DETAILED ACTION
Notice of Pre-AIA or AIA Status
1. 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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 12/10/2025 has been entered.
Election/Restrictions
2. Applicant's traversal of the prior restriction requirement in the reply filed on 26 June 2025 is acknowledged. The traversal is on the grounds that Groups III and IV are linked by a special technical feature not present in Blainey et al (International Patent Application No. WO-2019084046, published 2019-05-02). This is found persuasive given the amendments to claims 71 and 82, however upon further searching all of the limitations presented in independent claim 71 are found in Blainey et al (International Patent Application No. WO-2019084046, published 2019-05-02) in view of Cusanovich et al (Multiplex single-cell profiling of chromatin accessibility by combinatorial cellular indexing, Science, 348, 6237, 910-914, published 22 May 2015). Therefore there is no special technical feature present linking groups III and IV, and the restriction is still deemed proper.
Priority
3. Applicant’s claim for the benefit of a prior-filed application under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged. Applicant has not complied with one or more conditions for receiving the benefit of an earlier filing date under 35 U.S.C. 112 as follows:
The later-filed application must be an application for a patent for an invention which is also disclosed in the prior application (the parent or original nonprovisional application or provisional application). The disclosure of the invention in the parent application and in the later-filed application must be sufficient to comply with the requirements of 35 U.S.C. 112(a) or the first paragraph of pre-AIA 35 U.S.C. 112, except for the best mode requirement. See Transco Products, Inc. v. Performance Contracting, Inc., 38 F.3d 551, 32 USPQ2d 1077 (Fed. Cir. 1994).
An initial review of the disclosure of the prior-filed provisional application, Application No. 62/950,670, failed to provide adequate support or enablement in the manner provided by 35 U.S.C. 112(a) or pre-AIA 35 U.S.C. 112, first paragraph for one or more claims of this application.
Claim 96 is drawn to “…wherein there are no greater than 6 nucleotides between the 2 index sequences.” The provisional application does not describe or illustrate or suggest this limitation and therefore does not provide adequate support under 35 U.S.C. § 112 for the invention of claim 96. Therefore the effective filing date of claim 96 is 12/18/2020.
Claim Rejections - 35 USC § 112
4. The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
5. Claims 71-73, 81, 86-94 and 96-101 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
A. Amended claim 71 recites the phrase “wherein the indexed library comprises a plurality of modified target nucleic acids and are derived from DNA or protein of the cells or nuclei” however it is not made clear in the claim language nor in the applicant’s specification how the indexed library is, or how it could possibly be, derived from protein of the cells or nuclei. The metes and bounds of how this language is intended to limit the claim is unclear, and therefore the claim is indefinite.
B. Each claim not directly addressed in this rejection is rejected for being dependent on a previously rejected claim.
Claim Rejections - 35 USC § 103
6. 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.
7. 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.
8. Claims 71-73, 81, 86-94, 96, and 99-101 are rejected under 35 U.S.C. 103 as being unpatentable over Blainey et al (International Patent Application No. WO-2019084046, published 2019-05-02) in view of Cusanovich et al (Multiplex single-cell profiling of chromatin accessibility by combinatorial cellular indexing, Science, 348, 6237, 910-914, published 22 May 2015).
Regarding claim 71, Blainey teaches a method for sequencing a single cell (abstract, [0008] and [0314]), providing a library comprising specific cell barcodes (i.e., indexes; [008] and [0314]), wherein the indexed library comprises a population of RNA sequencing libraries (i.e., a plurality of modified nucleic acids ([0008] and [0314]), and wherein the modified target nucleic acids comprise a unique cell specific barcode (i.e., at least one index; [0008] and [0314]). Blainey teaches that these unique cell specific identifiers are produced in a combinatorial fashion by combining indices (i.e., the modified nucleic acid comprises two or more index sequence; [0118]) and that each such index has a distinct sequence (i.e., is unique [0118]). Blainey additionally teaches identifying the barcode (i.e., a marker specific index) associated with transcripts from a specific cell (i.e., a biological feature; [0008] and [0314]).
It is noted that the applicant’s specification defines a “marker specific index” as a “unique grouping of index sequences” associated with a biological feature ([00185]). This is consistent with the teaching of a cell specific barcode as taught by Blainey ([0008] and [0314]). It is additionally noted that without further limitation the phrase “…derived from DNA…” recited in amended claim 1 is interpreted to include RNA, because biologically RNA is transcribed from (i.e., derived from) DNA.
Blainey teaches the enrichment of a subgroup of transcripts associated with the biological feature of interest ([0009] and [0315]) and the amplification of this subgroup to prepare a library with increased representation of a particular cell barcode (i.e., marker index) compared to other nucleic acids that to not have that cell barcode ([0011] and [0315]). Blainey teaches that this enriched pool is then further sequenced ([0281] and [0315]).
Blainey specifically teaches that the indexed library is a library of RNA transcripts ([0008]-[0010]), Blainey does not teach that the index is associated with DNA of each cell or nucleus in the sample. However, Cusanovich teaches the single-cell indexing of transposase accessible chromatin (i.e., DNA) with dual indices to provide a unique cell barcode (FIG 1 and pg. 910 column 3 ¶ 1).
It would have been obvious to one having ordinary skill in the art to have simply substituted the uniquely indexed cDNA library taught by Blainey with the uniquely indexed ATAC library taught by Cusanovich to arrive at the instantly claimed invention with a reasonable expectation of success. The ordinary artisan would have been motivated to make this substitution because the method taught by Blainey discloses the enrichment of DNA molecules derived from RNA, which is directly analogous to the DNA library taught by Cusanovich. Blainey additionally teaches that any single cell library is used with their method, including DNA libraries such as those derived from ATAC-Seq ([0092]). In addition, one having ordinary skill in the art would have recognized that the known techniques in the cited references could have been combined with predictable results because the known techniques in the cited references predictably result in the single-cell sequencing of DNA libraries.
Regarding claim 72, Cusanovich teaches that the indexed libraries are derived from DNA (pg. 910 column 3 ¶ 3 and FIG 1).
Regarding claim 73, Cusanovich teaches the indexing of transposase accessible chromatin (pg. 910 column 3 ¶ 2). In this embodiment, the regions of “transposase accessible chromatin” are the claimed biological features.
Regarding claim 81, Blainey teaches that the barcodes are identified by sequencing ([0010] and [0171]).
Regarding claim 86, Blainey teaches preparing a library from a population of cells (i.e., multiple samples [0008]).
Regarding claim 87, Blainey teaches that samples comprise tissue samples ([0101]) and that samples come from different locations ([0092]).
Regarding claim 88, it is noted that applicant’s specification defines “targeted sequencing” as the isolation of genes or regions of interest by PCR or hybridization-based capture methods ([0028]). Blainey teaches the targeted sequencing of particular regions of interest by hybridization pull down to determine a cell specific barcode ([0185] and [0186]). It is again noted that the applicant’s specification defines a “marker index sequence” as a unique index associated with a biological feature.
Regarding claim 89, Blainey teaches a multiplexed second PCR enrichment step using a panel of unique cell barcodes ([0281]). This process would inherently require the determination of “more than one” marker index sequence.
Regarding claim 90, Blainey teaches enriching modified nucleic acids comprising the marker index sequence ([0281]).
Regarding claim 91, Blainey teaches the depletion of strands not amplified by a barcode-specific primer (i.e., not having the correct marker index sequence) by UDG and exonuclease digestion ([0165]).
Regarding claims 92 and 93, Cusanovich teaches that the combination of barcodes (i.e., unique indices) represents either a mouse or human nucleus (i.e., the species of the cell; pg. 911 column 2 ¶ 2).
Regarding claim 94, Blainey teaches that specific cells are different types of T cells (i.e., different cell classes).
Regarding claim 96, it is noted that the instant claims do not define any particular compartment nor do they specifically define what a “compartment-specific index” is. Therefore, given the breadth of this claim, any cell specific barcode taught by Blainey is a contiguous index of at least 2 compartment-specific indexes, wherein there are no greater than 6 nucleotides between the 2 index sequences (e.g., the 16 base pair cell barcode from [0295] is 2 contiguous 8 base pair indexes).
Regarding claims 99-101, Cusanovich teaches that providing the indexed library comprises processing nuclei from human and mouse cell lines (i.e., mammals) wherein the library is produced with the single-cell combinatorial indexing method ATAC-seq (i.e., assay for transposase-accessible chromatin; pg. 910 column 1 ¶ 1 and column 3 ¶ 4).
9. Claims 97 and 98 are rejected under 35 U.S.C. 103 as being unpatentable over Blainey et al (International Patent Application No. WO-2019084046, published 2019-05-02) and Cusanovich et al (Multiplex single-cell profiling of chromatin accessibility by combinatorial cellular indexing, Science, 348, 6237, 910-914, published 22 May 2015) as described in claim 71 above, and further in view of Wilson et al (Actionable Diagnosis of Neuroleptospirosis by Next-Generation Sequencing, N Engl J Med, 370, 25, 2408-2417, published 2014-06-19)
Regarding claims 97 and 98, Blainey teaches that a scRNA-seq library is prepared having cell specific barcodes (i.e., indexes [0008] and [0027]).
Blainey does not teach that the sample is a metagenomics sample obtained from an organism, wherein that organism is a mammal.
However, Wilson teaches a next-generation sequencing library prepared from cerebrospinal fluid from a human patient (i.e., an organism wherein the organism is a mammal; abstract) for the purpose of pathogen detection (i.e., metagenomics; pg. 2411, column 1, ¶ 2).
It would have been obvious to one having ordinary skill in the art to have prepared the next generation sequencing sample taught by Wilson with a sequencing method providing cell-specific barcodes as taught by Cusanovich (FIG 1 and pg. 910 column 3 ¶ 3), and to have enriched library members associated with the pathogen to perform a deeper follow-up sequencing analysis on those specific cells as taught by Blainey ([0278]). The ordinary artisan would have been motivated to make this combination in order to identify additional characteristics of an identified pathogen, such as antibiotic resistance. In addition, it would have been obvious to the ordinary artisan that the known techniques of the cited references could have been combined with predictable results, because the known techniques of the cited references predictably result in the preparation and analysis of sequencing libraries.
10. Claims 71-73, 81, 86-94, 96 and 99-101 are rejected under 35 U.S.C. 103 as being unpatentable over Cusanovich et al (Multiplex single-cell profiling of chromatin accessibility by combinatorial cellular indexing, Science348, 6237, 910-914, published 22 May 2015) in view of Blainey et al (International Patent Application No. WO-2019084046, published 02 May 2019).
Regarding claim 71, Cusanovich teaches a method of sequencing a single cell or nucleus (abstract) comprising providing an indexed library of each cell or nuclei in a sample (abstract; pg. 910 column 3 ¶ 3; FIG 1) wherein the indexed library comprises a plurality of modified target nucleic acids that are derived from nuclear DNA (the nucleic acids are ‘modified’ by transposition and PCR; pg. 910 column 3 ¶ 2 and 3). Cusanovich teaches that the target nucleic acids comprise a transposition index and a PCR index (i.e., two or more indexes; pg. 910 column 3 ¶ 2 and 3) and that the 2 or more indexes are unique (pg. 910 column 3 ¶ 1).
Cusanovich does not teach the limitations of instant steps (b) – (d).
However, Blainey teaches that certain genes (i.e., biological features) are used to identify subsets of cells based on the barcodes identifying those cells within sequencing libraries ([0088]). Blainey teaches that these barcodes are assembled in a combinatorial fashion by split-pool synthesis (i.e., the modified nucleic acids comprise two or more index sequences; [0118]), using these barcodes to enrich sequencing libraries comprising specific molecules of interest ([0088]) and that library enrichment boosts cDNAs derived from rare cells for resequencing (i.e., the sub-library comprises increased representation of the modified target nucleic acids; [0171]).
It would have been obvious to one having ordinary skill in the art to have modified the ATAC-seq library preparation taught by Cusanovich to have incorporated the library enrichment and resequencing steps taught by Blainey to arrive at the instantly claimed invention with a reasonable expectation of success. The ordinary artisan would have been motivated to make these modifications because Blainey specifically teaches that identifying and accessing rare cells is becoming increasingly important ([0006]), and Blainey specifically teaches that their methods are performed on ATAC-seq libraries (e.g., such as those taught by Cusanovich; [0092]). In addition, one having ordinary skill in the art would have recognized that the known techniques in the cited references could have been combined with predictable results because the known techniques in the cited references predictably result in the preparation of single-cell sequencing libraries.
Regarding claim 72, Cusanovich teaches that the indexed libraries are derived from DNA (pg. 910 column 3 ¶ 3 and FIG 1).
Regarding claim 73, Cusanovich teaches the indexing of transposase accessible chromatin (pg. 910 column 3 ¶ 2). In this embodiment, the regions of “transposase accessible chromatin” are the claimed biological features.
Regarding claim 81, Blainey teaches that the barcodes are identified by sequencing ([0010] and [0171]).
Regarding claims 86 and 87, Cusanovich teaches an indexed library that comprises human cells and mouse cells (i.e., multiple samples from different organisms; pg. 910 column 3 ¶ 4).
Regarding claim 88, it is noted that applicant’s specification defines “targeted sequencing” as the isolation of genes or regions of interest by PCR or hybridization-based capture methods ([0028]). Blainey teaches the targeted sequencing of particular regions of interest by hybridization pull down to determine a cell specific barcode ([0185] and [0186]). It is noted that the applicant’s specification defines a “marker index sequence” as a unique index associated with a biological feature.
Regarding claim 89, Blainey teaches a multiplexed second PCR enrichment step using a panel of unique cell barcodes ([0281]). This process would inherently require the determination of “more than one” marker index sequence.
Regarding claim 90, Blainey teaches enriching modified nucleic acids comprising the marker index sequence ([0281]).
Regarding claim 91, Blainey teaches the depletion of strands not amplified by a barcode-specific primer (i.e., not having the correct marker index sequence) by UDG and exonuclease digestion ([0165]).
Regarding claims 92 and 93, Cusanovich teaches that the combination of barcodes (i.e., unique indices) represents either a mouse or human nucleus (i.e., the species of the cell; pg. 911 column 2 ¶ 2).
Regarding claim 94, Blainey teaches that specific cells are different types of T cells (i.e., different cell classes).
Regarding claim 96, it is noted that the instant claims do not define any particular compartment nor do they specifically define what a “compartment-specific index” is. Therefore, given the breadth of this claim, any cell specific barcode taught by Blainey is a contiguous index of at least 2 compartment-specific indexes, wherein there are no greater than 6 nucleotides between the 2 index sequences (e.g., the 16 base pair cell barcode from [0295] is 2 contiguous 8 base pair indexes).
Regarding claims 99-101, Cusanovich teaches that providing the indexed library comprises processing nuclei from human and mouse cell lines (i.e., mammals) wherein the library is produced with the single-cell combinatorial indexing method ATAC-seq (i.e., assay for transposase-accessible chromatin; pg. 910 column 1 ¶ 1 and column 3 ¶ 4).
11. Claims 97 and 98 are rejected under 35 U.S.C. 103 as being unpatentable over Cusanovich et al (Multiplex single-cell profiling of chromatin accessibility by combinatorial cellular indexing, Science, 348, 6237, 910-914, published 22 May 2015) and Blainey et al (International Patent Application No. WO-2019084046, published 2019-05-02) as described in claim 71 above, and further in view of Wilson et al (Actionable Diagnosis of Neuroleptospirosis by Next-Generation Sequencing, N Engl J Med, 370, 25, 2408-2417, published 2014-06-19)
Regarding claims 97 and 98, Cusanovich in view of Blainey teaches that an indexed library is prepared having cell specific barcodes as discussed fully above and incorporated here.
Neither Cusanovich nor Blainey teach that the sample is a metagenomics sample obtained from an organism, wherein that organism is a mammal.
However, Wilson teaches a next-generation sequencing library prepared from cerebrospinal fluid from a human patient (i.e., an organism wherein the organism is a mammal; abstract) for the purpose of pathogen detection (i.e., metagenomics; pg. 2411, column 1, ¶ 2).
It would have been obvious to one having ordinary skill in the art to have prepared the next generation sequencing sample taught by Wilson with a sequencing method providing cell-specific barcodes as taught by Cusanovich (FIG 1 and pg. 910 column 3 ¶ 3), and to have enriched library members associated with the pathogen to perform a deeper follow-up sequencing analysis on those specific cells as taught by Blainey ([0278]) to arrive at the instantly claimed invention with a reasonable expectation of success. The ordinary artisan would have been motivated to make this combination in order to identify additional characteristics of an identified pathogen, such as antibiotic resistance. In addition, it would have been obvious to the ordinary artisan that the known techniques of the cited references could have been combined with predictable results, because the known techniques of the cited references predictably result in the preparation and analysis of sequencing libraries.
Response to Arguments
12. Applicant’s arguments, see pg. 2-4 of applicant’s remarks, filed 10 December 2025, with respect to the rejections of claims 71-73, 81, 86-94 and 96-101 under U.S.C. § 103 have been fully considered but are not found persuasive.
In applicant’s remarks pgs. 2-4, applicant argues that the motivation provided to combine Blainey and Cusanovich in the Office Action dated 19 September 2025 would change the principle operation of Blainey because Blainey teaches that the RNA transcriptome is an essential component in the identification of rare events. This is not found persuasive, because Blainey specifically teaches that any single cell library is used for their disclosed method, including ATAC-Seq libraries (such as those taught by Cusanovich), and that the libraries are composed of DNA molecules or RNA molecules. Therefore this argument is not found persuasive and the rejection is maintained.
Additionally, a new grounds of rejection is made based on Cusanovich in view of Blainey. As described fully above and incorporated here, the ordinary artisan would have additionally been motivated to modify Cusanovich using the disclosed method of Blainey to identify and enrich desired sequences based on their unique combinatorial index (i.e., their marker index sequence) to arrive at the instantly claimed invention with a reasonable expectation of success.
Conclusion
13. No claims are allowed.
14. Any inquiry concerning this communication or earlier communications from the examiner should be directed to BRIAN ELLIS YOUNG whose telephone number is (703)756-5397. The examiner can normally be reached M-T 0800 - 1630.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Heather Calamita can be reached on (571) 272-2876. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/BRIAN ELLIS YOUNG/Examiner, Art Unit 1684
/JULIET C SWITZER/Primary Examiner, Art Unit 1682
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