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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 January 23, 2026 has been entered.
Priority
The present application, filed October 29, 2021, is a 371 of PCT/US2020/044673, filed on August 1, 2020, and claims the benefit of U.S. Provisional Patent Application No. 62/881,936, filed August 1, 2019.
Claim Status and Action Summary
This action is in response to the papers filed on January 23, 2026.
Claims 1, 2, 4, 5, 7, 11, 13, 15,17, 18, 23, 24, 30, 32, 38, 40, 42, 46, 47, 49, 50, and 53 are pending.
Claim 3 was canceled in the amendment filed on January 23, 2026.
Claims 1, 2, 4, 5, 7, 11, 13, 15, 17, 18, 23, 24, 30, 32, 38, 40, 42, 46, 47, 49, 50, and 53 are under examination.
Any objections and rejections not reiterated below are hereby withdrawn.
Claim Interpretation
Claim 1 recites “a first and second orientation”.
For clarity, the examiner has interpreted the input library molecules recited in claim 1 as being of the form:
PNG
media_image1.png
720
1152
media_image1.png
Greyscale
The examiner has interpreted the molecules bound to the surface at a 5’ end or a 3’ end as follows:
PNG
media_image2.png
720
1152
media_image2.png
Greyscale
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.
Claims 1-2, 7, 11, 13, 15, 17-18, 23-24, 30, 32, and 53 are/remain rejected under 35 U.S.C. 103 as being unpatentable over Schmitt WO 2013/142389 A1 (published September 26, 2013) in view of Bentley et al. “Accurate whole human genome sequencing using reversible terminator chemistry.” Nature 456, 53–59 (2008).
Regarding claims 1, 2, and 11, Schmitt teaches a method of sequencing a double-stranded target nucleic acid molecule. (Schmitt, Abstract) Schmitt teaches that the target molecule may comprise a first adapter comprising a linker domain (i.e. a “U”-shaped or “hairpin” adapter) ligated to the first end of a double-stranded target nucleic acid, and a Y-shaped second adapter ligated to the second end of the double stranded target nucleic acid (i.e. a physically linked amplicon). (Schmitt, 0023 and embedded diagrams in 0032-0035)
PNG
media_image3.png
1043
750
media_image3.png
Greyscale
Schmitt teaches that the Y-adaptor comprises flow cell sequences FC1 and FC2 on each end of the molecule (i.e. the amplicons can bind to a flow cell in both a forward and reverse orientation), (Schmitt, 0023) and that a given sequence seen in Read 1 will have the sequence corresponding to the complementary DNA duplex strand seen in Read 2. (Schmitt, 0023) Schmitt further teaches that “the hairpin linker may additionally include one or more cleavable sequences” (Schmitt 0048) that may be cleaved after amplification to facilitate efficient sequencing (i.e. cleaving the physically linked amplicons to provide single stranded amplicons comprising information from one strand) (Schmitt 0050-0053).
PNG
media_image4.png
528
531
media_image4.png
Greyscale
Finally, Schmitt teaches a method for sequencing the double stranded target nucleic acid complex comprising U- and Y-shaped adapters wherein the complex is amplified by a method known in the art (i.e. bridge amplification) and sequenced using any method known in the art (expressly including “the Illumina sequencing platform”) (i.e. amplifying a physically linked nucleic acid complex on a surface). (Schmitt 0074)
Schmitt does not teach steps (b) and (f) directed to removing bridge amplification products bound to the surface in either the forward or reverse orientation.
However, Bentley et al (2008) teaches removal of amplicons bound to a surface in a first (forward or reverse) orientation, sequencing the remaining amplicons bound to a surface in a second orientation, repopulating the sequencing cluster, removing the amplicons bound to a surface in the second orientation, and sequencing the remaining amplicons bound to a surface in a first orientation. (Bentley, Figure 1c)
PNG
media_image5.png
300
395
media_image5.png
Greyscale
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to modify the method of massively parallel sequencing of physically linked nucleic acid complexes immobilized on a surface taught by Schmitt with the bridge amplification and paired end sequencing by synthesis workflow taught by Bentley. The ordinary artisan would have been motivated to modify the bridge amplification method of Schmitt because Bentley teaches this approach increases throughput, reduces costs, and generates high quality genomic data. (Bentley pg 53, column 1). The ordinary artisan would have had a reasonable expectation of success to modify the method of Schmitt with the bridge amplification method of Bentley.
Regarding claim 7, 13, and 23 Schmitt teaches comparing the sequences read from the two strands sharing a single molecule identifier (i.e. a UMI), identifying mismatches between the two reads, and generating a double-stranded consensus sequence (i.e. an error-corrected sequence). (Schmitt, Figure 3 and 0024)
Regarding claim 15, Schmitt teaches a UMI (i.e. a SMI) comprises a unique tag sequence (Schmitt, Figure 1 and 0012)
Regarding claims 17 and 18, Schmitt teaches differentiation between each strand using sequence read information (i.e. an SDE): “By virtue of the asymmetric nature of adapted fragments, two types of PCR products are produced from each capture event. Those derived from one strand will have the SMI sequence adjacent to flow-cell sequence 1 and the β SMI sequence adjacent to flow cell sequence 2. PCR products originating from the complementary strand are labeled reciprocally” (Schmitt 0012 and Figure 3)
Regarding claim 24, Schmitt teaches an adapter comprising a hairpin with a cleavable site (Schmitt 0048).
Regarding claim 30, Schmitt teaches an asymmetric adapter comprising distinct primer binding sites within the two non-complementary arms. (Schmitt 0050)
Regarding claim 32, Schmitt teaches a method for sequencing the double stranded target nucleic acid complex comprising U- and Y-shaped adapters wherein the complex is amplified by a method known in the art (i.e. bridge amplification) and sequenced using any method known in the art (expressly including “the Illumina sequencing platform”) (i.e. amplifying a physically linked nucleic acid complex on a surface). (Schmitt 0074)
Regarding claim 53, Schmitt teaches that self-hybridized nucleic acid complexes can be cleaved by a restriction enzyme. (Schmitt, 0048)
Response to arguments
The response asserts that the claimed invention is not obvious over Schmitt et al. in view of Bentley et al. because Bentley et al. does not teach sequencing methods utilizing an adapter comprising a hairpin loop structure and an adapter comprising two single stranded ends (i.e. “Y-shaped adapters”) that bind to a surface. This argument has been thoroughly reviewed and is not persuasive for the reasons which follow.
In response to Applicant's piecemeal analysis of the references, it has been held that one cannot show non-obviousness by attacking references individually where, as here, the rejections are based on combinations of references. In re Keller, 208 USPQ 871 (CCPA 1981).
As elaborated in the 103 rejection above, the combination of the teachings of Bentley et al., which describe sequencing methods wherein template molecules comprising Y-shaped adapters at both ends of a template molecule bind to a flow cell, and the teachings of Schmitt et al., which describe sequencing methods and adapters wherein template molecules comprise a Y-shaped adapter at one end of the molecule, and a “U-shaped” or hairpin adapter at the other end of the template molecule arrives at the method claimed by the present application. Furthermore, Schmitt et al. expressly teaches these adapters and methods of sequencing are useful for sequencing each strand of a double stranded input molecule for the purpose of detecting/eliminating PCR or sequencing errors based on comparing the first and second strand sequencing reads and that said adapters can be substituted for standard sequencing adapters (such as those taught by Bentley) (Schmitt., paragraph 0086). Therefore, the rejection is maintained.
As described above, Schmitt et al. teach methods of amplifying library molecules of essentially identical form to those employed in the claimed invention and cleaving the products to obtain “sequencable products” that maintain linked information from both strands of an original library molecule. Schmitt et al. do not explicitly teach that this amplification and cleavage step for preparing a library for sequencing occurs on a sequencing surface (i.e. by bridge amplification). Bentley et al. is cited above as describing suitable bridge amplification and cleavage methods that would have been widely known to those of ordinary skill in the art before the filing date of the claimed invention.
The further argument presented in the response that the ordinary artisan would not have been motivated to modify Schmitt with the teachings of Bentley because Bentley does not teach the double-stranded, linked template sequencing method claimed (and taught by Schmitt as described above) is not persuasive because Schmitt is cited in the 103 rejection for standard and widely known bridge amplification and cleavage methods that the ordinary artisan would have used to amplify and cleave the library molecules produced by Schmitt et al., as described in detail above, on a sequencing surface to obtain sequencing data from the libraries.
Claims 4-5, 38, 40, 42, 46-47, and 49-50 are rejected under 35 U.S.C. 103 as being unpatentable over Schmitt WO 2013/142389 A1 (published September 26, 2013) and Bentley et al. “Accurate whole human genome sequencing using reversible terminator chemistry.” Nature 456, 53–59 (2008) as applied to claims 1, 2, and 11 above, and further in view of Wong, et al. “A novel method for producing partial restriction digestion of DNA fragments by PCR with 5-methyl-CTP.” Nucleic acids research vol. 25(20), 4169-71 (1997).
This rejection has been updated as necessitated by the amendments to the claims.
Regarding claims 4 and 38, Schmitt teaches an embodiment wherein hairpin adapters (i.e. physically linked complex amplicons) may be cleaved prior to sequencing. (Schmitt 0052-0053) Neither Schmitt nor Bentley teach that said cleavage reaction does not go to completion (i.e. is inefficient). However, Wong (1997) teaches a method of partially digesting DNA fragments with a methylation-sensitive restriction endonuclease following a PCR reaction comprising a mixture of 5-methyl-dCTP (an anti-cleavage facilitator) and dCTP. (Wong, abstract) Wong further teaches “a partial restriction digestion pattern could be obtained by digesting the PCR products completely with methylation-sensitive restriction enzymes. This method eliminates the optimization steps needed for traditional partial digestion.” (Wong, page 1, column1)
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to produce a subset of physically linked nucleic acid complex amplicons (or oligonucleotide fragments thereof) by the method taught by Schmitt wherein a portion of the adapters comprising the linker domain comprises a methylated restriction site as taught by Wong. The ordinary artisan would have been motivated to combine these methods by the suggestion of Schmitt, “sequencing of a hairpin will be less efficient due to the self-complementarity present within the hairpin molecule.” (Schmitt 0053) As such, cleavage of a majority of physically linked complexes would have been expected to increase the efficiency of the sequencing reaction. Therefore the ordinary artisan would have been reasonably confident that inclusion of a subset of cleavage-resistant adapters would have eliminated the need to optimize the partial digestion of physically linked complexes, while providing at least one intact complex per cluster for regeneration of the clusters following the first (forward or reverse) sequencing read because Wong teaches a mixture of methylated and unmethylated substrates as a means of rapidly enabling partial restriction enzyme digestion. (Wong, page 1, column 1)
Regarding claim 5, Schmitt teaches comparing the sequences read from the two strands sharing a single molecule identifier (i.e. a UMI), identifying mismatches between the two reads, and generating a double-stranded consensus sequence (i.e. an error-corrected sequence). (Schmitt, Figure 3 and 0024)
Regarding claims 40 and 42, Bentley teaches that the cleavable site is in the linker domain (i.e. the primer binding domain) (Bentley, figure 1c) and that the cleavage facilitator comprises USER enzymes and Fpg (i.e. an endonuclease, a nickase, a nickase variant…). (Bentley, supplemental materials, page 4) Additionally, Schmitt teaches that the cleavable site in the linker domain (i.e. the hairpin adapter) may be cleaved by a restriction enzyme. (i.e. a cleavage facilitator; an endonuclease) (Schmitt, 0048-0053)
Regarding claims 46-47 and 50, Schmitt teaches an embodiment wherein hairpin adapters (i.e. physically linked complex amplicons) are cleaved prior to sequencing. Schmitt does not require that the cleavage is complete, as potential sequencing priming of the hairpin will be less efficient than the linearized products (Schmitt, 0053). Schmitt does not require that the cleavage is an inefficient process and does not teach that the amount of substrate (uncleaved complexes) can be scaled by controlling: the amount or concentration of a cleavage facilitator (i.e. an endonuclease), the length of time for which the substrate is exposed to the enzyme, or the presence of a reaction slowing or stopping reagent (an anti-cleavage facilitator, i.e. an inhibitor). However, these parameters (enzyme amount, concentration, duration of reaction, presence of inhibitors) are optimizable parameters for enzymatic digestion of a given substrate well known in the biochemical arts.
In fact, Wong teaches a method of partially digesting DNA fragments with a methylation-sensitive restriction endonuclease following a PCR reaction comprising a mixture of 5-methyl-dCTP (an anti-cleavage facilitator) and dCTP. (Wong, abstract) Wong further teaches “a partial restriction digestion pattern could be obtained by digesting the PCR products completely with methylation-sensitive restriction enzymes. This method eliminates the optimization steps needed for traditional partial digestion.” (Wong, page 1, column1)
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to produce a subset of physically linked nucleic acid complex amplicons (or oligonucleotide fragments thereof) by the method taught by Schmitt wherein a portion of the adapters comprising the linker domain comprises a methylated restriction site. The ordinary artisan would have been motivated to combine these methods by the suggestion of Schmitt, “sequencing of a hairpin will be less efficient due to the self-complementarity present within the hairpin molecule.” (Schmitt 0053) As such, cleavage of a majority of physically linked complexes would have been expected to increase the efficiency of the sequencing reaction. Therefore the ordinary artisan would have been reasonably confident that inclusion of a subset of cleavage-resistant adapters would have eliminated the need to optimize the partial digestion of physically linked complexes, while providing a population of intact complexes for regeneration of the clusters following the first (forward or reverse) sequencing read because Wong teaches a mixture of methylated and unmethylated substrates as a means of rapidly enabling partial restriction enzyme digestion. (Wong, page 1, column 1)
Regarding claim 49, Schmitt teaches cleavage of the physically linked nucleic acid complexes wherein the cleavable site “may be any appropriate restriction enzyme (i.e. an endonuclease) target sequence, or any other cleavable sequence.” (Schmitt 0048) (i.e. a dsDNA-specific endonuclease such as EcoRV) Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to try a restriction enzyme with activity on double stranded, but not single stranded DNA such as EcoRV. The ordinary artisan would have been motivated to choose a dsDNA-specific endonuclease in order to prevent unwanted cleavage of ssDNA reagents such as the flow cell oligos and sequencing primers. Therefore, the ordinary artisan would have had a reasonable expectation of success in choosing to linearize the physically linked nucleic acid complexes with a double strand-specific endonuclease.
Response to arguments
The response argues that the independent claims 1, 2, and 11 are not obvious over Schmitt and Bentley. This argument has been thoroughly reviewed and is not persuasive for the reasons discussed in the rejection of the independent claims above. The response does not provide any further arguments as to the patentability of the dependent claims. Therefore, the rejection is maintained.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claims 1-2, 7, 11, 13, 15, 17-18, 23, 24, 30, 32, and 53 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-4, 15, and 17 of U.S. Patent No. 12006545 (herein referred to as ‘545) in view of Schmitt WO 2013/142389 A1 (published September 26, 2013) and Bentley et al. “Accurate whole human genome sequencing using reversible terminator chemistry.” Nature 456, 53–59 (2008).
Regarding Claims 1, 2, 7, 11, 23, and 24, Claims 1, 15, and 17 of ‘545 teach a method for sequencing a plurality of hairpin adapter ligated (i.e. physically linked) double-stranded nucleic acid molecules comprising: cleaving at least one of the hairpin adapters with an endonuclease (i.e. a cleavage facilitator), cluster amplifying (i.e. amplifying clonal clusters on a surface) the nucleic acid complexes, sequencing at least a portion of the surface-bound molecules, grouping reads, and generating a consensus sequence for each group (i.e. comparing sequence information from both strands of an original double-stranded target nucleic acid to generate an error-corrected sequence). (‘545, claim 23)
‘545 does not claim removing physically linked complexes bound in a first orientation (forward or reverse) prior to sequencing complexes bound in a second orientation, regenerating the cluster in both orientations, and removing complexes bound in a second orientation prior to sequencing complexes bound in a first orientation. However, Bentley et al (2008) teaches removal of amplicons bound to a surface in a first (forward or reverse) orientation, sequencing the remaining amplicons bound to a surface in a second orientation, repopulating the sequencing cluster, removing the amplicons bound to a surface in the second orientation, and sequencing the remaining amplicons bound to a surface in a first orientation. (Bentley, Figure 1c)
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to modify the method of massively parallel sequencing of physically linked nucleic acid complexes immobilized on a surface claimed by ‘545 with the bridge amplification and paired end sequencing by synthesis workflow taught by Bentley. The ordinary artisan would have been motivated to modify the bridge amplification method of ‘545 because Bentley teaches this approach increases throughput, reduces costs, and generates high quality genomic data. (Bentley pg 53, column 1). The ordinary artisan would have had a reasonable expectation of success to modify the method of ‘545 with the bridge amplification method of Bentley.
Regarding claims 13 and 15, the claims of ‘545 teach grouping first and second strand sequence reads within the population of nucleic acid targets based at least in part on single molecule identifier sequences (i.e. a unique molecular identifier ; a tag sequence). (‘545 claim 14-15)
Regarding claims 17 and 18, the claims of ‘545 teach that the nucleic acid complexes may comprise Y-shape and hairpin adapters wherein the each of the two ends of the target molecule are ligated to a different adapter type. (‘545 claim 4) The claims of ‘545 do not teach that this structure comprises a “strand defining element”. However, Schmitt teaches differentiation between each strand using sequence read information (i.e. an SDE): “By virtue of the asymmetric nature of adapted fragments, two types of PCR products are produced from each capture event. Those derived from one strand will have the SMI sequence adjacent to flow-cell sequence 1 and the β SMI sequence adjacent to flow cell sequence 2. PCR products originating from the complementary strand are labeled reciprocally” (i.e. the association of asymmetric adapter sequences in read 1 and read 2 is the SDE) (Schmitt 0012 and Figure 3)
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to use the asymmetric nature of the Y-shaped adapters and hairpin adapters claimed by ‘545 and Schmitt to introduce strand-defining sequences into the nucleic acid complex. The ordinary artisan would have been motivated by the teaching of Schmitt “[amplicons] derived from one strand will have the SMI sequence adjacent to flow-cell sequence 1 and the β SMI sequence adjacent to flow cell sequence 2. PCR products originating from the complementary strand are labeled reciprocally” (Schmitt 0012) because the claims of ‘545 are directed to sequencing providing a consensus sequence for each strand of a double-stranded nucleic acid molecule (‘545, claim 21). The ordinary artisan would have had a reasonable expectation of success that inclusion of asymmetric SMIs taught by Schmitt in the sequencing scheme claimed by ‘545 would have facilitated differentiation between reads derived from each of the original strands.
Regarding claim 30, the claims of ‘545 teach that the one of the adapters may comprise a Y-shape (i.e. two noncomplementary arms with distinct primer binding sites) (‘545 claim 4).
Regarding claim 32, the claims of ‘545 teach that sequencing comprises cluster amplifying… on a sequencing substrate (i.e. flowing the complexes over the surface prior to amplifying on the surface) (‘545, claim 16)
Response to arguments
The response argues that the claims of the cited double patenting reference do not teach all of the elements of the current claims and that the current claims are not obvious over Schmitt and Bentley (alone and in combination) and therefore, the current claims are not obvious over the cited claims. This argument has been thoroughly reviewed and is not persuasive for the reasons discussed in the 103 rejection of the independent claims above. Therefore, the rejection is maintained.
Claims 4-5, 38, 40, 42, 46-47, 49, and 53 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-4, 15, and 17 of U.S. Patent No. 12006545 (herein referred to as ‘545) in view of Schmitt WO 2013/142389 A1 (published September 26, 2013), Bentley et al. “Accurate whole human genome sequencing using reversible terminator chemistry.” Nature 456, 53–59 (2008), as applied to claims 1, 2, and 11 above, and further in view of Wong, et al. “A novel method for producing partial restriction digestion of DNA fragments by PCR with 5-methyl-CTP.” Nucleic acids research vol. 25(20), 4169-71 (1997).
Regarding claims 4-5, and 38, the claims of ‘545 teach “sequencing at least a portion” [of the single-stranded amplicons], but does not teach that the cleavage reaction does not go to completion at each clonal cluster.
However, Wong (1997) teaches a method of partially digesting DNA fragments with a methylation-sensitive restriction endonuclease following a PCR reaction comprising a mixture of 5-methyl-dCTP (an anti-cleavage facilitator) and dCTP. (Wong, abstract) Wong further teaches “a partial restriction digestion pattern could be obtained by digesting the PCR products completely with methylation-sensitive restriction enzymes. This method eliminates the optimization steps needed for traditional partial digestion.” (Wong, page 1, column 1)
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to produce a subset of physically linked nucleic acid complex amplicons (or oligonucleotide fragments thereof) by the method taught by the claims of ‘545 wherein a portion of the adapters comprising the linker domain comprises a methylated restriction site as taught by Wong. The ordinary artisan would have been motivated to combine these methods by the suggestion of Schmitt, “sequencing of a hairpin will be less efficient due to the self-complementarity present within the hairpin molecule.” (Schmitt 0053) As such, cleavage of a majority of physically linked complexes would have been expected to increase the efficiency of the sequencing reaction. Therefore the ordinary artisan would have been reasonably confident that inclusion of a subset of cleavage-resistant adapters would have eliminated the need to optimize the partial digestion of physically linked complexes, while providing at least one intact complex per cluster for regeneration of the clusters following the first (forward or reverse) sequencing read because Wong teaches a mixture of methylated and unmethylated substrates as a means of rapidly enabling partial restriction enzyme digestion. (Wong, page 1, column 1)
Regarding claim 5, ‘545 claims comparing the sequences read from the two strands sharing a single molecule identifier (i.e. a UMI), identifying mismatches between the two reads, and generating a double-stranded consensus sequence (i.e. an error-corrected sequence). (‘545, claim 15)
Regarding claims 40 and 42, ‘545 claims that the cleavable site in the linker domain (i.e. the hairpin adapter) may be cleaved by a restriction enzyme. (i.e. a cleavage facilitator; an endonuclease) (‘545, claim 2)
Regarding claims 46-47 and 50, the claims of ‘545 teach that the hairpin adapters (i.e. physically linked complex amplicons) are cleaved prior to sequencing. (‘545, claim 2) The claims of ‘545 do not teach that the cleavage is inefficient or that the amount of substrate (uncleaved complexes) can be scaled by controlling: the amount or concentration of a cleavage facilitator (i.e. an endonuclease), the length of time for which the substrate is exposed to the enzyme, or the presence of a reaction slowing or stopping reagent (an anti-cleavage facilitator, i.e. an inhibitor). However, these parameters (enzyme amount, concentration, duration of reaction, presence of inhibitors) are optimizable parameters for enzymatic digestion of a given substrate well known in the biochemical arts.
In fact, Wong teaches a method of partially digesting DNA fragments with a methylation-sensitive restriction endonuclease following a PCR reaction comprising a mixture of 5-methyl-dCTP (an anti-cleavage facilitator) and dCTP. (Wong, abstract) Wong further teaches “a partial restriction digestion pattern could be obtained by digesting the PCR products completely with methylation-sensitive restriction enzymes. This method eliminates the optimization steps needed for traditional partial digestion.” (Wong, page 1, column 1)
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to produce a subset of physically linked nucleic acid complex amplicons (or oligonucleotide fragments thereof) by the method claimed by ‘545 wherein a portion of the adapters comprising the linker domain comprises a methylated restriction site. The ordinary artisan would have been motivated to combine these methods by the suggestion of Schmitt, “sequencing of a hairpin will be less efficient due to the self-complementarity present within the hairpin molecule.” (Schmitt 0053) As such, cleavage of a majority of physically linked complexes would have been expected to increase the efficiency of the sequencing reaction. Therefore the ordinary artisan would have been reasonably confident that inclusion of a subset of cleavage-resistant adapters would have eliminated the need to optimize the partial digestion of physically linked complexes, while providing a population of intact complexes for regeneration of the clusters following the first (forward or reverse) sequencing read because Wong teaches a mixture of methylated and unmethylated substrates as a means of rapidly enabling partial restriction enzyme digestion. (Wong, page 1, column 1)
Regarding claim 49, ‘545 claims cleaving the complex with an endonuclease (‘545, claim 3) Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to try a restriction enzyme with activity on double stranded, but not single stranded DNA such as EcoRV. The ordinary artisan would have been motivated to choose a dsDNA-specific endonuclease in order to prevent unwanted cleavage of ssDNA reagents such as the flow cell oligos and sequencing primers. Therefore, the ordinary artisan would have had a reasonable expectation of success in choosing to linearize the physically linked nucleic acid complexes with a double strand-specific endonuclease.
Regarding claim 53, the claims of ‘545 teach a cleavage site within a hairpin adapter is cleaved prior to sequencing. (i.e. the site is available when the amplicons are self-hybridized) (‘545, claim 3)
Response to arguments
The response argues that the claims of the cited double patenting reference do not teach all of the elements of the current claims and that the current claims are not obvious over Schmitt and Bentley (alone and in combination) and therefore, the current claims are not obvious over the cited claims. This argument has been thoroughly reviewed and is not persuasive for the reasons discussed in the 103 rejection of the independent claims above. Therefore, the rejection is maintained.
Claims 1-5, 7, 11, 13, 15, 17-18, 23, 24, 30, 32, 40, and 42 are rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1, 3, 10, 13, 17 and 19 of U.S. Patent No. 10760127 (herein referred to as ‘127) in view of Schmitt WO 2013/142389 A1 (published September 26, 2013), and Bentley et al. “Accurate whole human genome sequencing using reversible terminator chemistry.” Nature 456, 53–59 (2008).
Regarding claims 1-5, 11, 23, 24, 40, and 42, ‘127 claims a method of sequencing double-stranded DNA molecules comprising: amplifying asymmetrical adapter-ligated original strands by cluster amplification (i.e. amplification on a surface) (‘127, claim 10) wherein the adapters comprise a Y-shape or a U-shape, (‘127, claim 13) cleaving the adapters with a nuclease (‘127, claim 17), sequencing at least a portion of the surface-bound molecules, grouping reads by their distinct barcodes (i.e. UMIs), and generating a consensus sequence for each group (i.e. comparing sequence information from both strands of an original double-stranded target nucleic acid to generate an error-corrected sequence) (127, claim 1)
The claims of ‘127 do not teach cleaving at least a portion of the bound physically linked amplicons to provide subsets of single stranded amplicons. However, Schmitt teaches that “the hairpin (i.e. U-shaped) linker may additionally include one or more cleavable sequences” (Schmitt 0048) that may be cleaved after amplification to facilitate efficient sequencing (i.e. cleaving the physically linked amplicons to provide single stranded amplicons comprising information from one strand) (Schmitt 0050-0053). Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to combine the cleavable linker domain taught by Schmitt with the sequencing method claimed by ‘127. The ordinary artisan would have been motivated to combine these elements because of the teaching of Schmitt, “sequencing of a hairpin will be less efficient due to the self-complementarity present within the hairpin molecule.” (Schmitt 0053) As such, cleavage of a majority of physically linked complexes would have been expected to increase the efficiency of the sequencing reaction. Therefore, the ordinary artisan would have been reasonably confident that inclusion of a cleavage site in the hairpin linker of ‘127 would have improved the efficiency of sequencing the amplicons.
Furthermore, the claims of ‘127 do not claim removing physically linked complexes bound in a first orientation (forward or reverse) prior to sequencing complexes bound in a second orientation, regenerating the cluster in both orientations, and removing complexes bound in a second orientation prior to sequencing complexes bound in a first orientation. However, Bentley et al (2008) teaches removal of amplicons bound to a surface in a first (forward or reverse) orientation, sequencing the remaining amplicons bound to a surface in a second orientation, repopulating the sequencing cluster, removing the amplicons bound to a surface in the second orientation, and sequencing the remaining amplicons bound to a surface in a first orientation. (Bentley, Figure 1c)
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to modify the method of massively parallel sequencing of physically linked nucleic acid complexes immobilized on a surface claimed by ‘127 with the bridge amplification and paired end sequencing by synthesis workflow taught by Bentley. The ordinary artisan would have been motivated to modify the bridge amplification method of ‘127 because Bentley teaches this approach increases throughput, reduces costs, and generates high quality genomic data. (Bentley pg 53, column 1). The ordinary artisan would have had a reasonable expectation of success to modify the method of ‘127 with the bridge amplification method of Bentley.
Regarding claim 7, ‘127 claims comparing sequence information between the reads generated from each of the two strands to generate an error corrected sequence (127, claim 3)
Regarding claim 13, 15, and 17-18 ‘127 claims relating the two strands using an adapter sequence, a sequence length, sequence alignment information, and/or original strand information. (a UMI, an SDE) (‘127, claim 3)
Regarding claims 13 and 15, the claims of ‘127 teach grouping first and second strand sequence reads within the population of nucleic acid targets based at least in part on barcode sequences present in the adapters (i.e. a unique molecular identifier ; a tag sequence). (‘127 claim 3)
Regarding claims 17 and 18, the claims of ‘127 teach that the nucleic acid complexes may comprise Y-shape or hairpin adapters. (‘127 claim 13) The claims of ‘127 further teach that the method comprises associating and differentiating between the first strand sequences with the second strand sequence from a particular original nucleic acid complex based on an asymmetrical barcode sequence (i.e. a UMI and SDE). (‘127, claim 3)
Regarding claim 30, the claims of ‘127 teach that the adapters comprise asymmetrical double-stranded adapter molecules. (i.e. two noncomplementary arms with distinct primer binding sites) (‘127 claim 19).
Regarding claim 32, the claims of ‘127 teach “amplifying original strands comprises… cluster amplification” (‘127, claim 10) (i.e. flowing the complexes over the surface prior to amplification)
Regarding claims 40 and 42, ‘127 claims that the complexes are cleaved by a nuclease (‘127, claim 17)
Response to arguments
The response argues that the claims of the cited double patenting reference do not teach all of the elements of the current claims and that the current claims are not obvious over Schmitt and Bentley (alone and in combination) and therefore, the current claims are not obvious over the cited claims. This argument has been thoroughly reviewed and is not persuasive for the reasons discussed in the 103 rejection of the independent claims above. Therefore, the rejection is maintained.
Claims 1-5, 7, 11, 13, 15, 17-18, 23-24, 38, 40, and 42 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 8, 10, and 11 of U.S. Patent No. 12,241,123 (herein referred to as ‘123) in view of Schmitt WO 2013/142389 A1 (published September 26, 2013), and Bentley et al. “Accurate whole human genome sequencing using reversible terminator chemistry.” Nature 456, 53–59 (2008).
This rejection has been updated as necessitated by issuance of the previously cited copending application 17/392203 as U.S. Patent No. 12,241,123.
Regarding claims 1-4, 11, 24, 38, 40, and 42, the claims of ‘123 teach a method of sequencing a double stranded nucleic acid comprising: ligating a Y-adapter to a first end and a hairpin adapter to a second end (‘123, claim 1), generating amplicons of the resulting nucleic acid complex on a surface by bridge amplification (‘123, claim 8), and sequencing each strand. (‘123, claim 1)
The claims of ‘123 do not teach cleaving at least a portion of the bound physically linked amplicons to provide subsets of single stranded amplicons. However, Schmitt teaches that “the hairpin (i.e. U-shaped) linker may additionally include one or more cleavable sequences” (i.e. a restriction site) (Schmitt 0048) that may be cleaved (i.e. by an endonuclease) after amplification to facilitate efficient sequencing (i.e. cleaving the physically linked amplicons to provide single stranded amplicons comprising information from one strand) (Schmitt 0050-0053). Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to combine the cleavable linker domain taught by Schmitt with the sequencing method claimed by ‘123. The ordinary artisan would have been motivated to combine these elements because of the teaching of Schmitt, “sequencing of a hairpin will be less efficient due to the self-complementarity present within the hairpin molecule.” (Schmitt 0053) As such, cleavage of a majority of physically linked complexes would have been expected to increase the efficiency of the sequencing reaction. Therefore, the ordinary artisan would have been reasonably confident that inclusion of a cleavage site in the hairpin linker of ‘123 would have improved the efficiency of sequencing the amplicons.
The claims of ‘123 do not teach removing physically linked complexes bound in a first orientation (forward or reverse) prior to sequencing complexes bound in a second orientation, regenerating the cluster in both orientations, and removing complexes bound in a second orientation prior to sequencing complexes bound in a first orientation. However, Bentley et al (2008) teaches removal of amplicons bound to a surface in a first (forward or reverse) orientation, sequencing the remaining amplicons bound to a surface in a second orientation, repopulating the sequencing cluster, removing the amplicons bound to a surface in the second orientation, and sequencing the remaining amplicons bound to a surface in a first orientation. (Bentley, Figure 1c)
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to modify the method of massively parallel sequencing of physically linked nucleic acid complexes immobilized on a surface claimed by ‘123 with the bridge amplification and paired end sequencing by synthesis workflow taught by Bentley and the hairpin adapters taught by Schmitt. The ordinary artisan would have been motivated to modify the bridge amplification method of Schmitt because Bentley teaches this approach increases throughput, reduces costs, and generates high quality genomic data. (Bentley pg 53, column 1). The ordinary artisan would have had a reasonable expectation of success to modify the method of Schmitt with the bridge amplification method of Bentley.
Regarding claims 5,7, 13, 15, 17-18, and 23, the claims of ‘123 teach that the adapter molecules comprise one or more of: a sample barcode sequence, a molecular identifier sequence, or both (a SMI/UMI and an SDE) (‘123, claims 10-11)
The claims of ‘123 do not teach comparing the sequence reads derived from the two original strands to generate an error corrected consensus sequence. However, Schmitt teaches a method of “Duplex Consensus Sequencing” directed to a method that “greatly reduces errors by independently tagging and sequencing each of the two strands of a DNA duplex. As the two strands are complementary, true mutations are found at the same position in both strands. In contrast, PCR or sequencing errors will result in errors in only one strand.” (Schmitt, abstract)
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to compare the sequences of the two complementary strands generated by the method of ‘123 to construct an error-corrected consensus sequence. The ordinary artisan would have been motivated to combine these teachings because of the suggestion of Schmitt that “Duplex Consensus Sequencing… greatly reduces errors” [in deep sequencing genetically heterogenous mixtures] (Schmitt, abstract). Therefore, the ordinary artisan would have been reasonably confident that the sequences produced by ‘123 would have enabled such error-correction by comparison between the information generated from each strand.
Response to arguments
The response argues that the claims of the cited double patenting reference do not teach all of the elements of the current claims and that the current claims are not obvious over Schmitt and Bentley (alone and in combination) and therefore, the current claims are not obvious over the cited claims. This argument has been thoroughly reviewed and is not persuasive for the reasons discussed in the 103 rejection of the independent claims above. Therefore, the rejection is maintained.
Conclusion
No claim is allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to ZACHARY MARK TURPIN whose telephone number is (703)756-5917. The examiner can normally be reached Monday-Friday 8:00 am - 5:00 pm.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Winston Shen can be reached at 5712723157. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/Z.M.T./Examiner, Art Unit 1682
/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682