DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Office Action: Notice
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 2/18/2026 has been entered.
Claim Status
Claims 1-89 and 91-94 were previously cancelled. Claim 100 is newly amended (2/18/2026). No new matter was added. Thus, claims 90 and 95-114 are under examination (2/18/2026).
Priority
Claims are given a priority date of 10/4/2018, the effective filing date of US Provisional 62741466.
New Objections - Specification
The disclosure is objected to because of the following informalities (see MPEP § 608.01):
The disclosure is objected to because it contains embedded hyperlinks (3x) and/or other forms of browser-executable code in reference to shuffle nucleotide sequences (p. 30), non-limiting sequence generator (p. 59), and CDC site (p. 109). Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01.
Rejections Maintained
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 90 and 95-114 are rejected under 35 U.S.C. 103 as being unpatentable over Christians (WO 2017/165864 A1, see IDS filed 04/06/2022), hereinafter Christians in view of Jiang (Jiang et. al. 2011. Synthetic spike-in standards for RNA-seq experiments. Genome Res. September 2011. 21(9):1543-51), hereinafter Jiang and Wu (US10465232 B1; filed October 7, 2016), hereinafter Wu and further in view of Conti et al. (“An improved multiple-frequency method for measuring in situ target strengths”, Journal of Marine Science, 2005) hereinafter Conti. This rejection has been modified in response to Applicant’s amendments (2/18/2026).
Regarding claim 90, Christians teaches a method of quantifying a titer (e.g., determining abundance) of a target organism (e.g., pathogen) in a sample, comprising: a. providing a sample comprising the target organism, wherein the target organism comprises at least one target sequence (e.g. target nucleic acids, para. 150); b. providing a multi-analyte control (e.g., positive control) comprising known titers of at least three species of organisms (“11 pathogens,” see example 6, para. 436; c. mixing (e.g., “spiking”) a known amount (e.g., known concentrations) of the normalization control with the sample and with the multi-analyte control d. preparing high throughput (e.g., multiplexed) sequencing libraries from the sample and the multi- analyte control (e.g., producing sequencing libraries for the samples, positive controls); e. sequencing said libraries to produce a collection of sample reads and a collection of multi-analyte control reads; f. normalizing the collection of sample reads and the collection of multi-analyte control reads from (e) using the normalization controls (para. 179).
Regarding claim 95, Christians teaches a method, wherein the normalization control (e.g., spike-ins); comprises at least three groups of polynucleotides, wherein the polynucleotides within each group are of the same length (para. 152). The reference teaches multiple spike-in nucleic acids (specifically stating up to about 10) are added to a sample or reagent. Each different “spike-in” nucleic acid is a “group of polynucleotides” consistent with the instant claim.
Regarding claim 96, Christians teaches a method, wherein the lengths of all of the polynucleotides in the normalization control are the same (para. 133, para. 152, para. 170).
Regarding claim 97, Christians teaches a method, wherein the polynucleotides within each group of the normalization control are of a different length when compared to the polynucleotides within any other group of the normalization control (para. 152, para. 171).
Regarding claim 98, Christians teaches a method, wherein the lengths of the groups of polynucleotides in the normalization control are distributed in a linear sequence (para. 133, para. 152). Christians does not specifically recite the lengths of the groups of polynucleotides in the normalization control are “distributed in a linear sequence.” However, Christians describes “in some cases, the spike-in nucleic acids added to a sample or reagent are the same length.” Therefore, if the groups of polynucleotides in the normalization control are all the same length, the three points would make a linear sequence distribution or a line.
Regarding claim 99, Christians teaches a method, wherein the lengths of the polynucleotides in the at least three groups in the normalization control are between about 15 bp and about 50,000 bp (e.g., 100 bp, para. 93; para. 134-135). Christians does not inherently recite “the lengths of the polynucleotides in the groups of normalization controls being between about 15 bp and about 50,000 bp. However, Christians describes “the collection of synthetic nucleic acid species may collectively span the observable range of lengths of certain target nucleic acids. For example, the species may collectively span the lengths of disease-specific or pathogen-specific nucleic acids in a sample,” and “in some cases, the lengths of disease-specific or pathogen-specific nucleic acids in a sample may be in the range from about 40 to about 100 base pairs.” Therefore, the synthetic nucleic acid lengths would fall between 15 bp and 50,000 bp.
Regarding claim 100, Christians teaches a method, wherein the lengths of the polynucleotides in the at least three groups in the normalization control are between about 100 and 1200 bp (e.g., 100 bp, para. 93; para. 134-135).
Regarding claim 101, Christians teaches a method, wherein the polynucleotides within each group in the normalization control comprise a shared sequence (para. 133).
Regarding claim 102, Christians teaches a method, wherein the polynucleotides within each group in the normalization control do not comprise a shared sequence (para. 133).
Regard claim 103, Christian teaches a method, wherein each group of polynucleotides in the normalization control comprises at least three subgroups of polynucleotides (para. 152), wherein the polynucleotides within each subgroup comprise a shared sequence, and wherein the polynucleotides within each subgroup do not comprise a shared sequence as any other subgroup (e.g., “10 spike-in nucleic acids added to sample” para. 152, “same or different spike-in synthetic nucleic acids are introduced,” para. 162).
Regarding claim 104, Christians teaches a method, wherein at least one sequence in at least one group of polynucleotides in the normalization control comprises a component sequence (e.g., “specific sequence,” para. 3; para. 190).
Regarding claim 105, Christians teaches a method, wherein every sequence in every group of polynucleotides in the normalization control comprises a component sequence (para. 133). Christians does not specifically recite “component sequence.” However, the specification defines a “component sequence” to be “a portion or entirety of a polynucleotide found in a normalization control.” Thus, the teachings of Christian inherently provide spike-in synthetic nucleic acids that comprise “ a portion or entirety of a polynucleotide.”
Regarding claim 106, Christians teaches a method, wherein the sequence of at least one group of polynucleotides in the normalization control comprises an isolated sequence (e.g., “naturally-occurring,” 146; “comprise genomic nucleic acids such as human or pathogen genomic nucleic acids,” para. 147).
Regarding claim 107, Christians teaches a method, wherein the sequence of every group (e.g., “3 spike-in nucleic acids added to sample” para. 152) of polynucleotides in the normalization control comprises an isolated sequence (para. 147).
Regarding claim 108, Christian teaches a method, wherein the sequence of at least one subgroup (e.g., “2 spike-in nucleic acids added to sample” para. 152) of at least one group of polynucleotides in the normalization control comprises an isolated sequence (para. 147).
Regarding claim 109, Christians teaches a method, wherein the sequence of every subgroup (e.g., “4 spike-in nucleic acids added to sample” para. 152) of at least one group of the normalization control comprises an isolated sequence (para. 147).
Regarding claim 110, Christians teaches a method, wherein the sequence every subgroup of every group (e.g., “9 spike-in nucleic acids added to sample,” para. 152) in the normalization control comprises an isolated sequence (para. 147).
Regarding claim 111, Christians teaches a method, wherein the isolated sequence (para. 147) of each subgroup in the normalization control is not the same as the isolated sequence of any other subgroup (“same or different spike-in synthetic nucleic acids are introduced,” para. 162).
Regarding claim 112, Christians teaches a method, wherein the isolated sequence in the normalization control comprises a sequence of between about 6 bp and about 200,000 bp, between about 15 bp and about 50,000 bp, between about 500 bp and about 1500 bp, between about 100 bp and about 1200 bp, or between about 150 bp and about 600 bp (para. 137).
Regarding claim 113, Christians teaches a method, wherein the isolated sequence in the normalization control is isolated or derived from a virus, a bacterium, a fungus or a eukaryotic parasite (e.g., “viral particles,” para. 144; “pathogen genomic nucleic acids,” para. 147).
Regarding claim 114, Christians teaches a method, wherein the isolated sequence in the normalization control is not the same as at least one target sequence in a sequencing sample (para. 150).
Regarding claim 90, Christians fails to teach a method, wherein g. determining a relationship between normalized reads and the known titers in the multi-analyte control; and h. calculating a titer of the target organism in the sample using the relationship determined in (g). However, Jiang teaches a method, wherein g. determining a relationship between normalized reads and the known titers in the multi-analyte control (e.g., control RNAs; Col. 1, para. 2, pg. 1545); and h. calculating a titer of the target organism (e.g., transcript abundance) in the sample using the relationship determined in (g) (Col. 2, para. 1, pg. 1545, see Fig. 2). Christians further fails to teach a method, wherein the multi-analyte control is inactivated.
Christians and Jiang are considered to be analogous to the claimed invention because they both utilize spike-in controls for assay normalization. Therefore, it would have been obvious to have modified Christians to incorporate the teachings of Jiang to teach a method, wherein g. determining a relationship between normalized reads and the known titers in the multi-analyte control; and h. calculating a titer of the target organism in the sample using the relationship determined in (g) because “RNA standards allow one to determine if an RNA-seq assay accurately represents the composition of known input and to derive standard calibration curves that relate read counts to RNA concentration in the studied sample” (Col. 2, para. 2, pg. 1543). Furthermore, “external RNA standards are a powerful tool for routine assessment of RNA-seq experiments and during experimental and computational protocol development” (Col. 1, para. 2, pg. 1548).
Christians in view of Jiang fails to teach a method, wherein the organisms of the multi-analyte control have been inactivated.
However, Wu teaches a method, wherein the organisms of the multi-analyte control have been inactivated (Col. 7, para. 5).
Christians, Jiang and Wu are considered to be analogous to the claimed invention because they each utilize spike-in controls for assay normalization. Therefore, it would have been obvious to have modified Christians in view of Jiang to incorporate the teachings of Wu to teach a method, wherein the organisms of the multi-analyte control are inactivated because “the synthetic sequences used in the spike-in do not match any known organism, they will not generate any false positive hits when used in combination with naturally occurring DNA samples” (Col. 38, para. 3).
Regarding claim 100, Christians teaches a method, wherein the lengths of the polynucleotides in the at least three groups in the normalization control are between about 100 and 1200 bp (e.g., 100 bp, para. 93; para. 134-135). Christians in view of Jiang and Wu fails to teach determining the frequency of at least one species-specific target sequence from each of the species in the multi-analyte control via generation of a calibration curve.
However, Conti teaches that refinements have been made to the multiple-frequency method for rejecting overlapping echoes when making target-strength measurements with split-beam echosounders, simultaneously detected with two or more adjacent split-beam transducers of different frequencies, pass multiple-target rejection algorithms at each frequency, and characterize virtually identical three-dimensional target coordinates in order to translate the coordinates into a common reference system for comparison (Abstract). Further, Conti teaches it is possible to make high-quality, multiple-frequency measurements concurrent with echo-integration surveys for the purposes of taxa identification and the estimation of animal
Abundance (p. 1637, Column 1, Paragraph 1) via an on-axis system gain and a 3-D, curve fitting program (p. 1643, Column 1, Paragraph 2).
It would have been obvious to a person of ordinary skill in the art at the time of the invention to modify the multi-analyte sequencing workflow of Christians in view of Jiang and Wu, and further in view of Conti, because each reference addresses the same technical objective of improving quantitative accuracy in target measurement using known reference standards and measured signal data. Christians teaches preparing and sequencing samples together with multi-species controls of known concentrations and normalizing read counts. While, Wu teaches that organisms in such multi-analyte controls may be inactivated, which improves biosafety and handling reliability. Incorporating Wu’s inactivated organisms into Christians’ control system represents the substitution of a known safety feature for its intended purpose and would have been a predictable modification that does not alter the underlying sequencing and quantification framework.
Further, Jiang teaches a quantitative relationship between normalized sequencing read counts and known input concentrations and generating a calibration curve for calculating target abundance. Because Christians already provides known titers for multiple species within a control, a person of ordinary skill in the art would have been motivated to apply Jiang’s calibration curve methodology for Christians normalized read data in order to improve quantitative precision and enable calculation of target organism titer based on established read-to-concentration relationship. This combination reflects the application of a known quantification technique to an existing sequencing-based control system to obtain improved accuracy and standardization.
Conti further demonstrates the analytical principle that measured signal frequency can be correlated with known reference quantities using calibration modeling to determine target strength. Although arising in a different measurement context. Conti reinforces the well-established concept that frequency-derived signals may be translated into quantitative values through calibration curves in a multi-analyte context. A person of ordinary skill in the art would have recognized that normalized sequencing read frequency constitutes a measurable signal analogous to those described in Conti and would have reasonably applied similar calibration modeling techniques to sequencing output in order to enhance quantitative interpretation.
Further, a person of ordinary skill in the art would have had a reasonable expectation of succuss in making this combination because each reference relies on well-established molecular biology techniques, including sequencing, normalization, and computation analysis of read data. Jiang expressly demonstrates that normalized read counts correlate predictably with known input correlations, and Christians provides the known titers necessary to establish such a relationship. Applying established calibration modeling principles, as reinforced by Conti, to Christians’ multi-analyte control system would therefore have predictably yielded improved and standardized quantification of target organism abundance without requiring undue experimentation.
Applicant’s Response: The Applicant argues that the combination of Christians, Jiang, and Wu does not teach normalizing sample and multi-analyte control reads using normalization controls and determining a relationship between normalized reads and known titers of at least three species in the multi-analyte control, as required by independent claim 90. The Applicant further contends that Jiang’s teachings of relating read counts to RNA concentration using spike-in RNA standards is not equivalent to determining a relationship between normalized reads and organism titers in a multi-species control.
Examiner’s Response to Traversal: Applicant’s arguments have been carefully considered and are not found to be persuasive, as discussed below.
First, Christians teaches sequencing both sample and control material and normalizing read counts using spike-in controls (Paragraph 179). Under the broadest reasonable interpretation (BRI) (MPEP 2111), normalizing the collection of sample reads and the collection of multi-analyte control reads using the normalization controls reads on Christians’ normalization of sequencing reads based on spike-in standards added to the sample and control. The fact that Christians does not use identical terminology does not remove the teaching where the functional step is disclosed. Further, Christians teaches multi-analyte controls comprising multiple organisms (i.e., 11 pathogens), which necessarily includes at least three species. Therefore, the “at least three species” limitation is met by Christians. Even if Christians did not specify a minimum number, selecting three or more species from a disclosed plurality would have been an obvious design choice within the ordinary skill in the art, as optimizing number of reference organisms constitutes routine assay configuration (see MPEP 2144.05).
Second, although Christians does not expressly teach determining a relationship between normalized reads and known titers of at least three species, Jiang teaches deriving standard calibration curves that relate read counts to known input concentrations (Column 1, Paragraph 2, p. 1545). Under MPEP 2143 and KSR Int’l Co. v Teleflex Inc. 550 US 398 (2007), it is proper to combine references where each teaches part of the claimed invention and the combination yields predictable results. Jiang’s calibration curve expressly establishes a quantitative relationship between sequencing-derived read counts and known input amounts. Applying Jiang’s known calibration methodology to Christians’ multi-species control system would have predictably resulted in determining a relationship between normalized read frequency and known titers of the control species.
Specifically, the Applicant’s argument that Jiang relates read counts to RNA concentration rather than organism titer is unpersuasive. Under BRI, a known concentration of RNA derived from an organism is a measurable proxy for organism abundance in a sequencing-based assay. The claim does not require any specific biological mechanism linking RNA concentration and organism titer; rather it requires determining a relationship between normalized reads and known titers. Jiang teaches that read counts correlated with known input concentrations and may be used to generate calibration curves. Once Christians provides known titers for multiple species, applying Jiang’s calibration approach inherently yields the claimed relationship. A reference need not recognize the identical scientific rationale as long as it teaches the claimed structure or function (see MPEP 2141; In re Schreiber, 128 F. 3d 1473 (Fed. Cir. 1997)).
Also, Wu addresses the inactivation of organisms in multi-analyte controls, which Christians does not expressly teach. Substituting inactivated organisms as taught by Wu represents the predictable use of known safety measures in assay preparation and does not change the underlying quantification process. Such modification is consistent with MPEP 2144.05 (use of known technique to improve similar devices or methods).
With the addition of Conti to the obviousness rationale, Conti directly reinforces the obviousness rationale of the claimed relationship and calibration curve limitations. Conti teaches correlating measure signal frequency with known reference quantities and generating calibration models to quantify target strength (Abstract). Although arising in a different technical context, Conti demonstrates the general and well-established analytical principle that measurable signal frequencies may be translated into quantitative values using calibration curves. Under MPEP 2144.01 (analogous art), Conti is reasonably pertinent because it addresses the same problem of improving quantitative accuracy through calibration modeling. A person of ordinary skill in the art would have recognized that normalized sequencing read frequency is a type of measurable signal analogous to those in Conti and would have reasonably expected calibration modeling to yield improved quantitative results.
Accordingly, when Christians’ multi-species sequencing workflow is combined with Jiang’s calibration curve methodology and Wu’s inactivated controls, as reinforced by Conti’s signal-to-quantity modeling principles, the claimed steps of normalizing read and determining a relationship between normalized reads and known titers would have been obvious with a reasonable expectation of success.
Conclusions
No claim is allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to ELIZABETH ROSE LAFAVE whose telephone number is (703)756-4747. The examiner can normally be reached Compressed Bi-Week: M-F 7:30-4:30.
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/ELIZABETH ROSE LAFAVE/
Examiner, Art Unit 1684
/HEATHER CALAMITA/Supervisory Patent Examiner, Art Unit 1684