DETAILED ACTION
CONTINUED EXAMINATION UNDER 37 CFR 1.114 AFTER FINAL REJECTION
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 of RCE and amendment filed on August 29, 2025 have been entered. The claims pending in this application are claims 22-35. Claims 22-35 will be examined.
Claim Objections
Claim 22 is objected to because of the following informalities: (1) “different sub-samples” in line 3 of step (c) should be “the different sub-samples”; (2) “tagged adaptor-ligated DNA fragments” in step (d) should be “the tagged adaptor-ligated DNA fragments”; (3) “the top strand/or the bottom strand” in (i) (2) of step (d) should be “the top strand or the bottom strand”; (4) “of step (d) (ii) (1)” in step (e) should be “in (ii) (1) of step (d)”; and (5) “of step (d) (ii) (2)” in step (e) should be “in (ii) (2) of step (d)”.
Claim 24 is objected to because of the following informality: “tagged adaptor-ligated DNA fragments” should be “the tagged adaptor-ligated DNA fragments”.
Claim 28 is objected to because of the following informalities: (1) “the tagged adaptor-ligated DNA fragments from each of the tagged sub-samples of step (c) each comprise” should be “each of the tagged adaptor-ligated DNA fragments from each of the tagged sub-samples of step (c) comprises”; and (2) “each sequencing read of step (d) (i) further comprises” should be “each of sequencing reads in (i) of step (d) further comprises”.
Claim 29 is objected to because of the following informalities: (1) “one or more of the sequence tags that identifies the sub-sample used in step (c)” should be “one or more of the sequence tags that identify the sub-sample used in step (c)”; (2) “the tagged adaptor-ligated DNA fragments from each of the tagged sub-samples of step (c) each comprise” should be “each of the tagged adaptor-ligated DNA fragments from each of the tagged sub-samples of step (c) comprises”; and (3) “each sequencing read of step (d) (i) further comprises” should be “each of sequencing reads in (i) of step (d) further comprises”.
Appropriate correction is required.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 22, 24, 28, and 30-35 are rejected under 35 U.S.C. 103 as being unpatentable over Pushkarev et al., (US 2013/0157870 A1, published on June 20, 2013) in view of So et al., (US 2014/0287937 A1, priority date: February 21, 2013).
Regarding claims 22, 24, 28, and 30-35, since the specification defines “asymmetric adaptor” as “an adaptor that, when ligated to both ends of a double stranded nucleic acid fragment, will lead to a top strand that contains a 5’ tag sequence that is not the same as or complementary to the tag sequence at the 3’ end. Exemplary asymmetric adapters are described in: U.S. Pat. Nos. 5,712,126 and 6,372,434 and WO/2009/032167; all of which are incorporated by reference herein in their entirety. An asymmetrically tagged fragment can be amplified by two primers: one that hybridizes to a first tag sequence added to the 3’ end of a strand, and another that hybridizes to the complement of a second tag sequence added to the 5’ end of a strand. Y-adaptors and hairpin adaptors (which can be cleaved, after ligation, to produce a ‘Y-adaptor’) are examples of asymmetric adaptors” (see paragraph [0053] of US 2023/0065345 A1, which is US Publication of this instant case) and defines “minority variant” as “a variant that is present a frequency of less than 50%, relative to other molecules in the sample. In some cases, a minority variant may be a first allele of a polymorphic target sequence, where, in a sample, the ratio of molecules that contain the first allele of the polymorphic target sequence compared to molecules that contain other alleles of the polymorphic target sequence is 1:100 or less, 1:1,000 or less, 1:10,000 or less, 1:100,000 or less or 1:1,000,000 or less” (see paragraph [0073] of US 2023/0065345 A1, which is US Publication of this instant case ), Pushkarev et al., teach a method for determining, in the following order, if a potential sequence variation is in the top and bottom strands of the same DNA fragment, comprising: (a) ligating asymmetric adaptors (ie., hairpin adapters) to the 5’ end and the 3’ end of DNA fragments in a sample, thereby producing an adaptor-ligated sample; (b) splitting the adaptor-ligated sample of step (a) (eg., by partitioning) into a plurality of sub-samples, wherein each of the subsamples is placed in one container (ie., one well of a 96 well plate) and the different sub-samples are placed in separate containers (ie., different wells of one or two 96 well plates); (c) separately tagging adaptor-ligated DNA fragments of each of the different sub-samples of step (b) with one of a plurality of sequence tags that identify the tagging adaptor-ligated DNA fragments of the different sub-samples (ie., sequencing adapters unique to each well of two 96 well plates) of step (b) by polymerase chain reaction (PCR) using primers that have a 5’ tail that has a sub-sample identifier sequence (eg., A-primer-A which contains a barcode, see Figure 5A and paragraphs [0086] and [0087]), thereby producing tagged sub-samples; and (d) sequencing the tagged adaptor-ligated DNA fragments from each of the tagged sub-samples of step (c), or copies of the same, to produce sequence reads, wherein (i) each of the sequence reads comprises: (1) one of the plurality of sequence tags that identify the different sub-samples (ie., the barcode from A-primer-A) and (2) the sequence of at least part of the top strand or the bottom strand of a DNA fragment of the tagged adaptor-ligated DNA fragments from one of the sub-samples, (ii) the sequence reads comprises: (1) a plurality of sequence reads derived from the top strand of a DNA fragment of the tagged adaptor-ligated DNA fragments from one of the sub-samples, and
(2) a different plurality of sequence reads derived from the bottom strand of the seme DNA freemen (ii)(1) of step (d) (eg., the sequencing comprises obtaining paired end reads which read
two DNA sequences generated from opposite ends of the same fragmented DNA molecule) as recited in claim 22 wherein the method comprises: pooling the tagged sub-samples of step (c) prior to step (d) to produce a pooled sample, and wherein step (d) comprises sequencing the tagged adaptor-ligated DNA fragments of the pooled sample as recited in claim 24, the asymmetric adaptor of step (a) comprises a sample identifier sequence (eg., one or more identification elements, see paragraph [0024]) that identifies the sample to which the asymmetric adaptor is added; each of the tagged adaptor-ligated DNA fragments from each of the tagged sub-samples of step (c) comprises one of the sequence tags that identify the sub-samples and the sample identifier sequence; and each of sequencing reads in (i) of step (d) further comprises: the sample identifier sequence as recited claim 28, in step (b) the adaptor-ligated sample is split into at least 4 sub-samples (eg., in two 96 well plates) as recited in claim 30, the sample of step (a) comprises fragments of human genomic DNA as recited in claim 31, the sample of step (a) is obtained from a cancer patient as recited in claim 32, further identifying a minority variant sequence in the sequence reads (eg., identifying a polymorphism with a frequency greater than 1% or 10% or 20%) as recited in claim 33, the minority variant is a somatic mutation as recited in claim 34, and the PCR of step (c) is composed of 4 to 20 cycles (eg., see paragraph [0055]) as recited in claim 35 (see paragraphs [0007] to [0011], [0013], [0024], [0026], [0046], [0047], [0055], [0057], [0058], [0063], [0074], [0085] to [0087], [0107], [0113], [0114], [0116], [0131] to [0133], [0146], [0150], [0154], [0162], [0163], [0181], [0182], and [0227] to [0229], and Figures 1 and 5A).
Pushkarev et al., do not disclose that the 5’ tail of the primers does not hybridize to the adaptor-ligated sample, identifying a potential sequence variation in the plurality of sequence reads in (ii) (1) of step(d), and determining if the potential sequence variation is in the plurality of sequence reads in (ii) (2) of step (d) as recited in steps (c), (e), and (f) of claim 22. However, Pushkarev et al., teach that “the sequencing comprises obtaining paired end reads”, “[A]ccurate phasing of long sequences by the methods and systems described herein may increase variant calling accuracy, for example by using haploid error models. Further, methods and systems described herein allow for the phasing of low frequency variants not present in reference SNP chips” and “[O]ther embodiments include combining phasing approaches described herein with raw whole genome sequencing data, paired end data, or sequencing information from a close relative to establish accurate whole genome variant phasing. Hypermaping can be employed to increase fragment mapping accuracy. Phased genome blocks can be used to establish which variants are collocated on the one of the two chromosomes, whether both copies of a gene are affected by a mutation, or for any other suitable uses known in the art” (see paragraphs [0011], [0160] and [0161]) wherein the paired end reads read two DNA sequences generated from opposite ends of the same fragmented DNA molecule.
So et al., teach that a primer comprises a template binding region located at a 3’ region of the primer and a probe-binding located at a 5’ region of the primer wherein the template binding region hybridizes to a template nucleic acid, the probe-binding region comprises a unique sequence or barcode that does not hybridize to the template nucleic acid and is designed to avoid significant sequence similarity or complementarity to known genomic sequences of an organism of interest (see paragraphs [0211], [0212], [0215] and [0217]).
Therefore, it would have been prima facie obvious to one having ordinary skill in the art at the time the invention was made to have performed the method recited in claim 22 by PCR using primers that have a 5’ tail that has a sub-sample identifier sequence and does not hybridize to the adaptor-ligated sample, identifying a potential sequence variation in the plurality of sequence reads in (ii) (1) of step (d), and determining if the potential sequence variation is in the plurality of sequence reads in (ii) (2) of step (d) in view of the prior arts of Pushkarev et al., and So et al.. One having ordinary skill in the art would have been motivated to do so because Pushkarev et al., teach that “the sequencing comprises obtaining paired end reads” (see paragraph”, “[A]ccurate phasing of long sequences by the methods and systems described herein may increase variant calling accuracy, for example by using haploid error models. Further, methods and systems described herein allow for the phasing of low frequency variants not present in reference SNP chips” and “[O]ther embodiments include combining phasing approaches described herein with raw whole genome sequencing data, paired end data, or sequencing information from a close relative to establish accurate whole genome variant phasing. Hypermaping can be employed to increase fragment mapping accuracy. Phased genome blocks can be used to establish which variants are collocated on the one of the two chromosomes, whether both copies of a gene are affected by a mutation, or for any other suitable uses known in the art” (see paragraphs [0011], [0160] and [0161]) wherein the paired end reads read two DNA sequences generated from opposite ends of the same fragmented DNA molecule and So et al., have taught that a primer comprises a template binding region located at a 3’ region of the primer and a probe-binding located at a 5’ region of the primer wherein the template binding region hybridizes to a template nucleic acid, the probe-binding region comprises a unique sequence or barcode that does not hybridize to the template nucleic acid and is designed to avoid significant sequence similarity or complementarity to known genomic sequences of an organism of interest (see paragraphs [0211], [0212], [0125] and [0217]). One having ordinary skill in the art at the time the invention was made would have a reasonable expectation of success to perform the method recited in claim 22 using primers that have a 5’ tail that has a sub-sample identifier sequence and does not hybridize to the adaptor-ligated sample in order to avoid significant sequence complementarity to known genomic sequences of an organism of interest in the sample, and using phasing approaches combining with raw whole genome sequencing data, paired end data, or sequencing information from a close relative in view of the prior arts of Pushkarev et al., and So et al., such that a potential sequence variation in the plurality of sequence reads derived from the top strand of a DNA fragment of the tagged adaptor-ligated DNA fragments from one of the sub-samples in (ii) (1) of step (d) of claim 22 would be identified and if the potential sequence variation is in the plurality of sequence reads derived from the bottom strand of the same DNA fragment of the tagged adaptor-ligated DNA fragments from the one of the sub-samples in (ii) (2) of step(d) of claim 22 would be determined.
Claim 23 is rejected under 35 U.S.C. 103 as being unpatentable over Pushkarev et al., in view of So et al., as applied to claims 22, 24, 28, and 30-35 above, and further in view of Eltoukhy et al., (US 2016/0046986 A1, priority date: December 28, 2013) and Stapleton et al., (US 2016/0152972 A1, priority date: November 21, 2014).
The teachings of Pushkarev et al., and So et al., have been summarized previously, supra.
Pushkarev et al., and So et al., do not disclose grouping the plurality of sequence reads of step (d) wherein sequence reads having identical sequences, identical fragmentation breakpoints and the same sub-sample identifier sequence are placed in a group as recited in claim 23.
Eltoukhy et al., teach grouping a plurality of sequence reads into families based on
distinct molecular barcodes coupled to the plurality of polynucleotide molecules and similarities between the plurality of sequence reads wherein each family includes a plurality of nucleic acid sequences that are associated with a distinct combination of molecular barcodes and similar or identical sequence reads (see paragraphs [0034] and [0232]).
Stapleton et al., teach grouping nucleic acid sequence reads sharing the same barcode sequences (see paragraph [0132]).
However, it would have been prima facie obvious to one having ordinary skill in the art at the time the invention was made to have performed the method recited in claim 23 by grouping the plurality of sequence reads of step (d) wherein sequence reads having identical sequences, identical fragmentation breakpoints and the same sub-sample identifier sequence are placed in a group in view of the prior arts of Pushkarev et al., So et al., Eltoukhy et al., and Stapleton et al.. One having ordinary skill in the art would have been motivated to do so because the specification of this instant case defines “identical or near-identical fragmentation breakpoints” as “two molecules that have the same 5’ end, the same 3’ end, or the same 5’ and 3’ ends, where the differences are due to a PCR error, sequencing error, mapping or alignment error or somatic mutation” (see paragraph [0069] of US 2023/0065345 A1, which is US Publication of this instant case), Eltoukhy et al., teach grouping a plurality of sequence reads into families based on distinct molecular barcodes coupled to the plurality of polynucleotide molecules and similarities between the plurality of sequence reads wherein each family includes a plurality of nucleic acid sequences that are associated with a distinct combination of molecular barcodes and similar or identical sequence reads (see paragraphs [0034] and [0232]), and Stapleton et al., teach grouping nucleic acid sequence reads sharing the same barcode sequences (see paragraph [0132]). One having ordinary skill in the art at the time the invention was made would have a reasonable expectation of success to perform the method recited in claim 23 by grouping the plurality of sequence reads of step (d) based on similarities between nucleic acid sequences, similarities between fragmentation breakpoints, and similarities between sub-sample identifier sequences in view of the prior arts of Pushkarev et al., So et al., Eltoukhy et al., and Stapleton et al., in order to place the sequence reads of step (d) of claim 22 that have identical sequences, identical fragmentation breakpoints and the same sub-sample identifier sequence into the same group.
Claim 23 is rejected under 35 U.S.C. 103 as being unpatentable over Pushkarev et al., in view of So et al., as applied to claims 22, 24, 28, and 30-35 above, and further in view of Osborne et al., (US 2016/0289753 A1, priority date: December 2, 2013) and Stapleton et al., (US 2016/0152972 A1, priority date: November 21, 2014).
The teachings of Pushkarev et al., and So et al., have been summarized previously, supra.
Pushkarev et al., and So et al., do not disclose grouping the plurality of sequence reads of step (d) wherein sequence reads having identical sequences, identical fragmentation breakpoints and the same sub-sample identifier sequence are placed in a group as recited in claim 23.
Osborne et al., teach that sequence reads of copies of fragments that have the same fragmentation breakpoints and that are substantially identical sequences are grouped into read groups (see paragraph [0090]).
Stapleton et al., teach grouping nucleic acid sequence reads sharing the same barcode sequences (see paragraph [0132]).
Therefore, it would have been prima facie obvious to one having ordinary skill in the art at the time the invention was made to have performed the method recited in claim 23 by grouping the plurality of sequence reads of step (d) wherein sequence reads having identical sequences, identical fragmentation breakpoints and the same sub-sample identifier sequence are placed in a group in view of the prior arts of Pushkarev et al., So et al., Osborne et al., and Stapleton et al.. One having ordinary skill in the art would have been motivated to do so because Osborne et al., teach that sequence reads of copies of fragments that have the same fragmentation breakpoints and that are substantially identical sequences are grouped into read groups (see paragraph [0090]) while Stapleton et al., teach grouping nucleic acid sequence reads sharing the same barcode sequences (see paragraph [0132]). One having ordinary skill in the art at the time the invention was made would have a reasonable expectation of success to perform the method recited in claim 23 by grouping the plurality of sequence reads of step (d) based on similarities between nucleic acid sequences, similarities between fragmentation breakpoints, and similarities between sub-sample identifier sequences in view of the prior arts of Pushkarev et al., So et al., Osborne et al., et al., and Stapleton et al., in order to place the plurality of sequence reads of step (d) of claim 22 that have identical sequences, identical fragmentation breakpoints and the same sub-sample identifier sequence into the same group.
Claims 25 and 26 are rejected under 35 U.S.C. 103 as being unpatentable over Pushkarev et al., in view of So et al., as applied to claims 22, 24, 28, and 30-35 above, and further in view of Sanz Herranz et al., (US 2010/0310520 A1, published on December 9, 2010).
The teachings of Pushkarev et al., and So et al., have been summarized previously, supra.
Pushkarev et al., and So et al., do not disclose that the tagged adaptor-ligated DNA fragments sequenced in step (d) are selected by target enrichment as recited in claim 25 wherein the target enrichment is done by polymerase chain reaction as recited in claim 26.
Sanz Herranz et al., teach to amplify a sequenced nucleic acid fragment by PCR (see paragraph [0046]).
Therefore, it would have been prima facie obvious to one having ordinary skill in the art at the time the invention was made to have performed the methods recited in claims 25 and 26 wherein the tagged adaptor-ligated DNA fragments sequenced in step (d) are selected by target enrichment and the target enrichment is done by polymerase chain reaction in view of the prior arts of Pushkarev et al., So et al., and Sanz Herranz et al.. One having ordinary skill in the art would have been motivated to do so because Sanz Herranz et al., have successfully amplified a sequenced nucleic acid fragment by PCR (see paragraph [0046]). One having ordinary skill in the art at the time the invention was made would have a reasonable expectation of success to perform the methods recited in claims 25 and 26 by amplifying the tagged adaptor-ligated DNA fragments sequenced in step (d) of claim 22 using PCR in view of the prior art of Pushkarev et al., So et al., and Sanz Herranz et al., in order to selective target enrichment of the tagged adaptor-ligated DNA fragments sequenced in step (d) of claim 22.
Claim 27 is rejected under 35 U.S.C. 103 as being unpatentable over Pushkarev et al., in view of So et al., as applied to claims 22, 24, 28, and 30-35 above, and further in view of Druley et al., (US 2018/0002747 A1, priority date: January 23, 2015).
The teachings of Pushkarev et al., and So et al., have been summarized previously, supra.
Pushkarev et al., and So et al., do not disclose that the asymmetric adaptor is a Y adaptor as recited in claim 27.
Druley et al., teach that “[A]key feature of the adapter is to enable the unique amplification of the amplicon or product only without the need to remove existing template nucleic acid or purify the amplicons or products. This feature enables an ‘add only’ reaction with fewer steps and ease of automation. The adapter is attached to the 5’ and 3’ end of the amplicon or product. The adapter may be Y-shaped, U-shaped, hairpin-shaped, or a combination thereof” (see paragraph [0062]).
Therefore, it would have been prima facie obvious to one having ordinary skill in the art at the time the invention was made to have performed the method recited in claim 27 wherein the asymmetric adaptor is a Y adaptor in view of the prior arts of Pushkarev et al., So et al., and Druley et al.. One having ordinary skill in the art would have been motivated to do so because Druley et al., have shown that “[A]key feature of the adapter is to enable the unique amplification of the amplicon or product only without the need to remove existing template nucleic acid or purify the amplicons or products. This feature enables an ‘add only’ reaction with fewer steps and ease of automation. The adapter is attached to the 5’ and 3’ end of the amplicon or product. The adapter may be Y-shaped, U-shaped, hairpin-shaped, or a combination thereof” (see paragraph [0062]) and the simple substitution of one kind of asymmetric adaptor (ie., the hairpin adaptor taught by Pushkarev et al.,) from another kind of asymmetric adaptor (ie., the Y adaptor taught by Druley et al.,) during the process of performing the methods of claim 22, in the absence of convincing evidence to the contrary, would have been prima facie obvious to one having ordinary skill in the art at the time the invention was made since the hairpin adaptor taught by Pushkarev et al., and the Y adaptor taught by Druley et al., can be used for the same purpose and are exchangeable. One having ordinary skill in the art at the time the invention was made would have a reasonable expectation of success to perform the method recited in claim 22 using a Y adaptor in view of the prior arts of Pushkarev et al., So et al., and Druley et al..
Furthermore, the motivation to make the substitution cited above arises from the expectation that the prior art elements will perform their expected functions to achieve their expected results when combined for their common known purpose. Support for making the obviousness rejection comes from the M.P.E.P. at 2144.06, 2144.07 and 2144.09.
Also note that there is no invention involved in combining old elements is such a manner that these elements perform in combination the same function as set forth in the prior art without giving unobvious or unexpected results. In re Rose 220 F.2d. 459, 105 USPQ 237 (CCPA 1955).
Claim 29 is rejected under 35 U.S.C. 103 as being unpatentable over Pushkarev et al., in view of So et al., as applied to claims 22, 24, 28, and 30-35 above, and further in view of Bielas et al., (US 2015/0024950 A1, priority date: February 17, 2012).
The teachings of Pushkarev et al., and So et al., have been summarized previously, supra.
Pushkarev et al., and So et al., do not disclose that one or more of the sequence tags that identify the sub-sample used in step (c) contain a sample identifier sequence that identifies the sample of step (a), wherein the tagged adaptor-ligated DNA fragments from each of the tagged sub-samples of step (c) comprise one of the sequence tags that identify the sub-samples and the sample identifier sequence, and wherein each of the sequencing reads in (i) of step (d) further comprise the sample identifier sequence as recited in claim 29.
Bielas et al., teach that nucleic acid molecules include dual cyphers or barcodes in each end of the nucleic acid molecules (see paragraph [0015]).
Therefore, it would have been prima facie obvious to one having ordinary skill in the art at the time the invention was made to have performed the method recited in claim 29 wherein one or more of the sequence tags that identify the sub-sample used in step (c) contain a sample identifier sequence that identifies the sample of step (a), wherein the tagged adaptor-ligated DNA fragments from each of the tagged sub-samples of step (c) comprise one of the sequence tags that identify the sub-samples and the sample identifier sequence, and wherein each of the sequencing reads in (i) of step (d) further comprise the sample identifier sequence in view of the prior arts of Pushkarev et al., So et al., and Bielas et al., One having ordinary skill in the art would have been motivated to do so because Bielas et al., have shown that nucleic acid molecules include dual cyphers or barcodes in each end of the nucleic acid molecules (see paragraph [0015]) while Pushkarev et al., have shown that adaptors can contain one or more the same or different barcode sequences (see paragraphs [0058] and [0086]). One having ordinary skill in the art at the time the invention was made would have a reasonable expectation of success to perform the method recited in claim 29 by modifying the sequencing method taught by Pushkarev et al., using one or more of the sequence tags that contain a sample identifier sequence that identifies the sample of step (a) and a sub-sample identifier sequence in view of the prior arts of Pushkarev et al., So et al., and Bielas et al., in order to make the tagged adaptor-ligated DNA fragments from each of the tagged sub-samples of step (c) which comprise one of the sequence tags that identify the sub-samples and the sample identifier sequence such that each of the sequencing reads in (i) of step (d) would comprise the sample identifier sequence.
Response to Arguments
In page 6, second paragraph bridging to page 9, second paragraph of applicant’s remarks, applicant argues that “[A]pplicant respectfully submits that the combination of cited references is not sufficient to establish a prima facie case of obviousness with respect to the amended claims at least because the cited references, alone or in combination, does not teach each and every element recited in amended claim 22” and “[S]tep (d) requires sequencing tagged adaptor-ligated DNA fragments from the tagged sub-samples of (c), the sequence reads comprising a plurality of sequence reads from the top strand of a fragment and a different plurality of sequence reads from the bottom strand of the same fragment; step (e) requires identification of variations in the sequence reads of the top strand, and step (f) requires determining that the variation is also present in the bottom strand of the fragment. As
shown in FIG. 1 of the instant application, top strands and bottom strand sequences of a particular fragment are distinguishable based on the combination of the asymmetric adaptor ligated to the fragment in step (a); and the sequence tags that identify the sub-sample in which the fragment is contained, which are introduced in step (c). This combination allows for one to distinguish between top strands and bottom strands of a particular DNA fragment using sequence reads, as recited in steps (d)-(f) of amended claim 22. None of the cited references teach or suggest any means of distinguishing sequence reads corresponding to top and bottom strands of a DNA fragment, nor identifying sequence variations from sequence reads corresponding to each strand, as claimed. Pushkarev makes no mention of comparing, distinguishing between, or even identifying sequence reads corresponding to a particular strand of a double-stranded fragment, much less identifying sequence variations in the same and comparing the two. Instead, Pushkarev discloses a method for reconstructing longer sequences from shorter sequence reads by partitioning a sample containing long DNA molecules, fragmenting these long DNA molecules, and then ligating adaptors to the resulting partitioned fragments prior to sequencing. This method does not allow for any distinction between the top and bottom strands of a particular sequence, as the information has already been lost with the fragmenting step. None of the other cited references remedy this deficiency. As a second matter, Applicant respectfully submits that one of ordinary skill in the art would not be motivated to modify the method of Pushkarev as proposed by the Examiner because the proposed modification would render the prior art invention unsatisfactory for its intended purpose3; moreover, the resulting modification would still not arrive at the method of the instant claims. As noted in the Amendment dated February 27, 2025, the Examiner’s proposed modification to ligate adaptors to DNA prior to partitioning the sample (as recited in the instant claims) would result in Pushkarev’s method not working as intended because the adaptor ligated molecules (i.e., the long DNA molecules) would not be the molecules that would ultimately be sequenced; instead, non-adaptor ligated fragments would be sequenced, and the longer sequences could not be reconstructed. The Examiner has also not articulated a reason why the disclosures of the other cited references, alone or in combination, would have provided a person of ordinary skill in the art with a reason to have modified the methods of Pushkarev to ligate adaptors prior to partitioning the sample. Furthermore, even if, arguendo, one of ordinary skill in the art were motivated to modify Pushkarev against its intended purpose, which Applicant does not concede, one would still not arrive at the method of the instant claims. Step (d) of amended claim 22 recites sequencing of ‘tagged adaptor-ligated DNA fragments’; as noted supra, the Examiner’s modification of Pushkarev does not result in adaptor-ligated DNA fragments being sequenced, as the adaptors would instead be ligated to non-partitioned, non-fragmented long DNA molecules. Moreover, as explained supra, the Examiner's modification of Pushkarev’s method still does not allow for any method of grouping sequence reads into top strand groups and bottom strand groups, as recited in step (d) of instant claim 22. This deficiency is not remedied by any of the other cited references. Accordingly, the combination of cited references is not sufficient to establish a prima facie case of obviousness with respect to the amended claims”.
The above arguments have been fully considered but they are not persuasive toward the withdrawal of the rejection.
First, since it is known that “[U]nlike single-read sequencing, paired-end sequencing allows users to sequence both ends of a fragment and generate high-quality, alignable sequence data” (see “Advantages of paired-end and single-read sequencing”) and Pushkarev et al., teach that “the sequencing comprises obtaining paired end reads” (see paragraph [0011]), the paired end reads taught by Pushkarev et al., read two DNA sequences generated from opposite ends of the same fragmented DNA molecule. Since “[A]ccurate phasing of long sequences by the methods and systems described herein may increase variant calling accuracy, for example by using haploid error models. Further, methods and systems described herein allow for the phasing of low frequency variants not present in reference SNP chips” and “[O]ther embodiments include combining phasing approaches described herein with raw whole genome sequencing data, paired end data, or sequencing information from a close relative to establish accurate whole genome variant phasing. Hypermaping can be employed to increase fragment mapping accuracy. Phased genome blocks can be used to establish which variants are collocated on the one of the two chromosomes, whether both copies of a gene are affected by a mutation, or for any other suitable uses known in the art” (see paragraphs [0160] and [0161]), the paired end data or sequencing information taught by Pushkarev et al., at least includes two DNA sequences generated from opposite ends of the same fragmented DNA molecule and applicant’s arguments “[N]one of the cited references teach or suggest any means of distinguishing sequence reads corresponding to top and bottom strands of a DNA fragment, nor identifying sequence variations from sequence reads corresponding to each strand, as claimed. Pushkarev makes no mention of comparing, distinguishing between, or even identifying sequence reads corresponding to a particular strand of a double-stranded fragment, much less identifying sequence variations in the same and comparing the two” and “the Examiner's modification of Pushkarev’s method still does not allow for any method of grouping sequence reads into top strand groups and bottom strand groups, as recited in step (d) of instant claim 22” are incorrect.
Second, although applicant argues that “[T]he Examiner has also not articulated a reason why the disclosures of the other cited references, alone or in combination, would have provided a person of ordinary skill in the art with a reason to have modified the methods of Pushkarev to ligate adaptors prior to partitioning the sample”, since Figure 1 of Pushkarev et al., teaches steps, “Ligate amplification adapter” and then “dilution to 500 molecules/well”, Pushkarev et al., teach “ligate adaptors prior to partitioning the sample” as argued by applicant. Furthermore, since Pushkarev et al., teach that “[T]he resulting pool of amplified molecules were fragmented into a sequencing library using Nextera DNA transposase, and sequencing adapters with barcodes unique to each well were incorporated through limited cycle PCR. The library was then sequenced. After sequencing, reads were separated according to the barcodes and original long fragments were assembled using developed assembly algorithms” (see paragraph [0227] and Figure 1) and the primer “A-primer-A” used for PCR in Figure 5A of Pushkarev et al., contains a barcode (see paragraph [0087]), the adaptor ligated molecules are sequenced and applicant’s arguments “the adaptor ligated molecules (i.e., the long DNA molecules) would not be the molecules that would ultimately be sequenced; instead, non-adaptor ligated fragments would be sequenced, and the longer sequences could not be reconstructed” and “the Examiner’s modification of Pushkarev does not result in adaptor-ligated DNA fragments being sequenced, as the adaptors would instead be ligated to non-partitioned, non-fragmented long DNA molecules” are incorrect.
Conclusion
No claim is allowed.
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/FRANK W LU/
Primary Examiner, Art Unit 1683
January 13, 2026