks fails to teach or disclose this element.
`
`9143702_1.docx
`
`— 12—
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`Attorney Docket No.: 42534—710302
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`A1414
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`A1414
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`Case 1:20-cv-01580-LPS Document 41-1 Filed 03/05/21 Page 19 of 528 PageID #: 8919
`Case 1:20-cv-01580-LPS Document 41-1 Filed 03/05/21 Page 19 of 528 PageID #: 8919
`US. Application No: 15/076,565
`Non-Final Office Action Mailed on February 9, 2017
`Amendment and Response Filed on May 9, 2017
`
`0
`
`
`
`rou in the se uence reads ma:S arks does not teach or disclose claim 1 ed in e
`
`
`
`
`
`produced from each tagged parent polynucleotide into families
`
`Claim 1(f) as amended recites:
`
`“D grouping the sequence reads mapped in e) into families based at least on
`the barcode sequences of the sequence reads, each of the families comprising
`sequence reads comprising the same barcode sequence, whereby each of the
`families comprises sequence reads amplified from the same tagged parent
`polynucleotide.”
`
`The Office Action, on pages 17—18, refers to page 5, column 1 of Sparks as showing this
`
`element. Sparks states there, “The slide was processed on an Illumina HiSeq 2000TM to produce a 56
`
`base locus—specific sequence and a 7 base sample tag sequence from each cluster.” Thus, the
`
`sequence of Sparks included a 56—base sequence from the original DNA molecule and a 7—base
`
`sequence from the sample tag.
`
`Sparks states, on page 5, col. 1, “NNNNNNN represents a 7 base sample index enabling 96
`
`sample multiplexed sequencing.” Accordingly, Sparks recognizes that a_ll the molecules in any one
`
`sample, rather than different individual molecules in the same sample, are indexed using the same 7—
`
`
`base sample tag. Because a_ll molecules in a single sample bear the same sample index, different
`
`original molecules in the sample cannot be distinguished from each other based on the tag, that is,
`
`the sample index cannot be used to group sequence reads originating from a parent polynucleotide.
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`Further, while Sparks uses the 56—base sequence to group sequence reads by locus (i.e., to
`
`identify one locus among the plurality of 384 loci), this information cannot be used to group
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`sequence reads produced from a single parent molecule into a family, as Sparks does not teach any
`
`way to distinguish reads having the same sequence as originating from the same or difi‘erent parent
`
`molecules. Thus, Sparks does not, and cannot, group a set of sequence reads produced from each
`
`9143702_1.docx
`
`— 13—
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`Attorney Docket No.: 42534—710302
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`A1415
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`A1415
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`

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`Case 1:20-cv-01580-LPS Document