DETAILED ACTION
The amendment filed on 11/07/2025 has been entered.
Claims 1 and 19 were amended in the claim set filed on 11/07/2025.
Claim 3 is canceled.
Claims 1-2, 4-15, 18-21, and 23 are currently pending and under examination.
Response to the Arguments
Applicant’s arguments regarding previous rejections of claims 1-2, 4-15 and 18 under 35 U.S.C. 102 have been fully considered and are persuasive. The 35 U.S.C. 102 rejections documented in the previously mailed non-final have been withdrawn in light of applicants claim amendments and arguments on Pg. 5. As necessitated by amendment and after consideration and search, new grounds of rejections for claims 1-2, 4-15 and 18 are made, as documented below, under the 35 U.S.C. 103 rejections in this office action on Pg. 3-8.
Applicant’s arguments regarding previous rejections of claims 19-21, and 23 under 35 U.S.C. 103 have been fully considered and are not persuasive. The 35 U.S.C. 103 rejections documented in the previously mailed non-final have been maintained and revised in light of applicants claim amendments and arguments on Pg. 6. As necessitated by amendment, revised rejections for claims 19-21, and 23 are made, as documented below, under the 35 U.S.C. 103 rejections in this office action on Pg. 10-14.
After further consideration of claim amendments to claim 1 the instant application and claim 1 of copending Application No. 17/628861 the provisional Nonstatutory Double Patenting rejection documented in the previously mailed non-final have been withdrawn.
The rejections for claims 1-2, 4-15, 18-21, and 23 are documented below in this Final Office Action are necessitated by claim amendments filed on 11/07/2025.
Priority
This application 17/620,077 is a 371 national phase of PCT/US2020/056904 filed on 10/22/2020, which claims benefit of 62/924,590 filed on 10/22/2019. Accordingly, the priority date of instant claims is determined to be 10/22/2019, the filing date of 62/924,590.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
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.
Claims 1-2, 4-15 and 18 are rejected under 35 U.S.C. 103 as being unpatentable over Reuter in view of Promega Corporation. (“Promega” (2014). Choosing the Right Reverse Transcriptase. Choosing the right reverse transcriptase. Archived on 09/20/2014.https://web.archive.org/web/20140920192559/http://www.promega.com/resources/pubhub/choosing-the-right-reverse-transcriptase/).
Reuter discloses methods, compositions, and kits for simultaneously generating RNA and DNA sequencing libraries are disclosed. In particular, the disclosed methods allow high-throughput amplification and sequencing of both RNA and DNA from a single sample source without the need to divide the sample to separate nucleic acids from each other. All of the preparation steps from harvesting nucleic acids from cells or tissue to preparing RNA and DNA sequencing libraries can be performed with the RNA and DNA pooled together. This technology streamlines generation of DNA and RNA sequencing data in combination and makes possible comprehensive genomic and transcriptomic profiling of a cell population concurrently (Abstract).
Regarding claim 1, Reuter teaches a method comprising “sequencing RNA and DNA”, “e) ligating a 3′ oligonucleotide adapter comprising a 3′ common priming site for RNA-specific amplification to the 3′ end of each RNA fragment …; f) ligating a 5′ oligonucleotide adapter comprising a 5′ common priming site for RNA-specific amplification to the 5′ end of each RNA fragment …; g) adding reverse transcriptase such that a plurality of cDNA fragments is produced from the plurality of RNA fragments”, and “ j) sequencing the DNA amplicons and the cDNA amplicons, wherein the DNA index sequence is used to identify DNA sequences and the RNA index sequence is used to identify RNA sequences. All steps of the method may be performed with the RNA and DNA nucleic acids pooled together.” (e.g., Para. 8). Thus, Reuter teaches a method of analyzing nucleic acid sequences, comprising: providing a sample comprising DNA and RNA; reverse transcribing the RNA with a primer comprising a barcode to produce cDNA while maintaining the DNA in the sample; sequencing the DNA and the cDNA together; and differentiating the sequenced DNA and the sequenced cDNA using the barcode of the primers.
Regarding claim 1, Reuter teaches a method wherein “denaturation step is typically effected by heat, but may alternatively be carried out using alkali, followed by neutralization” (e.g., Para. 50; Para. 8 see above). “denaturation step is typically effected by heat, but may alternatively be carried out using alkali, followed by neutralization” reads on avoiding heating of the sample. Furthermore, the method in para. 8 recited above does not explicitly recite heating the sample therefore the method reads on avoiding heating of the sample. Thus, Reuter suggests a method wherein the DNA is maintained in the sample by avoiding heating of the sample to denature the DNA prior to and during reverse transcription of the RNA.
Although Reuter does not explicitly teach a method wherein the DNA is maintained in the sample by avoiding heating of the sample prior to and during the reverse transcription of the RNA it is suggested by Reuter and further evidenced by Promega. Promega teaches “Optimal enzyme activity and maximum cDNA length occur at 42–48°C, but the reaction temperature can range from 25°C to 58°C” (e.g., Para. 3). “the reaction temperature can range from 25°C to 58°C” (e.g., Para. 3)” reads on avoiding heating of the sample prior to and during the reverse transcription of the RNA. Thus, Reuter and Promega suggest a method wherein the DNA is maintained in the sample by avoiding heating of the sample to denature the DNA prior to and during reverse transcription of the RNA.
Reuter and Promega are both considered to be analogous to the claimed invention because they are in the same field of using reverse transcriptase to synthesize cDNA. Therefore, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of maintaining DNA in the sample by avoiding heating of the sample to denature the DNA prior to and during the reverse transcription of the RNA as taught by Reuter to incorporate the method of reverse transcription reaction from a range of 25 degrees C as taught by Promega with a reasonable expectation to avoid heating prior to and during reverse transcription. Furthermore, these claim elements were known in the art and one of skill in the art could have combined these elements by known methods with no change in their respective functions, and the combination would have yielded the predictable outcome of maintaining the DNA.
The teachings of Reuter and Promega are documented above in the rejection of claim under 35 U.S.C. 103. Claims 2, 4, 5, 9-13, 15 and 18 depends on claim 1. Claims 7 and 8 depend on claim 6, which depends on claim 5, which depends on claim 1. Claim 14 depends on claim 13, which depends on claim 1.
Regarding claim 2, Reuter teaches a method wherein “a primer may further comprise a “tail” comprising additional nucleotides at the 5′ end of the primer that are non-complementary to the template” (e.g., Para. 50). Thus, Reuter teaches a method wherein the barcoded primer comprises a random sequence.
Regarding claim 4, Reuter teaches a method further comprising “ c) fragmenting the DNA … d) fragmenting the RNA … (e.g., Para. 8). Thus, Reuter teaches a method further comprising fragmenting the DNA and the RNA.
Regarding claim 5, Reuter teaches a method “comprising tagging of DNA (i.e., tagmentation)” (e.g., Para. 74; Figure 1a). Thus, Reuter teaches a method further comprising tagmenting the DNA and the cDNA.
Regarding claim 6, Reuter teaches a method wherein “tagging of DNA (i.e., tagmentation) can be accomplished by treatment of the nucleic acids with a Class 2 transposase (e.g., Para. 74)” Thus, Reuter teaches a method wherein the tagmentation comprises use of a transposase.
Regarding claim 7, Reuter teaches a method wherein “the transposase may be a Tn5 transposase” (e.g., Para. 8). Thus, Reuter teaches a method wherein the transposase comprises a Tn5 transposase.
Regarding claim 8, Reuter teaches a method wherein “c) … wherein the transposase catalyzes cleavage of the DNA into a plurality of DNA fragments and ligation of the oligonucleotide adapter to each DNA fragment at the 5′ ends of its DNA strands; (e.g., Para. 8). Thus, Reuter teaches a method wherein the transposase adds an adapter sequence to the DNA and the cDNA.
Regarding claim 9, Reuter teaches a method further comprising “strand displacement amplification” (e.g., Para. 51) and “any enzyme capable of producing primer extension products, for example, E. coli DNA polymerase I, Klenow fragment of DNA polymerase I, T4 DNA polymerase, thermostable DNA polymerases isolated from Thermus aquaticus (Taq)” (e.g., Para. 78). Thus, Reuter teaches a method further comprising conducting end repair of the DNA and the cDNA with a strand displacing polymerase.
Regarding claim 10, Reuter teaches a method further comprising “ Poly(A) or poly(C), or other non-complementary nucleotide extensions may be incorporated“ (e.g., Para. 85). Reuter teaches a method further comprising “ligation of the oligonucleotide adapter to each DNA fragment at the 5′ ends of its DNA strand” (e.g., Para. 8). Thus, Reuter teaches a method further comprising conducting A-tailing and adapter ligation to the DNA and/or the cDNA.
Regarding claim 11, Reuter teaches a method wherein “adapters may comprise, … a common priming site to allow massively parallel sequencing and/or amplification of DNA or cDNA ... In addition, adapters may comprise indexing/barcoding sequences to identify the sample source” (e.g., Para. 53). Thus, Reuter teaches a method wherein the barcoded primer comprises an adapter sequence.
Regarding claim 12, Reuter teaches a method wherein “h) amplifying the plurality of DNA fragments with a set of DNA indexing primers … i) amplifying the plurality of cDNA fragments with a set of RNA indexing primers (e.g., Para. 8). Thus, Reuter teaches a method further comprising adding a sample-specific index to the DNA and/or the cDNA.
Regarding claim 13, Reuter teaches a method further comprising “identifying a mutation in the DNA or RNA” (e.g., Para. 16). Thus, Reuter teaches a method further comprising determining a mutation in the DNA and determining whether the RNA comprises the mutation.
Regarding claim 14, Reuter teaches a method wherein the “sample may comprise a genetically aberrant cell, cancer cell” (e.g., Para. 9). Thus, Reuter teaches a method wherein the sample comprises a tumor or cancer sample.
Regarding claim 15, Reuter teaches a method comprising “wherein the DNA index sequence is used to identify DNA sequences, and the RNA index sequence is used to identify RNA sequences” (e.g., Para. 8). Reuter teaches a method further comprising “the DNA and RNA are obtained from a biological sample comprising a eukaryotic cell (e.g., animal, plant, fungus, or protist), prokaryotic cell (e.g., bacterium or archaeon), or a virus” (e.g., Para. 9). Thus, Reuter teaches a method further comprising identifying a pathogen DNA in the sample and a pathogen RNA in the sample, wherein the pathogen comprises a bacterium, a fungus, or a virus.
Regarding claim 18, Reuter teaches a method further comprising “The cell may be a live cell or a fixed cell” and “the DNA and RNA are obtained from a biological sample comprising a eukaryotic cell (e.g., animal, plant, fungus, or protist), prokaryotic cell (e.g., bacterium or archaeon), or a virus. (e.g., Para. 9). Thus, Reuter teaches a method further comprising identifying a microbe in the sample based on the sequenced DNA and identifying whether the microbe is alive, or dead based on the sequenced cDNA.
Response to Arguments
Applicant' s arguments filed 11/07/2025 (Pg. 5) with respect to claims 1-2, 4-15 and 18 do not apply to the new grounds of rejections. To clarify some instances argued in the response filed 11/07/2025 see responses to each argument made by Applicant below:
Applicants’ argument: “Without conceding in the basis of rejection and solely to expedite the prosecution of this application, Applicant has amended claim 1 to incorporate elements of claim 3, which is not rejected under 35 U.S.C. §102. See Office Action, pg. 2. Accordingly, amended claim I is novel over Reuter.” (Pg. 5)
Response: Applicant's arguments filed 11/07/2025 have been fully considered but they are not persuasive. As necessitated by amendment, see revised rejection towards claim 1 under 35 U.S.C. 103.
Applicants’ argument: “Promega fails to cure the above-mentioned deficiencies because the cited reference does not disclose "a method of analyzing nucleic acid sequences, comprising: providing a sample comprising DNA and RNA; reverse transcribing the RNA with a primer comprising a barcode to produce cDNA while maintaining the DNA in the sample; sequencing the DNA and the cDNA together; and differentiating the sequenced DNA and the sequenced cDNA using the barcode of the primers, wherein the DNA is maintained in the sample by avoiding heating of the sample to denature the DNA prior to and during the reverse transcription of the RNA" as recited in amended claim 1.” (Pg. 6)
Response: Applicant's arguments filed 11/07/2025 have been fully considered but they are not persuasive. In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). Furthermore, Promega is used as evidence to further support the notion that avoiding heating during the reverse transcription of RNA can be accomplished with a reasonable expectation of success.
Applicants’ argument: “As such, a person having ordinary skill in the art (PHOSITA) would not be motivated to avoid heating of the sample if they knew the reaction could effectively operate over a wide range of temperatures” (Pg. 6-7)
Response: In response to applicant’s argument that there is no teaching, suggestion, or motivation to combine the references, the examiner recognizes that obviousness may be established by combining or modifying the teachings of the prior art to produce the claimed invention where there is some teaching, suggestion, or motivation to do so found either in the references themselves or in the knowledge generally available to one of ordinary skill in the art. See In re Fine, 837 F.2d 1071, 5 USPQ2d 1596 (Fed. Cir. 1988), In re Jones, 958 F.2d 347, 21 USPQ2d 1941 (Fed. Cir. 1992), and KSR International Co. v. Teleflex, Inc., 550 U.S. 398, 82 USPQ2d 1385 (2007). In this case, Reuter teaches a method wherein “denaturation step is typically effected by heat, but may alternatively be carried out using alkali, followed by neutralization” (e.g., Para. 50; Para. 8 see above). “denaturation step is typically effected by heat, but may alternatively be carried out using alkali, followed by neutralization” reads on avoiding heating of the sample. Furthermore, the method in para. 8 recited above does not explicitly recite heating the sample therefore the method reads on avoiding heating of the sample. Thus, Reuter suggests a method wherein the DNA is maintained in the sample by avoiding heating of the sample to denature the DNA prior to and during reverse transcription of the RNA.
Claims 19-21, and 23 are rejected under 35 U.S.C. 103 as being unpatentable over Reuter in view of Rahman et al. (“Rahman” (2018). Association mapping from sequencing reads using k-mers. eLife, 7, e32920.).
Regarding claim 19, Reuter teaches a method comprising “sequencing of DNA or cDNA may comprise paired-end sequencing or single-read sequencing… sequencing comprises performing a next-generation sequencing (NGS) technique... the method further comprises assembling DNA sequences or cDNA sequences to produce longer sequences. In certain embodiments, overlapping sequence reads are assembled into contigs or full or partial contiguous sequences of a nucleic acid of interest (e.g. DNA gene, intergenic regulatory region, or RNA coding or non-coding transcript (e.g., Para. 15; Para. 16; Para. 17). Reuter teaches a method comprising “The present invention relates to a method for simultaneously generating RNA and DNA sequencing libraries from the same sample” (Para. 7). “RNA and DNA” and “DNA or cDNA” read on a first and a second sample. Furthermore, Reuter teaches a method comprising “…the method further comprises identifying a mutation in the DNA or RNA. A mutation may comprise, for example, an insertion, a deletion, or a substitution. For example, a mutation may include a single nucleotide variation, gene fusion, translocation, inversion, duplication, frameshift, missense, nonsense, or other mutation associated with a phenotype or disease of interest. …the method further comprises detecting genomic copy number variation. …the method further comprises performing transcriptome quantification or isoform analysis.” (Para. 16-18). Thus, Reuter teaches a method for analysis of nucleic acid sequences, comprising: providing nucleic acid sequence reads for a first sample and a second sample; identifying a differential sequence between the reads the first and second samples; and wherein the first sample and the second sample is from a same subject.
Reuter does not explicitly teach “separating the reads of the first sample and the second sample into k-mers; comparing the k-mers of the first sample to the k-mers of the second sample; and identifying a statistical difference between the k-mers of the first and second samples.”
Rahman discloses an alignment free method for association studies of categorical phenotypes based on counting k-mers in whole-genome sequencing reads, testing for associations directly between k-mers and the trait of interest, and local assembly of the statistically significant k-mers to identify sequence differences.
Regarding claim 19, Rahman teaches a method comprising “sequencing reads from
two sets of samples”, “count k-mers in reads from each sample”, “k-mers with significantly
different counts in two sets are detected” and “overlapping k-mers are assembled into sequences to get a sequence, shown side by side, for each associated locus. The sequences may correspond to a SNP (underlined) in which case corresponding sequence may be detected in the other group” (e.g., Pg. 3, Figure 1 legend and Figure 1, shown below).
PNG
media_image1.png
875
858
media_image1.png
Greyscale
Thus, Reuter and Rahman teach a method for analysis of nucleic acid sequences, comprising: a first sample and a second sample; separating the reads of the first sample and the second sample into k-mers; comparing the k-mers of the first sample to the k-mers of the second sample; separating the reads of the first sample and the second sample into k-mers; comparing the k-mers of the first sample to the k-mers of the second sample; identifying a statistical difference between the k-mers of the first and second samples, thereby identifying a differential sequence between the reads of the first and second samples, wherein the first sample and the second sample is from the same subject.
Reuter and Rahman are both considered to be analogous to the claimed invention because they are in the same field of genetic analysis by mapping sequencing reads. Therefore, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of analysis of nucleic acid sequences as taught by Reuter to incorporate the method of separating the reads of the first sample and the second sample into k-mers; comparing the k-mers of the first sample to the k-mers of the second sample; and identifying a statistical difference between the k-mers of the first and second samples as taught by Rahman and provide a method according to the limitations of claim 19. Doing so would allow for an alignment free method for association mapping of sequencing reads with the expectation of more power and less ambiguity (Rahman, Pg. 13, Discussion).
The teachings of Reuter and Rahman are documented above in the rejection of claim 19 under 35 U.S.C. 103. Claims 20-21 and 23 depend on claim 19.
Regarding claim 20, Rahman teaches a method comprising “k-mers of length 31 which is the longest k-mer length which can be efficiently represented on 64-bit machines” (e.g., Pg. 4 Para. 1, Counting k-mers). Thus, Reuter and Rahman teach a method wherein each of the k-mers comprises a sequence length of about 10, 25, 50, 75, 100, 125, 150, 250, or a range defined by any two of the aforementioned integers, or more, nucleotides.
Regarding claim 21, Rahman teaches a method comprising “overlapping k-mers are assembled into sequences to get a sequence, shown side by side, for each associated locus. The sequences may correspond to a SNP (underlined) in which case corresponding sequence may be detected in the other group” (e.g., Pg. 3, Figure 1 legend; Figure 1, shown above). Thus, Reuter and Rahman teach a method further comprising performing a local de novo assembly to expand a length of a differential sequence, and/or identifying a genome region associated with the differential sequence. Thus, Reuter and Rahman teach a method wherein the nucleic acid sequence reads are provided by a method that includes sequencing DNA and cDNA together, and wherein the analysis comprises analyzing a DNA and an RNA in the first sample simultaneously.
Regarding claim 23, Reuter teaches a method wherein “a method for simultaneously generating RNA and DNA sequencing libraries from the same sample” (e.g., Para. 7) and “the method may be performed with the RNA and DNA nucleic acids pooled together” (e.g., Para. 8). Thus, Reuter and Rahman teach a method suggests a method wherein the nucleic acid sequence reads are provided by a method that includes sequencing DNA and cDNA together, and wherein the analysis comprises analyzing a DNA and an RNA in the first sample simultaneously.
Response to Arguments
Applicant' s arguments filed on 11/07/2025 (Pg. 6) with respect to claims 12-15,18-19, 24-27, 37, 47, 52, 92 and 120-121 have been considered but are not persuasive. To clarify some instances argued in the response filed 11/07/2025 that remains relevant to the revised 103 rejections documented in this Final Office Action, examiner’s responses to each relevant argument made by Applicant are provided below:
Applicants’ argument: “amended claim 19 is patentable over Reuter and Rahman, alone or in combination, because the cited references fail to teach each and every element of amended claim 19” (Pg. 7).
Response: Applicant's arguments filed 11/07/2025 have been fully considered but they are not persuasive. As necessitated by amendment, see revised rejection of claim 19 above.
Applicants’ argument: “As a result, the method described in Rahman centers on analyzing multiple groups of metagenomic samples. Instead, the instant application provides that the claimed method "requires . . just enough sequencing depth to determine a statistical difference between sequence motifs of comparable sample types" by sequencing "a tumor and matched normal sample from the same individual.”
Response: Applicant's arguments filed 11/07/2025 have been fully considered but they are not persuasive. As necessitated by amendment, see revised rejection of claim 19 above. Furthermore, In response to applicant's argument that the references fail to show certain features of the invention, it is noted that the features upon which applicant relies (i.e., “the claimed method "requires . . just enough sequencing depth to determine a statistical difference between sequence motifs of comparable sample types" by sequencing "a tumor and matched normal sample from the same individual.”) is not recited in the rejected claim(s). Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993).
Conclusion of Response to Arguments
In view of the amendments, revised and new grounds of rejections, and responses to arguments are documented in this Final Office Action. No claims are in condition for allowance.
Conclusion
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to KENDRA R VANN-OJUEKAIYE whose telephone number is (571)270-7529. The examiner can normally be reached M-F 9:00 AM- 5:00 PM.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Winston Shen can be reached at (571)272-3157. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/KENDRA R VANN-OJUEKAIYE/Examiner, Art Unit 1682
/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682