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 .
Continued Examination Under 37 CFR 1.114
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 4/29/2026 has been entered.
Applicant’s arguments and amendments have been thoroughly reviewed and considered. Claims 46-65 are pending and are examined on the merits herein.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 6/3/2026 is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Response to Applicant’s Arguments and Amendments
Claims 46-54 and 59-61 were rejected under 35 U.S.C. 103 as being unpatentable over Evans et al. (US 2017/0355984 A1) in view of Peng et al. (Nature Scientific Reports, 2019).
Claims 55-58 and 62-65 were rejected under 35 U.S.C. 103 as being unpatentable over Evans et al. (US 2017/0355984 A1), in view of Peng et al. (Nature Scientific Reports, 2019), and further in view of Zhao et al. (US 2019/0085384 A1).
Regarding the 35 USC 103 Rejections, Applicant argues that the cited references previously used to reject claim 46 (Evans in view of Peng) do not read on the newly amended claim (Remarks, pages 7-8). The Examiner agrees, as Peng teaches the removal of UMI nucleotides in their trimming protocol (see paras. 33-34 of the Final Rejection mailed 3/4/2026).
Thus, the previously set forth 35 USC 103 Rejections have been withdrawn. However, see new grounds of rejection below.
Claim Objections
Claim 46 is objected to because of the following informalities: it is recommended to alter the term “adapter-ligated sample polynucleotides” in line 2 of step (a) to read “adapter-ligated sample nucleic acids”, as sample nucleic acids are described earlier in the step. Additionally, in line 2 of step (d), “removing of one” should read “removing . Appropriate correction is required.
Claim Rejections - 35 USC § 112(b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 46-65 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Regarding claim 46, step (a) of the method states that adapters are ligated to sample nucleic acids, where this ligation creates a library, and wherein only some of the adapters need have a unique molecular identifier. This means that there is no requirement that each adapter have a unique molecular identifier. Then the library is amplified and sequenced, which would produce sequence reads where some reads do not have a unique molecule identifiers, corresponding to those adapters which did not have said identifiers. However, in step (d), for each sequencing read, trimming between the unique molecular identifier and the sample nucleic acid must occur. However, this could not occur for those sequences that do not have the unique molecular identifiers. Therefore, it is unclear how, in instances where not every adapter has a unique molecular identifier, the requirements for step (d) would be possible to complete. This renders the scope of the claim indefinite.
Additionally for claim 46, the phrase “at least some” in step (a) is also generally indefinite, as it is unclear what fraction of the adapters would need to contain unique molecular identifiers in order to meet the “at least some” requirement. Applicant is pointed to terms that may convey a similar meaning but would not be considered indefinite, such as “one or more,” and “most.” It is noted that if Applicant amends the “at least some” language, said language would likely also need to be similarly amended in line 1 of claim 47.
Claims 47-65 are rejected based on their dependence on rejected claim 46.
Claim Interpretation
In instant claim 51, the phrase “terminal adapter region” is not defined by the instant specification. Thus, any region at the end of an adapter can be considered terminal, and therefore this region is considered inherent to the structure of an adapter.
In instant claim 61, step (iii) is not specific about how the merging of tagged unaligned and aligned reads should occur. In the instant specification, this appears to simply involve utilizing the UMI tags originally associated with the unaligned sequence reads with the aligned sequence reads before the consensus sequences are created (page 85, para. 2). Thus, any prior art which utilizes UMI tags and aligned sequence reads in the formation of a consensus sequence will be considered to meet this limitation.
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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
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 46-54 and 59-61 are rejected under 35 U.S.C. 103 as being unpatentable over Evans et al. (US 2017/0355984 A1) in view of Viailly et al. (BMC Bioinformatics, 2021).
Evans teaches methods of sequencing related to genetic variants using sequencing adaptors, with the goal of identifying sequencing errors (Abstract). The sample nucleic acid can be genomic DNA (paras. 187, 657, 661, and 666; instant claim 60). The sequencing method involves sequencing amplified first and second strands of a duplex nucleic acid molecule, where said molecule is ligated to a sequencing adapter prior to being amplified (paras. 9 and 20). The sequencing adapter is double-stranded, where each strand contains a molecular barcode region that is complementary to the corresponding region on the second strand (para. 138 and Figure 1A, characters 10 and 10’). Evans notes that the molecular barcode can be a unique molecular identifier (para. 5; instant claims 47 and 54). The sequencing adapter additionally comprises a duplex region on each strand, where the duplex regions are complementary to one another (para. 138 and Figure 1A, characters 20 and 20’). These would correspond to the yoke regions of the instant claims. Non-complementary regions (reference characters 30 and 30’) are also included on the adapters (instant claim 52). As noted above, terminal adapter regions are considered inherent to the structure of an adapter, and so would be included at each end of the sequencing adapter (instant claim 51). The number of unique molecular identifiers used can be at least 8 and not more than 48 (para. 140; instant claims 48-50). The edit distance between the two unique molecular identifiers can be 2 or more, which would equate to a Hamming distance of at least 2 (para. 141; instant claim 59).
In performing the method of Evans, consensus sequences can be generated using sets of strand reads, where the sets are compiled based on the similarity of their unique molecular identifiers (para. 194). When two sequencing adapters are used, the sequence identity of the read is also considered (para. 196). In this case, the sequences can also be aligned to a reference region to create a consensus sequence (paras. 187, 194, and 197). As shown in Figures 8-9, when compiling consensus reads, errors in single reads that are introduced during amplification can be noted and removed (paras. 202-203). Figures 9B-9C show further ways to note errors arising from non-natural means (i.e. not true variants), and Evans states, “If the error was a true variant in the original duplex nucleic acid, then both Strand 1 and Strand 1′ would include the variant. Comparing the consensus sequence for Strand 1 with the consensus sequence for Strand 1′ allows for identification of an error at that position, as only one of the consensus sequences include the error,” (para. 204). Thus, Evans provides a way to distinguish between errors generated during amplification and mutants. True variants can be identified with the method of Evans, such as single nucleic polymorphisms (para. 181). Example 4 of Evans also shows the detection of SNPs (para. 661-662).
Evans also teaches that at least some of their adapter sequences can contain overhangs, which would be a sequence between a UMI and a target sequence (e.g. paras. 22, 52, and Figure 2). These overhangs can exist on both the first and second strands of the duplex adaptor, can be complementary to one another, and are constant among the duplex nucleic acid molecule adaptors used (paras. 22, 142, and 185). Para. 124 notes that these overhangs generally contain 1-2 nucleotides, though there does not appear to be a strict length limit. This paragraph also notes that the overhangs can be directly ligated to the target nucleic acids. Para. 185 notes that the use of such overhangs promotes efficient ligation and limits ligation errors such as self-ligation, and so would be useful to include on the duplex nucleic acid molecules.
However, though Evans teaches that whole sequencing reads can be removed if they are thought to contain amplification or sequencing errors (e.g. para. 202), Evans does not teach trimming of the bases in each sequence read.
Regarding claims 46 and 61, Viailly teaches the use of unique molecular identifiers during NGS analyses related to copy number variations (Abstract). During their library construction, each genomic DNA target sequence was fragmented, end repaired, and then ligated with UMI adaptors before undergoing library preparation (page 4, “Sample collection and sequencing”). Figure 1 shows the resulting sequence read types – note that R2 fastq sequences contain a UMI, followed by the common sequence, followed by the sample DNA sequence. In processing the sequence reads, the common sequences were trimmed (meaning the UMI sequences were left intact) before the UMI sequences were extracted from the read constructions (page 4, “Library sequencing and bioinformatics pre-processing’). Figure S1 provides additional details regarding this process. Raw reads were first trimmed of common sequences using sequence comparison. UMIs were then extracted from reads and added to the end of each read name (thus acting as a tag). Then reads were aligned with a reference genome to produce Binary Alignment Map (BAM) files that are then used for CNV and tumor analysis (see Figure S1 caption and “Prerequisites” on page 5).
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use the processing methods detailed by Viailly in combination with the consensus read determinations of Evans. Firstly, Viailly teaches sequence reads that contain UMI sequences followed by a common sequence, which is similar in structure to the UMI followed by the constant overhang sequences of Evans. In Evans, the utility of the constant overhang sequences is stated to be in ensuring efficient and accurate ligation, and these sequences are not noted to be particularly useful in sequencing and subsequent analysis steps. In Viailly, the common sequences similarly do not serve a purpose during downstream methods, as they are removed before reads are tagged with extracted UMIs. The ordinary artisan would see the utility of this removal, namely so that upon UMI extraction, each sequence read is only labeled with the relevant and accurate UMI information, which would be particularly useful when filtering/deduplicating sequence reads, as is taught in Evans para. 240-241.
Additionally, both Evans and Viailly teach similar methods for organizing reads via UMIs. However, by trimming the common sequences and then extracting the UMIs, as is done in Viailly, and then later appending the UMIs onto their appropriate sequences, this also allows for the ignoring of errors that may occur in these trimmed/extracted sequences that are not relevant to the targeted sequencing results. MPEP 2143 I (A) states, “The rationale to support a conclusion that the claim would have been obvious is that all the claimed elements were known in the prior art and one skilled in the art could have combined the elements as claimed by known methods with no change in their respective functions, and the combination yielded nothing more than predictable results to one of ordinary skill in the art.” The processing methods of Viailly described above still allow for the identification of variants via sequencing methods, as sequencing reads are aligned/compared to a reference sequence and then further analyzed, and once these alignments are made, the consensus sequence determinations described by Evans could be performed. Thus, the combination of Evans in view of Viailly would still result in the formation of consensus sequences while identifying sequence errors that can occur during amplification and sequencing, thereby producing predictable results. There would also be a reasonable expectation of success for the ordinary artisan as combining these methods would simply add additional downstream analyses to the method of Evans that would not affect the adapter design or the amplification and sequencing methodologies of the reference.
Thus, claims 46-52, 54, and 59-61 are prima facie obvious over Evans in view of Viailly.
Regarding claim 53, Evans does not explicitly teach how long the non-barcode duplex regions of their adapters (i.e. the yoke regions) may be. However, the reference does generally teach that the barcodes may be of any length, and provides specific teachings of 2-24 bases in length (para. 143). In the provided examples, the molecular barcodes were 12-14 nucleotides in length (paras. 653-654 and 656). From these teachings, it would be prima facie obvious to make the yoke regions of the adapters around or shorter than 10-12 bases in length in the method of Evans in view of Viailly, depending on the length of the overhang region between the UMI and the sample nucleic acid, as this would still keep the entire duplex region under the taught length of 24 nucleotides, while also allowing for barcodes of a length typically taught in Evans. This length of barcode is shown by Evans to be successful (e.g. 653-655 and 656-658), and so the ordinary artisan would be motivated to use said length, and by making the yoke region of such a length as to keep the entire duplex region under the stated maximum for the barcode in particular embodiments, this can ensure that no adverse effects related to adapter length are seen in the method of Evans in view of Viailly.
Thus, claim 53 is prima facie obvious over Evans in view of Viailly.
Claims 55-58 and 62-65 are rejected under 35 U.S.C. 103 as being unpatentable over Evans et al. (US 2017/0355984 A1), in view of Viailly et al. (BMC Bioinformatics, 2021), and further in view of Zhao et al. (US 2019/0085384 A1).
Evans in view of Viailly teaches the methods of claims 46-54 and 59-61, as noted above. Evans also teaches that a variety of barcodes (UMIs) may be used (e.g. the 96 barcodes of para. 97 and Figure 3) and teaches that the balance of nucleotides among a given set of barcodes can be relatively equal (para. 148 and Figures 4-5). Evans also teaches the use of a large range of unique molecular identifiers, from 2 to 384 (para. 140). However, neither Evans nor Viailly explicitly teaches the specific UMIs shown in instant claims 55-58 and 62-65.
Zhao teaches methods for making and using UMIs to determine sequences of interest (Abstract). Zhao particularly focuses on non-random UMIs, which are predefined for a particular application, and rules can be used to generate particular UMI sequences, such as where the sequences can differ by a particular number of nucleotides (e.g. 2, 3, or 4; para. 74). Zhao teaches that UMIs can be 5 base pairs long (para. 324), and the references also provides examples of UMIs that are 6 and 7 nucleotides in length (paras. 74 and Table 4). Many of the sequences in Table 4 comprise the claimed UMI sequences – for example, CTAAGGA, AAGGATG, GACAAC, CCGATA, TCGTGTG, ATTGTCG, and CTTGGC, where the bolded portions show the claimed UMI sequences. Thus, Zhao explicitly teaches utilizing 6 of the UMIs recited in the instant claims. It is noted that the instant claims state the UMIs may comprise the listed sequences, and so can include additional nucleotides. Zhao also teaches elsewhere in the reference that UMI sequences may be 5-7 nucleotides in length (para. 77). Zhao further teaches additional selection criteria for the set of UMIs, including wherein the set of UMIs excludes sequences having three or more consecutive identical bases, excludes sequences having a combined number of guanine and cytosine bases smaller than 2 and larger than 4, excludes sequences having a same base at the last two positions, and excludes sequences having a thymine base at the last position (paras. 16-19). Zhao further teaches that these UMIs are configured to identify individual nucleic acid molecules in a sample for multiplex massively parallel sequencing (para. 4), and teaches that purposefully designed, non-random unique molecular identifiers allow for simpler manufacturing (para. 168).
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use the UMI criteria established by Zhao in the method of Evans in view of Viailly. Evans and Zhao teach very similar adapter structures (e.g. see Figure 1B of Zhao and Figure 1A of Evans) but Evans does not explicitly teach particular UMI sequences. Zhao teaches deliberate rules for creating UMIs that would lead to successful use by the ordinary artisan, and so the ordinary artisan would be motivated to use the design criteria described by this reference. MPEP 2143 I (B) states, “The rationale to support a conclusion that the claim would have been obvious is that the substitution of one known element for another yields predictable results to one of ordinary skill in the art.” Substituting the UMI structure of Zhao for that of Evans in view of Viailly would yield predictable results, as said result would simply be known UMI sequences that could be used for grouping sequence reads, as is done in Evans in view of Viailly. As noted above, Zhao teaches the use of 6 of the specifically claimed UMI sequences. A UMI with 5 nucleotides would have a possible 1,024 combinations, a UMI with 6 nucleotides would have a possible 4,096 combinations, and a UMI with 7 nucleotides would have a possible 16,384 combinations (utilizing the logic from Zhao para. 74). Using all or some of the selection criteria of Zhao would further limit the number of possible UMIs that can be used. As there are a finite number of UMIs that can be created, it would be obvious to one of ordinary skill in the art to develop other sequences beyond the ones explicitly enumerated by Zhao and to arrive at the other sequences listed in instant claims 55-58 and 62-65 that are not explicitly taught by Zhao - this would be a simple substitution of one UMI for another. It is also noted that no evidence of critical or unexpected results for the use of a particular UMI sequence has been described by Applicant. Additionally, it would have been obvious to one of ordinary skill in the art to select a set of unique molecular identifiers as taught by Zhao, as Zhao teaches that purposefully designed non-random UMIs allow for simpler manufacturing. Ten or more unique molecular identifiers could be used in this set, in accordance with the guidance provided by Evans.
Thus, claims 55-58 and 62-65 are prima facie obvious over Evans, in view of Viailly, and further in view of Zhao.
Conclusion
No claims are currently allowable.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to FRANCESCA F GIAMMONA whose telephone number is (571)270-0595. The examiner can normally be reached M-Th, 7-5pm.
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/FRANCESCA FILIPPA GIAMMONA/Examiner, Art Unit 1681
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