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 on July 14, 2026 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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 July 14, 2026 has been entered.
Status of Claims / Response to Amendment
This office action is in response to an amendment filed on July 14, 2026.
Claims 1, 5-8, 13-22, 24 and 26-28 were previously pending. Applicant cancelled claims 20-21; added new claims 29-32.
Claims 1, 5-8, 13-19, 22, 24 and 26-32 are currently pending and under consideration.
No rejection has been overcome by amendment. 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 -- Updated in View of Amendment
For the instant claims 1, 5-8, 13-19, 22, 24 and 26-32 in this application, the applicant claims priority of US provisional application NO. 61/915,435, which has a filling date on 12/12/2013.
Response to Arguments
Applicant's arguments filed on July 14, 2026 have been fully considered.
Claim Rejections - 35 USC § 102
In the prior Office Action (Final Office Action- 04/14/2026):
Claims 1, 7, 13-14, 16-18, 20-22, 24, 26 and 28 are rejected under 35 U.S.C. 102(a)(2) as being anticipated by Zhang (WO2014043140A1).
These rejections are maintained 1 in this Office Action for reasons below.
Applicant argues that the rejections above should be withdrawn (Remarks, page 5-7). Applicant's arguments have been fully considered but are not found persuasive.
The limitation at issue is "providing amplicons of a sample nucleic acid molecule in a first pool and a second pool."
Applicant asserts that "Zhang does not disclose providing amplicons of a sample nucleic acid molecule, such as amplicon products of an artificial amplification event, in first and second pools" and it instead teaches separating the strands of a sample molecule (chromosomal DNA from a cell) into separate amplification chambers. (Remarks, pp. 5-6).
This argument is not persuasive. As discussed in the claim interpretation and reiterated here, under BRI, "amplicon" encompasses single strands of genomic DNA that are products of natural DNA replication. Applicant does not dispute this interpretation in the remarks.
The term "sample nucleic acid molecule" is not defined in the claim or the disclosure with any feature that distinguishes it " from any nucleic acid in the art. Therefore, under BRI the term "sample nucleic acid molecule" encompasses any nucleic acid molecule, including genomic DNA copied during DNA replication. Further, the claimed method does not require the sample nucleic acid itself being provided. The limitation only requires providing amplicons, in other words, copies that derived from a sample nucleic acid molecule.
Accordingly, the cellular genomic DNA strands in Zhang qualify as "amplicons of a sample nucleic acid molecule" because they are copies generated from a DNA template in DNA replication.
Applicant is also reminded that claim 1 does not recite an "artificial amplification event," nor does the claim require any step of generating the recited amplicons from the sample nucleic acid molecule. The step of "providing" is broad and recited at a high level of generality. For example, "providing" could encompass placing a 96-well plate comprising pools of amplicons on a lab bench.
Zhang's teaching of providing genomic DNA strands in separate pools (Fig. 4 ; Claim 1; [0054], “evenly distribute single-stranded DNA molecules to 24 amplification chambers for independent amplification”; [0067]) therefore fully meets the limitation of "providing amplicons of a sample nucleic acid molecule in a first pool and a second pool."
Claim Rejections - 35 USC § 103
In the prior Office Action (Final Office Action- 04/14/2026):
Claims 5-6, 8, 15, 19 and 27 are rejected under 35 U.S.C. 103 as being unpatentable over Zhang (WO2014043140A1), in view of Samuels (WO2012112804A1).
Regarding the prior rejections under 35 USC § 103, Applicant does not provide any additional basis for traversing the rejection beyond the same argument made against Zhang above. For the same reasons discussed above, these arguments are not persuasive against the § 103 rejection.
For the reasons above, Applicant's arguments are unpersuasive. Accordingly, the rejections are maintained.
Claim Interpretation -- Updated in View of Amendment
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, claim 1 recites a "sample nucleic acid molecule," without further define this term. The application's disclosure does not define a "sample nucleic acid molecule" with any structural feature that could distinguish this term from any nucleic acid molecule known in the art.
Therefore, under BRI, "sample nucleic acid molecule" is interpreted to encompass any nucleic acid molecule, as any nucleic acid molecule can be present in, or obtained from, a sample.
For the purpose of applying prior art, newly added claim 31 recites the term "drop track," which is not expressly defined in the application's disclosure by any required structural features. FIG. 5 of the specification illustrates an embodiment of a drop track, which is a fluid channel that allows droplets to flow within:
"Figure 5 illustrates an embodiment in which a drop track 208 is used in conjunction with electrodes 207 to facilitate merging of a portion of the second fluid 205 with the droplet 201." (p. 25, lines 3-4)
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Accordingly, in light of the specification and under BRI, a "drop track" is interpreted as a fluid channel capable of flowing droplets.
For the purpose applying prior art, claim 1 and its dependent claims recite the term "amplicon," which is not expressly defined in the application's disclosure.
The specification describes amplicon as products of amplification (page 28, lines 30-31 to page 29, line 1):
“The term “amplification” as used herein generally refers to the production of substantially identical copies of a nucleic acid sequence (typically referred to as “amplicons”).”
According to the specification, amplification encompasses linear amplification using only one of forward or reverse primer in a pool, thereby generating single stranded products. Alternatively, both forward and reverse primers are present in the same pool, thereby generating double-stranded products:
"Embodiments of the invention may involve splitting the sample into two or more pools and using linear amplification and primers to create forward and reverse strand products of a nucleic acid comprising a target locus. In one embodiment only one of either the forward or the reverse primer for a given loci in a given pool, but in some cases it may be advantageous to have both primers present in one or more pools in equal or asymmetric abundances." (page 10, lines 11-16)
"In the described embodiments, a linear amplification reaction is carried out in each pool by, for example, using only a one member of a pair of primer species so that a first strand product is produced but no copies of the first strand product are produced. Linear amplification is well known in the art, an example of which may be found in “DNA linear amplification,” Chih Long Liu, Bradley E. Bernstein and Stuart L. Schreiber, Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford St., Cambridge, Mass., 02138, USA." (page 15, lines 24-29)
Thus, in light of the specification, the term "amplicon" encompasses both single-stranded amplification products and double-stranded amplification products.
The common meaning for this term aligns with interpretation from the specification:
"An amplicon is a piece of DNA formed as the product of natural or artificial amplification events. For example, it can be formed via polymerase chain reactions (PCR) or ligase chain reactions (LCR), as well as by natural gene duplication." See Wikipedia (Amplicon - Wikipedia; Archived Feb 28, 2012 on WaybackMachine).
Thus, under BRI and in light of the specification, the term "amplicon" is understood to encompass single stranded and double-stranded products of both natural or artificial amplification events, including genomic DNAs, which are products of natural DNA replication.
Regarding all claims, terms such as "first" and "second" are interpreted as adjectives for identification purposes to distinguish between repeated instances of an element or limitation, and do not impose any additional features, such as any specific temporal limitation, any sequential order of steps, or any structural or composition differences. See 3M Innovative Props. Co. v. Avery Dennison Corp., 350 F.3d 1365 (Fed. Cir. 2003)2.
For the purpose of applying prior art, claim 1 recites "first unique sequence tag" and "second unique sequence tag," which are terms not expressly defined by the application's disclosure. The specification does not provide any definition for the term "unique sequence tag" or any description of structural features, that can distinguish this term from any sequence known in the art.
Thus, under BRI, the term "unique sequence tag" is interpreted to encompass any sequence, such as a barcode sequence.
For the purpose of applying prior art, claim 8 recites "wherein the forward and reverse strand amplicons further comprise a pool identification tag and a first universal portion."
The application's disclosure does not define the terms "pool identification tag" and "first universal portion" with any structural feature or characteristics that could distinguish these terms from any sequences or molecular biology assay elements, that can be comprised by "forward and reverse strand amplicons" (interpreted under BRI as any nucleic acids).
Therefore, under BRI, the terms "pool identification tag" and "first universal portion" are interpreted to encompass any nucleic acid sequences or elements that can be comprised by or attached to a nucleic acid molecule (e.g. biotin tag, linker, etc. ).
For the purpose of applying prior art, claim 1 recites:
"conducting a first amplification reaction in the first pool to enrich for forward strand amplicons" and
"conducting a second amplification reaction in the second pool to enrich for reverse strand amplicons."
The application's disclosure does not expressly define the term "enrich." Thus, under BRI and based on the commonly understood meaning by those skilled in the art, the term "enrich" is understood to encompass any approach that increases the presence of targets of interest in a sample. This can be achieved by increase the number of targets, such as through target amplification 3; isolating targets from non-targets, such as via hybridization capture 2; or depleting non-targets to reduce background 4.
Maintained 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)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 1, 7, 13-14, 16-18, 22, 24, 26 and 28 are rejected under 35 U.S.C. 102(a)(2) as being anticipated by Zhang (WO2014043140A1- Accurate genome sequencing of single cells by single-stranded amplification and sequencing ; effective filing date September 10, 2013).
Regarding claim 1, Zhang teaches a method comprising the steps of:
providing amplicons of a sample nucleic acid molecule in a first pool and a second pool (Claim 1; FIG. 4; [0054], “evenly distribute single-stranded DNA molecules to 24 amplification
chambers for independent amplification”; [0067]);
conducting a first amplification reaction in the first pool to enrich for forward strand amplicons comprising a first unique sequence tag without enriching for reverse strand amplicons in the first pool (Claim 1; FIG. 4; [0054], [0067]; [0037] complementary strands are placed in separate solutions for independent amplification, thus the solution comprising forward strand of a DNA would not comprise the reverse strand and does not amplify the reverse strand);
conducting a second amplification reaction in the second pool to enrich for reverse strand amplicons comprising a second unique sequence tag without enriching for forward strand amplicons in the second pool (Claim 1; FIG. 4; [0054], [0067]; [0037]);
sequencing the forward strand amplicons to produce forward strand sequence reads and sequencing the reverse strand amplicons to produce reverse strand sequence reads (Claim 1; FIG. 4; [0054], [0067]; [0037]);
finding a variant in the forward strand sequence reads and the reverse strand sequence reads ([0028] “the mutations are called only when sequencing data from
the two strands are consistent… The concept of obtaining sequence information from both the Watson and Crick strands of a DNA molecule was commonly used”); and
identifying the variant as a true variant ([0026]; [0028]).
Regarding claim 7, Zhang teaches the forward and reverse strand amplicons are produced by a polymerase extension reaction ([0054] multiple displacement amplification).
Regarding claim 13, Zhang teaches variant is associated with a disease ([0057]).
Regarding claim 14, Zhang teaches the disease is cancer ([0057]).
Regarding claim 16, Zhang teaches identifying a second variant as a false variant when the second variant is found among one of, but not the other of, the forward strand sequence reads and the reverse strand sequence reads ([0026] lines 5-8; [0043]).
Regarding claim 17, Zhang teaches the second variant is an amplification or sequencing error ([0043]).
Regarding claims 18 and 24, they are anticipated by Zhang because they do not further limit the claimed method.
Claim 18 recites :"wherein the sample nucleic acid molecule is derived from formalin-fixed, paraffin-embedded tissue."
Claim 24 recites: "wherein the nucleic acid molecule is from a blood sample."
Per MPEP 2111.04, a wherein clause can limit a method claim if it contributes meaning and purpose to the manipulative steps. In this instant case, the wherein clauses do not limit the method claim because the base claim 1 does not recite any step of obtaining or deriving nucleic acid molecules. As such, this clause merely describes a source of the nucleic acid molecules, without modifying any step of the claimed method. Therefore, these claim languages are descriptive statements without any associated active steps and do not distinguish the claims from the prior art.
Regarding claim 22, Zhang teaches the sequencing is sequencing-by- synthesis ([0042] Illumina™ sequencing,).
Regarding claim 26, Zhang teaches the first unique sequence tag identifies the forward strand sequence reads and the second unique sequence tag identifies the reverse strand sequence reads (([0067] line 16).
Regarding claim 28, Zhang teaches the forward strand amplicons comprise first double-stranded nucleic acids and wherein the reverse strand amplicons comprise second double-stranded nucleic acids ([0059] lines 1-3; [0041]).
Maintained 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.
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 5-6, 8, 15, 19 and 27 are rejected under 35 U.S.C. 103 as being unpatentable over Zhang (WO2014043140A1- Accurate genome sequencing of single cells by single-stranded amplification and sequencing ; effective filing date September 10, 2013), in view of Samuels (WO2012112804A1- Compositions and methods for molecular labeling; Published August 23 2012).
A) The teachings of Zhang are recited above and applied as for base claim 1.
Zhang teaches methods for strand-specific amplification and sequencing in microfluidic reactors, in which two complementary strands of a double-stranded nucleic acid are separated for independent amplification and sequencing, thereby enabling discrimination of true mutations from in vitro amplification errors (e.g., [0028]).
Regarding claim 8, Zhang teaches that, following strand-specific amplification, sequencing libraries are prepared for Illumina sequencing, including the addition of pool identification tags ([0067] line 16) to the strand-specific amplicons. Although Zhang does not explicitly describe library preparation in detail, such as further reciting a first universal portion, this feature is implicit in the context of sequencing library generation, which routinely requires the incorporation of sequencing adaptors comprising common sequences shared across all library fragments, this is supported by Samuels.
Samuels teaches methods for analyzing nucleic acids by compartmentalizing the nucleic acid amplicons (e.g., droplet encapsulation) with barcode libraries, followed by analysis approaches such as sequencing (entire document; summary of the invention; FIGs 13-14 for examples).
Samuels teaches that sequencing library preparation protocols, including the addition of adaptors, are well known in the art:
"[S]equencing library preparation protocols well known to those skilled in the art. For example, the amplified genetic material can be sheared/fragmented using methods well known to those of ordinary skill in the art, and adaptors can be ligated onto the ends of the fragments to be utilized, for example in direct sequencing, or in an enrichment process." (page 56, para 3, lines 4-7)
Samuels further teaches the use of a universal barcode library that can be combined with sequencing platform adaptor libraries, such as amplification products directly incorporated into the workflow of any given sequencing platform, such as Illumina. This approach can minimize PCR bias:
"A barcode library can also be made to include a sticky-end adapter specific for a sequencing platform. In certain embodiments, a construct is made that includes a sequencing platform N-mer and a sticky-end N-mer. A library of these constructs can be made. Separately, a universal barcode library as discussed above can be made. The, the universal barcode library can be combined with the sequencing platform adapter library by means of the sticky ends in view of a particular application. Thus products of any analysis discussed herein can be adapted to go directly into the workflow of any given sequencing platform (e.g. sticky-ended Illumina adaptors to anneal/ligate onto either the primer library or the output from a targeted sequencing run, so that it could be hybridized directly onto their flow cell. A different sticky-end adaptor set could be used for 454, etc.). This approach can minimize PCR bias." (page 46, para 2)
Accordingly, a skilled artisan would have found it prima facie obvious before the effective filing date of the claimed invention to apply Samuels's teaching of utilizing a universal barcode library to the library preparation step of Zhang, to allow direct compatibility with Illumina sequencing platform and to reduce PCR bias, as suggested by Samuels.
The person of ordinary skill would have had a reasonable expectation of success in combining these teachings because the references are technically compatible and provide complementary teachings in the field of sequencing. Both references teach Illumina sequencing. Specifically, Samuels teaches an improved approach for generating sequencing libraries for Illumina platform, which is used in Zhang. Samuels further teaches that the use of a universal barcode library minimizes PCR bias, which directly addresses a known issue identified in Zhang ꟷ namely, that "false positives generated by polymerase errors alone greatly outnumber the true mutations" ([0019]). Zhang teaches PCR amplification in library generation ([0060]). Thus, a skilled artisan would understand that Samuels's approach that reduces polymerase chain reaction (PCR) bias would have been desirable and applicable in Zhang's methods utilizing DNA polymerase in PCR.
Regarding claims 5-6, they are obvious as Zhang teaches "a variety of amplification techniques can be used" in its methods ([0041]), and Samuels teaches exponential amplification comprises PCR (Figure 14, PCR).
Regarding claim 15, Samuels teaches the forward and reverse strand amplicons further comprise a sequencing adaptor (page 56, para 3, lines 3-7).
Regarding claim 19, Samuels teaches the amplicons are produced by linear amplification (page 55, para 4, lines6-9).
Regarding claim 27, Samuels teaches the first unique sequence tag and the second unique sequence tag identify the forward and reverse strand sequence reads as corresponding to the sample nucleic acid molecule (page 52, para 2, lines 6-9; FIG 43).
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.
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 1, 5-8, 13-19, 22, 24, 26-32 are rejected under 35 U.S.C. 103 as being unpatentable over Samuels (WO2012112804A1- Compositions and methods for molecular labeling; Published August 23 2012), in view of
Kirsch (S. Kirsch, & C.A. Klein, Sequence error storms and the landscape of mutations in cancer, Proc. Natl. Acad. Sci. U.S.A. 109 (36) 14289-14290, doi.org/10.1073/pnas.1212246109 (2012));
Meacham (Meacham et al. Identification and correction of systematic error in high-throughput sequence data. BMC Bioinformatics 12, 451 (2011). doi.org/10.1186/1471-2105-12-451); and
Kurn (US20110105364A1 - Compositions and methods for targeted nucleic acid sequence selection and amplification ; Published on 2011-05-05).
A) Regarding claim 1, the claimed method represents an obvious combination of a known genomic variant identification sequencing method ( disclosed in Samuels), with known strand-specific sequencing data analysis approach ( disclosed in Kirsch and Meacham), and a known strand-specific amplification approach ( disclosed in Kurn).
Samuels teaches methods for analyzing biological samples by compartmentalizing samples (e.g., droplet encapsulation) with barcode libraries, followed by analysis approaches such as sequencing (see summary of the invention) for identifying real variant (page 70-71; Fig. 43).
Regarding claim 1, Samuels teaches a method comprising the steps of:
providing amplicons of a sample nucleic acid molecule in a first pool and a second pool (Figure 13, shows a first pool and second pool for amplification; page 11“Figure 13 shows barcode addition for amplification-based single molecule haplotyping with universal PCR barcodes.”; page 47, lines 16-18, “the target locus can be pre-amplificated using a single pair of PCR primers that flanks the entire locus, before appropriate loading of the sample into droplets for amplification and barcoding”);
conducting a first amplification reaction in the first pool to enrich for forward strand amplicons comprising a first unique sequence tag (Figure 13-14; ; page 52, para 2, lines 1-3; unique barcode: page 52, para 2, lines 6-9; page 41, para 2; page 51, para 3);
conducting a second amplification reaction in the second pool to enrich for reverse strand amplicons comprising a second unique sequence tag (Figure 13-14; ; page 52, para 2, lines 1-3; unique barcode: page 52, para 2, lines 6-9; page 41, para 2; page 51, para 3);
sequencing the forward strand amplicons to produce forward strand sequence reads and sequencing the reverse strand amplicons to produce reverse strand sequence reads (page 52, para 2, lines 6-7; page 11, lines 3-4 “schematic depicting an example of barcode labeled strands in a droplet before sequencing (in droplet) and after sequencing (in bulk)”; page 47, para 4, lines 4-5, barcode that will enable post-sequencing correlation to the target strand);
identifying the variant as a true variant (page 70-71; Fig. 43).
Regarding the limitation “finding a variant in the forward strand sequence reads and the reverse strand sequence reads,” Samuels teaches strand-specific labeling of nucleic acids for sequencing (page 52, para 2, lines 6-7; page 11, lines 3-4 “schematic depicting an example of barcode labeled strands in a droplet before sequencing (in droplet) and after sequencing (in bulk)”; page 47, para 4, lines 4-5, barcode that will enable post-sequencing correlation to the target strand); it also teaches identifying a true variant (page 70-71; Fig. 43 ) in nucleic acid sequencing, particularly by enabling the discrimination between true SNPs and sequencing errors.
Specifically, Samuels teaches using barcodes on differently labeled molecules to determine whether a detected base change is real or an artifact of some kind. For instance, on the same strand of the same molecule (e.g., top strand of Fragment A in Fig. 43), the nucleotide base at a specific position should be the same (e.g., base “C” at position X). Therefore, if reads with the same molecule barcode presents a small fractions of reads with different nucleotide base at a specific position, it is likely not a true variant but rather a technical error.
Thus, Samuels teaches identifying the variant as a true variant (e.g., SNP) via nucleic acid molecule-specific barcoding in a sequencing method, it teaches distinguishing reads between nucleic acid fragments of the same molecule, which could comprise both strands, and does not explicitly teach specifically investigating a variant in both the forward and reverse stands. However, this feature would have been obvious in view of the knowledge in the prior art, as supported by Kirsch and Meacham.
Kirsch, a review article titled “Sequence error storms and the landscape of mutations in cancer” teaches that in determining whether a variant represents a true biological mutation or an amplification error, the approach of comparing the presence of a variant on both DNA strands is well-established in the art (see FIG. 1). This is based on the principle that the true mutation occurs on both DNA strands, thus by comparing both the forward and reverse strands, only the bases that are present on both strands are identified as true mutation (see FIG. 1, DCS).
Based on this principle, Meacham teaches an improved approach for the identification and correction of systematic errors in sequencing data analysis, specifically applicable to the identification of SNPs (entire document, Abstract for example).
Meacham teaches finding a variant in the forward strand sequence reads and the reverse strand sequence reads; and identifying the variant as a true variant (page 3, left-hand col, lines 11-17).
Meacham specifically highlights types of errors in sequencing (Figure 1), including systemic error that affects SNP calling methods as this type of error escapes traditional error identification approaches and cannot be distinguished from true SNPs (page 2).
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Meacham then teaches a solution to address the false-positive problem in SNP calling, by comparing sequencing reads from both strand directions:
"Due to the small fragment size in methyl-Seq experiments many of the mate-pair reads overlapped, providing for each such location two base calls sequenced from the same DNA molecule (Figure 1) albeit from different directions. We made use of this to distinguish between base-call errors and true heterozygosity calls in the following manner: each pair of bases originating from a single mate-pair and sequencing the same position was denoted a reference-pair if both calls agreed with the reference genome, a SNP-pair if both calls disagreed with the reference genome and agreed among themselves, and an error-pair if one of the calls agreed with the reference genome but the other did not." (page 3, left-hand col)
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 combine the nucleic acid sequencing method with strand-specific labeling for SNP analysis, taught by Samuels, with the teachings of Kirsch and Meacham regarding the improved method to more accurately identity true variants through strand-based comparison of sequencing reads.
All three references are in the same or overlapping fields of sequencing-based variant detection and provide complementary teachings related to variant identification.
Samuels teaches sequencing methods for variant (e.g. SNP) identification with labeling schemes that preserve strand-specific information, while Kirsch and Meacham suggest improvement in sequencing data analysis for identifying systematic errors in variant calling by comparing the two strands at the same genomic location, thereby improve variant calling accuracy. Therefore, a skilled artisan would have been motivated to apply the strand-comparing analysis method in Kirsch and Meacham to the strand-specific sequencing reads generated using Samuels's teaching as a logical extension to reduce false positives in SNP calling, as suggested by Meacham.
The person of ordinary skill would have had a reasonable expectation of success in combining these teachings because they are technically compatible and address the same objective ꟷ accurate variant identification from sequencing data. Samuels already enables strand-resolved analysis by preserving strand-specific information in sequencing reads, which supports the strand-based variant comparison taught by Kirsch and Meacham. Meacham teaches its data analysis approaches in detail, further supporting the feasibility of such combination. Moreover, both references describe sequencing workflows that are well-established in the field (e.g. Illumina sequencing). Doing so would have yielded the predictable result of improved SNP calling accuracy by identifying and filtering out false variants that exhibit strand-specific bias, as suggested by Meacham.
Additionally, this combination would have been obvious as it represents the KSR principle of predictable use of prior art elements (i.e., strand-based variant comparison for error identification of Kirsch and Meacham) according to a known method (i.e., strand-specific sequencing of Samuels) to yield predictable results (i.e., more accurate SNP calling). (See MPEP §2143).
Regarding the limitations:
“conducting a first amplification reaction in the first pool to enrich for forward strand amplicons comprising a first unique sequence tag without enriching for reverse strand amplicons in the first pool;”
conducting a second amplification reaction in the second pool to enrich for reverse strand amplicons comprising a second unique sequence tag without enriching for forward strand amplicons in the second pool,"
Samuels teaches enriching forward/reverse strand amplicons in a first/second pool by utilizing PCR amplification with both a reverse and forward primer in each pool (Figure 13; page 52, para 1). Although the combined teaching of Samuels, Kirsch and Meacham do not explicitly teach generating amplicons of one strand without producing amplicons of the other strand in each of two pools, this feature would have been obvious in view of Kurn.
Kurn teaches methods for preparing target polynucleotides in a strand-specific manner for sequencing (entire document; [0098] for example), and highlights that its methods are useful for preparing target polynucleotides for sequencing while performing amplifications in droplets ([0094] for example).
Kurn discloses a linear amplification method referred to as single primer isothermal amplification (SPIA) ([0073]; [0120]; Figure 4), which uses a single primer to enable generation of multiple copies of a strand-specific sequence region of interest without producing amplicons of the opposite strand. A skilled artisan would readily appreciate that in a strand-specific SPIA reaction using a single primer for linear amplification, only the specific strand of interest will be amplified, with no copies from the opposite strand.
Kurn further highlights the benefits of SPIA, such as reduced complexity in oligonucleotide design and manufacturing and desirable fidelity of copy number quantification ([0073]).
Accordingly, 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 modify the combined teachings of Samuels, Kirsch and Meacham ꟷ wherein strand-specific nucleic acid sequencing is used for accurate SNP identification ꟷ by performing strand-specific amplification of forward and reverse strands in separate pools using SPIA with a single primer as taught by Kurn, without generating copies of the opposite strand in each pool, instead of using PCR amplification with both primers as in Samuels.
All of the references are in the same field of nucleic acid assays, specifically sequencing methods. Samuels teaches strand-specific sequencing and discloses that amplification in a pool (e.g., droplet) may be performed using either linear amplification or PCR (page 53, para 4, lines 6-9). Kurn teaches a specific linear amplification approach, SPIA, for strand-specific sequencing and notes that its method is useful for performing amplifications in droplets. Meacham and Kirsch teach data analysis approaches that require strand-specific sequencing reads.
Therefore, the person of ordinary skill would have had a reasonable expectation of success in combining the references because they contain overlapping and complementary teachings in strand-specific sequencing methods.
The skilled artisan would have been motivated to do so because Kurn highlights benefits of SPIA, such as reduced complexity and providing desirable fidelity.
Such modification would have been an obvious substitution of one known element (i.e., linear SPIA amplification) for another (i.e., PCR amplification) to obtain predictable results (i.e., strand-specific sequencing results), consistent with the principles set forth in KSR. See MPEP 2141.
B) Regarding claims 5-6, Samuels teaches exponential amplification comprises PCR (Figure 14, PCR).
Regarding claim 7, Samuels teaches the forward and reverse strand products are produced by a polymerase extension reaction (Figure 12; page 51-52).
Regarding claim 8, Samuels teaches the forward and reverse strand products further comprise a pool identification tag (page 52, para 2, lines 6-9; page 41, para 2; page 51, para 3; page 48, para 2, as the forward and reverse primer each has barcode, this unique pair of barcodes labels the pool or droplet) and a first universal portion (Figure 14, “universal ends”; Page 8, para 3, unique N-mer and functional N-mer, functional N-mer comprise: sequence specific primers, random N-mer (pool identification), universal primer ; page 4, para 1).
Regarding claim 13, Samuels teaches the variant is associated with a disease (page 62, para 3, disease-associated copy number variation).
Regarding claim 14, Samuels teaches the disease is cancer (page 70, para 2).
Regarding claim 15, Samuels teaches incorporating sequencing adaptors into the forward and reverse strand amplicons (page 70, lines1-2, page 69, para 4).
Regarding claim 16, Meacham teaches identifying a variant as a false variant when the variant is found among one of, but not the other of, sequence reads from the forward strand and sequence reads from the reverse strand (Figure 1, systematic error).
Regarding claim 17, Meacham teaches wherein the second variant is an amplification or sequencing error (Figure 1) .
Regarding claim 18, it is obvious over the combined teachings of Samuels, Meacham, Kirsch and Kurn because it does not further limit the claimed method.
Claim 18 recites :"wherein the sample nucleic acid molecule is derived from formalin-fixed, paraffin-embedded tissue."
Per MPEP 2111.04, a wherein clause can limit a method claim if it contributes meaning and purpose to the manipulative steps. In this instant case, the wherein clause does not limit the method claim because the base claim 1 does not recite any step of obtaining or deriving nucleic acid molecules. As such, this clause merely describes a source of the nucleic acid molecules, without modifying any step of the claimed method. Therefore, this claim language is interpreted as descriptive statement without any associated active steps and do not distinguish the claims from the prior art.
Regarding claim 19, Samuels teaches the amplicons are produced by linear amplification (page 55, para 4, lines6-9).
Regarding claim 22, Samuels teaches sequencing-by- synthesis (page 8, para 2, lines1-2).
Regarding claim 24, it is obvious over the combined teachings of Samuels, Meacham, Kirsch and Kurn because it does not further limit the claimed method.
Claim 24 recites: "wherein the nucleic acid molecule is from a blood sample."
Per MPEP 2111.04, a wherein clause can limit a method claim if it contributes meaning and purpose to the manipulative steps. In this instant case, the wherein clause does not limit the method claim because the base claim 1 does not recite any step of obtaining or deriving nucleic acid molecules. As such, this clause merely describes a source of the nucleic acid molecule, without modifying any step of the claimed method. Therefore, this claim language is descriptive statements without any associated active steps and do not distinguish the claim from the prior art.
Regarding claim 26, Samuels teaches the first unique sequence tag identifies the forward strand sequence reads and the second unique sequence tag identifies the reverse strand sequence reads (page 109, lines 3-6, barcodes that enable post-sequencing correlation to target strand).
Regarding claim 27, Samuels teaches the first unique sequence tag and the second unique sequence tag identify the forward and reverse strand sequence reads as corresponding to the sample nucleic acid molecule (page 52, para 2, lines 6-9; FIG 43).
Regarding claim 28, Kurn teaches the forward strand amplicons comprises first double-stranded nucleic acids and wherein the reverse strand amplicons comprise second double- stranded nucleic acids (Figure 4-7, illustrating strand-specific amplification of a forward strand comprising a double-stranded product. Thus a skilled artisan would have readily understand that amplification of the reverse strand would have yielded similar double-stranded product).
Regarding claim 29, Samuels teaches the first pool and the second pool are contained within a first droplet and a second droplet, respectively (Figure 13B).
Regarding claim 30, Samuels teaches after providing the droplets, adding reagents for amplification into the first droplet and the second droplet (page 3, lines 17-18 “Once formed, droplets containing the target material can be merged with droplets containing other reagents”).
Regarding claim 31, Samuels teaches adding the reagents is done using a drop track in conjunction with electrodes (p.22, lines 17-18 “After formation of the sample droplet from the first sample fluid, the droplet is contacted with a flow of a second sample fluid stream”; p.23, lines 4-5 “each incoming droplet of first sample fluid is merged with the same amount of second sample fluid”; p. 23., line 6, 8-10 “an electric charge is applied to the first or second sample fluids… Electric charge may be created in a sample fluid within the carrier fluid using any suitable technique, for example, by placing the first and second sample fluids within an electric field”; p.23, lines 22-26, “an electric field is produced by applying voltage across a pair of electrodes, which may be positioned on or embedded within the fluidic system (for example, within a substrate defining the channel or microfluidic channel), and/or positioned proximate the fluid such that at least a portion of the electric field interacts with the fluid.” ).
Regarding claim 32, Samuels teaches amplicons are products of an artificial amplification event (page 47, lines 16-18, “the target locus can be pre-amplificated using a single pair of PCR primers that flanks the entire locus, before appropriate loading of the sample into droplets for amplification and barcoding”)).
Prior Art
Below are relevant prior art not used in rejection but pertinent to the claims or disclosure.
Yurkovetsky (US20120219947A1 - Methods for forming mixed droplets; published 2012-08-30) also teaches a microfluidic droplet track that allows droplet merging and forces droplets into a “squashed” conformation, see [0043].
Conclusion
No claims are allowed.
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/TIAN NMN YU/Examiner , Art Unit 1681
1 Maintained except for cancelled claims 20-21.
2 Holding that "first pattern" and "second pattern" is equivalent to "Pattern A" and "Pattern B": The use of the terms "first" and "second" is a common patent-law convention to distinguish between repeated instances of an element or limitation. See, e.g., Anchor Wall Sys., Inc. v. Rockwood Retaining Walls, Inc., 340 F.3d 1298, 1304 (Fed. Cir. 2003) ("first and second sidewall surfaces"); Springs Window Fashions LP v. Novo Indus., L.P., 323 F.3d 989, 992 (Fed. Cir. 2003) ("first and second opposed ends"). In the context of claim 1, the use of the terms "first . . . pattern" and "second . . . pattern" is equivalent to a reference to "pattern A" and "pattern B," and should not in and of itself impose a serial or temporal limitation onto claim 1."
3 see Tewhey et al. Microdroplet-based PCR enrichment for large-scale targeted sequencing. Nat Biotechnol 27, 1025–1031 (2009), (introduction);
See also Ware et al., Next generation sequencing for clinical diagnostics and personalised medicine: implications for the next generation cardiologist. Heart. 2012 Feb;98(4):276-81. doi: 10.1136/heartjnl-2011-300742. Epub 2011 Nov 29. PMID: 22128206. (Fig 2)
4 see Feehery et al. A method for selectively enriching microbial DNA from contaminating vertebrate host DNA. PLoS One. 2013 Oct 28;8(10):e76096. doi: 10.1371/journal.pone.0076096. PMID: 24204593; PMCID: PMC3810253 (entire document).
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