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 . Applicant’s amendment filed 11/25/2025 is acknowledged. Claim 1 has been amended. Claims 3, 7, 16, 18, 20, 23 and 28-34 have been canceled. Claims 1-2, 4-6, 8-15, 17, 19, 21-22, 24-27 are pending. All of the amendment and arguments have been thoroughly reviewed and considered.
Any rejection not reiterated in this action has been withdrawn as being obviated by the amendment of the claims.
This action is made Final.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Previous Rejections
The claimed objection directed to the claims 1 and 34 is withdrawn in view of Applicant’s amendment and cancellation of the claims. The claim rejection under 35 USC 112(b) directed to the claims 33 and 34 is withdrawn in view of Applicant’s cancellation of the claims. The prior art rejection under 35 USC 103 directed to the claims 1-6, 8-15, 17, 19, 21-22, 24-28, 32-34 as being unpatentable over Rasolonjatovo in view of Rigatto and further in view of Illumina is withdrawn in view of the new grounds of rejections necessitated by applicant’s amendment of the claims.
New Ground(s) of Rejection
THE NEW GROUND(S) OF REJECTIONS WERE NECESSITATED BY APPLICANT’S AMENDMENT OF THE CLAIMS:
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.
Claim(s) 1-2, 4-6, 8-15, 17, 19, 21-22, 24-27 is/are rejected under 35 U.S.C. 103 as being unpatentable over Rasolonjatovo et al in view of Vuyisich et al (US 20190153438, May 2019, effective filing date Nov. 2017) in view of Rigatto et al {Rigatto, in view of Illumina (https://www.illumina.com/content/dam/illumina-marketing/documents/products/other/miseq-overclustering-primer-770-2014-038.pdf (Citations of Rasolonjatovo, Rigatto and Illumina have been cited in prior Office action).
Regarding 1-2, 4-6, 8-15, 17, 19, 21-22, 24-27, Rasolonjatovo teaches a cluster counting methodology which allows a user to image template molecules hybridized to a solid support such as a flowcell surface prior to cluster amplification actually occurring. After analysis of the images, one is able to estimate how many clusters would be made if those molecules were taken on to sequencing. A decision can then be made as to how to process the flowcell. If the estimated density is within a desired range, then the molecules simply need to be 1.sup.st extended and amplified, for example using bridge amplification. If a lower density is desired, then the original molecules can be stripped off (dehybridized), and a lower concentration of template used to seed clusters. If a higher density is desired, then the original molecules can be 1.sup.st extended, and a second template mix used to increase the number of clusters made. In cases where the library concentration is very low it is possible to carry out additional rounds of hybridizing and extension as required. This will enable users to more reliably obtain required target cluster densities, so optimizing throughput on sequencing platforms and minimizing wastage and further improving the user experience.
Prior to sequencing, the target DNA is tagged and fragmented. This can be done using Illumina's Nextera technology where tagging and fragmentation is carried out in a single step often referred to as tagmentation. Transposomes simultaneously fragment the DNA and add adapter sequences to the ends. The tagmented (tagged and fragmented) DNA is then amplified by limited cycle PCR which also add indexes and sequencing primer sequences required for cluster formation. In sequencing by synthesis (SBS) methods, libraries are constructed and, regardless of the library construction method, libraries submitted for SBS will generally consist of a sequence of interest flanked on either side by adapter constructs. On each end, these adapter constructs have flow cell binding sites which allow the library fragment to attach to a flow cell surface. The constructs also contain several other primer binding sites including sequencing binding sites e.g. SBS3 /SBS12.
Before attachment to the flowcell, library fragments are denatured, and thus a single-stranded copy of the library fragment is copied by extension. The P5' (SEQ ID NO 1) and P7' (SEQ ID NO 2) regions of single-stranded library fragments anneal to their complementary oligonucleotides which are immobilized on the flowcell surface. Usually at this stage, the flowcell oligonucleotides act as primers and a strand complementary to the library fragment is synthesized. The original strand is washed away, leaving behind fragment copies that are covalently attached to the flowcell surface and turned into a clonal population, for example many-fold copies of each fragment are generated by using bridge amplification. However, the present method allows the hybridized strands to be imaged or counted at the single molecule level, prior to cluster formation. It has surprisingly been found that this determination of the number of single molecule hybridized strands enables the general prediction of what cluster numbers will be obtained after bridge amplification thus allowing possible corrections to be made to ensure maximum use of the flow cell is achieved (see pages 13 and 14).
Rasolonjatovo et al additionally teach once an estimate of cluster numbers has been obtained, a decision can be made as to whether correction of underloading or overloading is required. For example, a flowcell user would look for an optimum cluster density of 1 to 1.3 million clusters per mm.sup.2. If the estimated cluster density is within the desired range, then a first extension is carried out to copy the molecules onto the flowcell surface, before standard cluster amplification and processing. If the estimated cluster density is too high, adjustment can be made by stripping off the original molecules (for example by washing with sodium hydroxide and rehybridizing at a lower template concentration. The system then proceeds to first extension to copy the molecules onto the flowcell surface, before standard cluster amplification and processing (page 1-4, 16 and 18; See also Claims 1, 10). Rasolonjatovo et al teach wherein the PCR performed in the method may encompass limited cycles of PCR (see page 2).
With regards to the limitations concerning seeding a flow cell with a known concentration of polynucleotide for at least 1 minute, Rasolonjatov et al teaches that flow cell user look for optimum cluster density of 1 to 1 to 1.3 million clusters per mm2 based on an estimation of the desired cluster range. The reference teaches that corrections can be made by lower concentration of the library for second hybridization (page 16). Thus, implying that the concentration of the library is seeded at a known concentration.
Nonetheless in a method for preparing polynucleotide libraries for use in e.g., a flow cell in sequencing platforms [0111], [0139] and [0150]), Vuyisich et al teach that a plurality of libraries can have known amounts or concentration of nucleic acid, and/or have amounts of nucleic acid in know molar ratios. These libraries can be used to produce pooled libraries [0125]. Vuyisich et al teach these pooled sample can be used to populate a sequencing flow cell [0139]. This it would have been obvious to seed the flowcell with a know concentration of polynucleotides for at least one minute for the obvious benefit of allowing for binding before sequencing start as suggested by Vuyisich.
While Rasolonjatovo and Vuyisich mentions concentrations of template used to seed cluster for evaluating cluster efficiency, the reference does not expressly teach reseeding a flow cell with polynucleotide to analyze seeding efficiency of the flow cell. However, the Rasolonjatovo teaches lowering concentration to correct flow cell if desired clustered are not obtain, which suggest a step of reseeding the flowcell for seeding efficiency.
Nonetheless, Riggatti teaches a process of seeding concatemer DNA molecule of Figure 9A onto a flow cell (page 10 and figure 9B) and seeded DNA molecules to P5 primers and p7 primers (Figure 5, pages 11 and 12 and Figure 7, pages 15-16). Riggatti teaches to increase sequencing accuracy, creating molecules by linear amplification of a particular surface bound library molecule prior to clonal amplification, the molecules created by said linear amplification remains in close proximity (page 4, last paragraph). In the linear amplification process, only the original seeded DNA molecules are used as templates to produce new molecules. Because only the original seeded DNA molecules are used as templates, errors that may be introduced during linear amplification do not get propagated (page 12, last paragraph). The reference teaches Concatemer DNA molecule 925 may be used to seed cluster growth as described with reference to Figure 9B. The linear copies are all formed by copying the original circular molecule and therefore any mis-incorporation events happening during the RCA step are not propagated. Figure 9B illustrate a side view of a sequencing structure 950 and shows an example of a process of seeding concatemer DNA molecule of Figure 9A onto a flow cell. Sequencing structure 950 includes a solid support 955. In one example, solid support 955 is a flow cell. Bound on the surface of solid support 955 is a plurality of P5 oligonucleotide primers 960 and a plurality of P7 oligonucleotide primers 965. Concatemer DNA molecule 925 is seeded onto the surface of solid support 955 and hybridizes to P5 oligonucleotide primers 960. Cluster growth is initiated by an initial extension of most if not all linear copies contained in concatemer DNA molecule 925. A standard cluster amplification process may then be used (if required) to provide sufficient material for subsequent sequencing (pages 17 and 18) see also claims which discuss reseeding complementary strands.
While both Rasolonjatovo et al in view of Vuyisich et al and Riggatto et al discloses amplification of the polynucleotide on the flow cell, the references do not expressly teach wherein the amplification is qPCR or that the flowcell is a patterned flow cell.
In a general teaching for optimizing cluster density on Illumina sequencing system, Illumina teaches that qPCR is the most effective method of library quantification when paired with a standard of similar range which is useful for detecting clustering efficiency. The reference teaches that this technique is powerful as it measures only functional library fragments rather than all DNA species within a sample (e.g., primer dimers, free nucleotides, library fragment) (page 7). Illumina teaches that analyzing clyster density can be performed on patterned flow cells systems which consist of a nanowell substrate with billions of ordered wells. The reference teaches that the uniform cluster sizes enable optimal spacing and increased cluster density. In fact, over clustering is not possible on patterned flow cells (page 8).
It would have been prima facie obvious to one of ordinary skill in the art at the time of the effective filing date of the claimed invention to have been motivated to have combined the teachings of Rasolonjatovo et al in view of Vuyisich et al with the teaching of Riggatti and Illumina because the combination of the references all deal with increasing sequencing accuracy by analyzing cluster efficiencies. The ordinary artisan would have been motivated to incorporate qPCR and pattern flow cell sequencing systems as taught by Illumina into the methods of Rasolonjatovo, Vuyisich et al and Riggatti for the benefit of increasing cluster density to improve sequencing accuracies as suggested by Illumina.
Conclusion
8. No claims are allowed. Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
9. Any inquiry concerning this communication or earlier communications from the examiner should be directed to CYNTHIA B WILDER whose telephone number is (571)272-0791. The examiner can normally be reached Flexible.
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/CYNTHIA B WILDER/Primary Examiner, Art Unit 1681