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 .
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 12/03/2025 was filed after the mailing date of the Non-Final Office Action on 09/04/2025. The submission is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Status of claims / Response to Amendment
This office action is in response to an amendment filed on December 3, 2025.
Claims 1-2 and 4-21 were previously pending. Applicant amended claims 1-2, 6-7, 10 and 17; cancelled claim 14.
Claims 1-2, 4-13 and 15-21 are currently pending, with claim 20 withdrawn.
Claims 1-2, 4-13, 15-19 and 21 are under consideration.
All of the previously presented objections and rejections have been withdrawn as being obviated by the amendment of the claims.
This office action contains new grounds of 102 and 103 rejections necessitated by Applicant's amendments. Although the claims were previously rejected as being unpatentable over the same reference(s), Applicant's amendments have necessitated the inclusion of new grounds of rejections in this Office action.
Applicant' s amendments and arguments have been thoroughly reviewed, but are not persuasive to place the claims in condition for allowance for the reasons that follow.
This office action contains new grounds for rejection necessitated by amendment.
Priority
The priority date of the instant claims 1-2, 4-13, 15-19 and 21 is July 13, 2020, filling date of the US provisional application NO. 63/051,149.
Claim Interpretation
In evaluating the patentability of the claims presented in this application, claim terms have been given their broadest reasonable interpretation (BRI) consistent with the specification, as understood by one of ordinary skill in the art, as outlined in MPEP§ 2111.
For the purpose of applying prior art, regarding all claims, terms such as "first" and "second" are interpreted as labels for identification purposes and do not imply any specific priority or sequential order of steps. This interpretation aligns with the claim language, as evidenced by claim 7/6/1, which recites only a "second solid support" is mentioned, without reference to a first solid support. This interpretation is made so the claim can be examined.
For the purpose of applying prior art, claim 1 recites a term "molecular label," which is not defined in the specification.
Because the application's disclosure does not define the term "molecular label" with any required structural features, under BRI this term is interpreted to encompass any type of molecular label, including any detectable label known in the field of molecular biology such as nucleic acid barcode sequences, fluorescent moieties, or other functional groups such as biotin, methylation, or antibodies.
New Grounds of Rejections - 35 USC § 102
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 the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
The following are new grounds of rejections necessitated by Applicant's amendments. Although the claims were previously rejected as being unpatentable over the same reference(s), Applicant's amendments have necessitated the inclusion of new grounds of rejections in this Office action. It is noted that, to the extent that they apply to the present rejection; Applicant's arguments are addressed following the rejection.
Claims 1-2, 4-11 and 15-19 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Chang (US20180346970A1, Published on 2018-12-06, cited in IDS US Patent Documents #26, filed on 12/11/23), as evidenced by Illumina Marketing Document (Optimizing Cluster Density on Illumina Sequencing Systems (2016), Pub. No. 770-2014-038, Published on 11 April 2016).
Chang teaches methods for labeling nucleic acids in samples using molecular barcodes with barcoded control nucleic acids, and generating control library data using next-generation sequencing (entire document).
Regarding claim 1, Chang teaches a method for labeling nucleic acid targets in a sample, comprising:
(a) barcoding copies of a nucleic acid target (Fig.10; [0669]”mRNA”) with a first plurality of oligonucleotide barcodes to generate a plurality of barcoded nucleic acid molecules each comprising a sequence complementary to at least a portion of the nucleic acid target([0669] reverse transcription of mRNA ‘1030’ generates extension products using probe of beads ‘1020’comprising barcode and mRNA as templates),
wherein at least 10 oligonucleotide barcodes of the first plurality of oligonucleotide barcodes comprise different molecular label sequences from each other([0013] lines 19-24, the barcodes of the bead can comprise molecular label sequences selected from at least 1000 or 10000 different molecular label sequences), and
wherein each barcoded nucleic acid molecule of the plurality of barcoded nucleic acid molecules comprises a first universal sequence and a molecular label (see FIGs. 1-2);
(b) after step (a), providing one or more barcoded control nucleic acids (FIG 10A; [0669-0670] barcoded extension products of control particle oligonucleotides 1025 in 1045 b are pooled with barcoded cDNA of mRNA in 1045 a),
wherein the number of copies of each of the one or more barcoded control nucleic acids is predetermined ([0668] lines 4-6).
Chang teaches separating target nucleic acids and control nucleic acids into different droplets ([0668]lines 9-13), performing reverse transcription (RT) with barcodes for target and control nucleic acid separately (FIG. 10; [0669]), and subsequently pooling the RT extension products together ([0670]lines1-3), thereby providing the barcoded control nucleic acid to the barcoded target nucleic acids. Therefore, Change fully teaches the claimed method steps with the specified order for steps a and b.
Chang further teaches (c) generating a sequencing library comprising
a plurality of nucleic acid target library members and
a plurality of control nucleic acid library members ([0670]line 3; [0046]; [0494]; [0503]lines 3-8),
wherein generating a sequencing library comprises:
attaching sequencing adaptors to the plurality of barcoded nucleic acid molecules generated in step (a), or products thereof, to generate the plurality of nucleic acid target library members ([0670]line 3; [0046]; [0494]; [0503]lines 3-8; [0513-0514]); and
attaching sequencing adaptors to the one or more barcoded control nucleic acids provided in step (b), or products thereof, to generate the plurality of control nucleic acid library members([0670]line 3; [0046]; [0494]; [0503]lines 3-8; [0513-0514]);
(d) obtaining sequencing data comprising a plurality of sequencing reads of one or more nucleic acid target library members and a plurality of sequencing reads of one or more control nucleic acid library members ([0670]); and
(e) determining the presence of a workflow failure, wherein the workflow failure consists of a failure in sequencing library generation ([657] low efficiency in library preparation is a failure; [0394]; [0676] “The number of cells that are captured and go through the library preparation successfully can be determined,” with the opposite holding true for the cells that fail in sequencing library generation).
Regarding claim 2, Chang teaches barcoding copies of a nucleic acid target with the first plurality of oligonucleotide barcodes comprises: contacting copies of the nucleic acid target with the first plurality of oligonucleotide barcodes (FIG. 2, 216), wherein each oligonucleotide barcode of the first plurality of oligonucleotide barcodes comprises a first universal sequence, a molecular label, and a target-binding region capable of hybridizing to the nucleic acid targe (FIG.2); and extending the first plurality of oligonucleotide barcodes hybridized to the copies of the nucleic acid target to generate a plurality of barcoded nucleic acid molecules each comprising a sequence complementary to the at least a portion of the nucleic acid target (FIG. 2, 224).
Regarding claim 4, Chang teaches a sample comprises of a plurality of single cells, comprising, prior to contacting copies of the nucleic acid target with the first plurality of oligonucleotide barcodes: partitioning the plurality of single cells to a plurality of partitions ([0455]lines 2-4 ; FIG. 2), wherein a partition of the plurality of partitions comprises a single cell from the plurality of single cells; lysing the single cell after the partitioning step (FIG2, 216); and in the partition comprising the single cell, contacting copies of the nucleic acid target with the first plurality of oligonucleotide barcodes (FIG. 2, 216; [0455]).
Regarding claim 5, Chang teaches a first plurality of oligonucleotide barcodes associated with a first solid support, the method comprising associating the first solid support with the single cell in the sample, and wherein a partition of the plurality of partitions comprises a single first solid support ([0455]; FIG. 2).
Regarding claim 6, Chang teaches one or more barcoded control nucleic acids are generated by: contacting a predetermined number of copies of one or more control nucleic acids with a second plurality of oligonucleotide barcodes (FIG 8A1), wherein each oligonucleotide barcode of the second plurality of oligonucleotide barcodes comprises a first universal sequence (FIG 8A1, 815u ; [0055]), a control label (FIG 8A1, 815c; [0055]), and a target-binding region (FIG 8A1, 815t; [0055]) capable of hybridizing to the one or more control nucleic acids; and extending the second plurality of labeled oligonucleotides hybridized to the one or more control nucleic acids to generate a predetermined number of copies of one or more barcoded control nucleic acids each comprising a sequence complementary to the at least a portion of the one or more barcoded control nucleic acids (FIG 8A1, RT).
Regarding claim 7, Chang teaches a second plurality of oligonucleotide barcodes associated with a second solid support and/or one or more barcoded control nucleic acids are associated with a second solid support (FIG 8A1).
Regarding claim 8, Chang teaches each of the barcoded control nucleic acids comprise one or more of a first universal sequence (FIG 8A1, 815u; [0055]), a control label (FIG 8A1, 815c; [0055]), and a target-binding region (FIG 8A1, 815t; [0055]).
Regarding claim 9, Chang teaches one or more barcoded control nucleic acids ([0622] lines 18-20; [0628]; [0055]) comprises at least 2 different barcoded control nucleic acids.
Regarding claim 10, Chang teaches one or more barcoded control nucleic acids is homologous to genomic sequences of a species, wherein the species is a nonmammalian species, and wherein the non-mammalian species is a phage species ([630]).
Regarding claim 11, Chang teaches each of the plurality of sequencing reads of the plurality of barcoded nucleic acid molecules, or products thereof, comprise (1) a molecular label sequence, and (2) a subsequence of the nucleic acid target; and each of the plurality of sequencing reads of the plurality of barcoded control nucleic acid molecules, or products thereof, comprise (1) a control label sequence (FIG 13 C is the analysis from sequencing data, which indicates molecular label, subsequence of nucleic acid target, and control label sequence are present in the sequence as they are required by this figure).
Regarding claim 15, Chang teaches presence of a failure in barcoding copies of the nucleic acid target is determined by the ratio of sequencing reads of the one or more control nucleic acid library members to sequencing reads of the one or more nucleic acid target library members exceeding a predetermined barcoding threshold, and wherein the predetermined barcoding threshold is at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 ([0658], lines4-6, predetermined barcoding threshold is 5).
Regarding claim 16, Chang teaches determining the copy number of the nucleic acid target ([0655]; [0659] ;Table 4; FIGS. 53A-D; FIGS. 52A-C) in the sample based on the plurality of sequencing reads of one or more nucleic acid target library members.
Regarding claim 17, Chang teaches determining the copy number of the nucleic acid target in the sample comprises determining the copy number of the nucleic acid target in the sample based on the number of molecular labels ([0655]lines15-17, lines36-18; [0423]) with distinct sequences, complements thereof, or a combination thereof, associated with the one or more nucleic acid target library members, or products thereof.
Regarding claim 18, Chang teaches presence of a failure in barcoding copies of the nucleic acid target is determined by the ratio of the predetermined number of copies of the one or more barcoded control nucleic acids to the copy number of the nucleic acid target in the sample exceeding a predetermined barcoding threshold, and wherein the predetermined barcoding threshold is at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 ([0658], lines4-6, predetermined barcoding threshold is 5).
Regarding claim 19, Chang teaches obtaining sequencing data comprising a plurality of sequencing reads of a predetermined number of one or more spike-in library members, wherein the presence of a failure in sequencing library generation is determined by the ratio of sequencing reads of the predetermined number of the one or more spike-in library members to sequencing reads of the one or more control nucleic acid library members exceeding a predetermined library generation threshold, and wherein the predetermined library generation threshold is at least 1 ([501] Illumina sequencing). While Chang does not explicitly disclose the use of spike-in library members with a pre-determined library generation threshold, Chang teaches the sequencing method is Illumina sequencing. It is well-known in the art that Illumina sequencing method utilizes the PhiX library spike-in as sequencing control with a pre-determined library generation threshold, as evidenced by Illumina Marketing Document ( Page 7, c. the summary Tab, % Aligned; page 9: recommended spike in threshold is 1 percent).
Response to Arguments
Applicant's arguments regarding the previously cited reference Chang have been fully considered but are not found persuasive.
First, Applicant argues that Chang does not teach the newly added limitation to claim 1: "determining the presence of a workflow failure, wherein the workflow failure consists of a failure in sequencing library generation" (Remarks, page 14-15).
This argument is not persuasive. As discussed in the rejection above, Chang anticipates claim 1 and fully teaches this limitation. Specifically, para. [0657] of Chang teaches determining the efficiency of sequencing library preparation:
"[0657] For example, if the control particle has 100 control particle oligonucleotides with a particular control barcode sequence and the number of barcode sequences (e.g., molecular labels) with distinct sequences associated with the control barcode sequence ( e.g., the number of control particle oligonucleotides with the control barcode sequence that survive the library preparation process) is 80, then the efficiency of the library preparation ( e.g., reverse transcription, amplification, etc.) is 80%. Thus, data from different library preparations can be compared by normalizing using the library preparation efficiency."
Thus, a skilled artisan would readily understand that low efficiency in library generation constitutes a workflow failure, indicating that little nucleic acid survived the library generation process. Furthermore, steps such as reverse transcription and amplification are well-known components of the sequencing library generation process, as supported by para. [0513], [0515-0521], and [0675] of Chang. Additionally, paragraph [0676] of Chang teaches that “[t]he number of cells that are captured and go through the library preparation successfully can be determined.” This demonstrates that determining success in library preparation is explicitly supported in Chang, with the opposite holding true for cells that fail in sequencing library generation.
Applicant also asserts that "[i]n contrast to Chang, the presently claimed barcoded control nucleic acids are not barcoded in parallel with the target nucleic acids, but instead are provided after the barcoding step." (Remarks, page 15)
This argument is not persuasive as the distinguishing feature upon which the argument relies upon is not claimed.
Chang teaches the limitation "after step (a), providing one or more barcoded control nucleic acids," as discussed in detail in the rejection above.
It is noted that the negative feature upon which Applicant's assertion is based (i.e., that barcoded control nucleic acids are not barcoded in parallel with the target nucleic acids) is not recited in the claim. Additionally, the specification does not describe any method that expressly excludes barcoding control nucleic acids in parallel with the target nucleic acids.
New Grounds of 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 12-13 are rejected under 35 U.S.C. 103 as being unpatentable over Chang, in view of Fu (Fu et al. Molecular indexing enables quantitative targeted RNA sequencing and reveals poor efficiencies in standard library preparations. Proc Natl Acad Sci U S A. 2014 Feb 4;111(5):1891-6. PMID: 24449890; PMCID: PMC3918775.), as evidenced by Illumina Technical Note (Illumina Technical Note: Sequencing; Pub. No. 770-2011-022, Published on 12/01/14 by Illumina, Inc).
A) The teachings of Chang are recited above and applied as for base claim 1.
Regarding claim 12, Chang teaches determining a sequencing status of the one or more control nucleic acid library members in the sequencing data, wherein the sequencing status of the one or more control nucleic acid library members in the sequencing data is saturated sequencing or under sequencing ([0656]; [0657];[0658]), and wherein the under sequencing status ([0657] example of under sequencing with predetermined threshold) is determined by the one or more control nucleic acid library members having a number of sequencing reads less than a predetermined saturation threshold.
However, Change does not specifically teach in detail a case of saturated sequencing, and how the saturated sequencing status is determined using a pre-determined threshold.
B) Fu teaches a method for molecular indexing for quantitative target RNA sequencing, using a set of barcoded control nucleic acid library to monitor the efficiency of library construction (abstract).
Fu teaches a specific case of saturated sequencing, wherein the saturated sequencing status (Table 3; Page 1894, right hand col, lines13-15) is determined by the one or more control nucleic acid library members having a number of sequencing reads at or greater than a predetermined saturation threshold; and the predetermined saturation threshold (Page 1894, right hand col, lines13-15) is a number at least 1.1-fold greater than the predetermined number of copies of the one or more barcoded control nucleic acids (Page 1894, right hand col, lines13-15, 20 fold saturation).
Fu also suggests that the 20 fold saturation threshold should be implemented because : "At this sequencing depth, effectively all unique molecules in the library have been sampled, and very little new information will be obtained by additional sequencing." (page 1894, right hand col, line 15-7 to page 1895, left hand col, line 1)
C) It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the method for labeling nucleic acids using molecular barcodes taught by of Chang with the teachings of determining a saturated sequencing status using a saturation threshold disclosed by Fu because both references are in the overlapping field of nucleic acid sequencing and library construction. A skilled artisan in the field of molecular biology, specifically in sequencing technologies, would have naturally considered these references together when seeking to optimize sequencing processes. The person of ordinary skill would have had a reasonable expectation of success in applying Fu's specific saturation threshold concept to the method of Chang because these teachings are technically compatible: Chang teaches the similar concept of under sequencing but only lacks specificity in the case of saturated sequencing. Doing so would yield the predictable result of effectively determining the sequencing status of nucleic acid libraries, particularly in case of saturated sequencing. The skilled artisan would have been motivated to do so because a saturation threshold of 20 fold is optimal for ensuring that all unique molecules in the library have been sampled, providing a clear benchmark for sequencing efforts, as suggested by Fu. This is beneficial for enhancing the reliability and efficacy of the sequencing process described in Chang.
D) Regarding claim 13, which depends from claim 12. All limitations in claim 13 are taught by the combination of Chang. and Fu, as evidenced by Illumina Technical Note.
The combination of Chang and Fu teach the steps to determine sequencing status and motivation to reach saturated sequencing status, as discussed in the previous sections A-C. While the combination of Chang and Fu do not explicitly disclose that if sequencing status is the under sequencing status, the step of obtaining sequencing data should be repeated until the sequencing status is the saturated sequencing status, both the methods of Chang ([501] Illumina sequencing) and Fu(page 1895, materials and methods) teach Illumina sequencing as the sequencing method. The approach of repeated sequencing is well-known in the art especially in the application of Illumina sequencing, as evidenced by Illumina Technical Note (page 2, left hand col, when to sequence more). Therefore, the combination of Chang and Fu teach and suggest all elements of claim 13.
Claim 21 is rejected under 35 U.S.C. 103 as being unpatentable over Chang , in view of Seifi (Seifi et al. Amplification of GC-rich Putative Mouse PeP Promoter using Betaine and DMSO in Ammonium Sulfate Polymerase Chain Reaction Buffer. Avicenna J Med Biotechnol. 2012 Oct;4(4):206-9. PMID: 23408119; PMCID: PMC3558226) , as evidenced by Picelli (Picelli et al. Full-length RNA-seq from single cells using Smart-seq2. Nat Protoc 9, 171–181 (2014). doi.org/10.1038/nprot.2014.006).
A) The teachings of Chang are recited above and applied as for base claims 1 and 2.
Regarding claim 21, Chang teaches extending the first plurality of oligonucleotide barcodes by reverse transcription (RT) extension reaction ([0666] for example), but it does not specifically teach 1,2-propanediol.
RNA secondary structure is a known problem in reverse transcription reactions, which could cause premature termination of elongation. Betaine is a well-known additive that can enhance reverse transcription efficiency by reducing RNA secondary structure, especially in GC-rich regions, as evidenced by Picelli (page 171, right-hand col, para 2).
Seifi teaches that in addition to Betaine, other reaction additives such as 1, 2-propanediol are also useful for amplification of GC-rich region, providing the same benefit as betaine such as preventing secondary structure, and increase the annealing chance of primers (page 1, left-hand col, para 2).
Therefore, it would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to include 1, 2-propanediol, disclosed in Seifi, as an reaction additive to reduce secondary structure and enhance the reverse transcription reaction taught by Chang because these references are in the same or overlapping field of nucleic acid assay development and they have complementary teachings. Using reaction additives in reverse transcription to mitigate undesirable secondary structures and improve reaction efficiency has been a well-known technique in the art, as evidenced by Picelli. And Seifi teaches 1, 2-propanediol is an additive that can provide the benefit of preventing secondary structure, and increase the annealing chance of primers, which are highly appealing to reverse transcription reactions involving annealing primers to RNA templates that are prone to forming secondary structures.
Therefore, this combination would have been obvious as it represents the KSR principle of predictable use of prior art elements (i.e., 1, 2-propanediol as an reaction additive, taught by Seifi) according to a known method (i.e., performing reverse transcription reaction in Chang) to yield predictable results (i.e., reduced secondary structure, and increased annealing chance of primers in reverse transcription reaction). (See MPEP §2143).
Conclusion
No claims are allowed.
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.
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/TIAN NMN YU/Examiner , Art Unit 1681 /AARON A PRIEST/Primary Examiner, Art Unit 1681