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 .
2. 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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 08 October 2025 has been entered.
Claim Rejections - 35 USC § 103
3. 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.
4. Claims 1, 4 and 9-11, and 17-23 are rejected under 35 U.S.C. 103 as being unpatentable over Regev et al (US Patent Application No. US 20180030515 A1, published 2018-02-01) in view of Di et al (RNA sequencing by direct tagmentation of RNA/DNA hybrids, PNAS, 117(6), 2886-2893, published 11 February 2020), Zhu et al (ATAC-seq with unique molecular identifiers improves quantification and footprinting, BioRxiv, doi: https://doi.org/10.1101/2020.10.22.351478, published 23 October 2020), Hatori et al (Particle-templated emulsification for microfluidics-free digital biology, Analytical Chemistry, 90, 9813-9820, published 23 July 2018), Wu et al (International Patent Application No. WO 2018136248 A1, published 2018-07-26), and Smith et al (UMI-tools: modeling sequencing errors in unique molecular identifiers to improve quantification accuracy, Genome Research, 27, 491-499, published 2017-01-18).
Regarding claim 1, Regev teaches a mixture of cells comprising RNA and a plurality of beads ([0119]), and that the beads comprising individual primers (i.e., capture probes) that share the same cell barcode ([0118]). Regev teaches partitioning the cells and barcode beads (i.e., the template particles) into droplets ([0119]) by shearing two liquid phases to form an emulsion ([0114]) and that the droplets comprise individual cells (i.e., one cell) and one distinctly barcoded microparticle ([0190]), lysing the cells to release their RNA, and breaking the emulsion after capturing the mRNA onto the template particles ([0119]). Regev additionally teaches reverse transcribing the RNAs ([0119]) to generate RNA/cDNA hybrids on the template particles (i.e., double stranded polynucleotides; [0118]).
Regev does not teach that the cells comprising RNA and the template particles are in a single mixture, nor do they teach fragmenting the double-stranded polynucleotides at random locations with a transposon comprising a random N-mer to result in tagmented polynucleotides with unique labels and the cell barcode, wherein the unique labels are defined by different combinations of the random transposon locations and the random N-mers.
However, Hatori teaches a method of producing emulsions comprising either one or zero cells and a single bead per droplet. Hatori teaches that these droplets are prepared from a mixture comprising cells (cells inherently comprise RNA) and template particles (pg. 9818, column 2, ¶ 2).
Di teaches the random fragmentation of double-stranded polynucleotides (e.g., the RNA/cDNA hybrids generated in the method taught by Regev) to result in tagmented polynucleotides (abstract, FIG 1, and pg. 2887 column 1 ¶ 2) and amplifying the tagmented polynucleotides to create amplicons (FIG 1, FIG 2, and pg. 2888 column 1 ¶ 3). Di further teaches the sequencing of the amplicons to obtain sequencing reads (pg. 2889, column 1, ¶ 2).
Neither Regev, Hatori, nor Di, alone or in combination, teach that the transposon comprises a random N-mer and that the tagmented nucleotides comprise unique labels that are defined by different combinations of the random transposon locations and the random N-mers.
However, Zhu teaches the incorporation of UMI sequences (i.e., random N-mers; Supplementary Table 1) into Tn5 transposon complexes (pg. 5 ¶ 1), which are then incorporated into the tagmented polynucleotides (pg. 6 ¶ 2). This, in combination with the random tagmentation of the double-stranded polynucleotides as taught by Di, inherently produces tagmented polynucleotides with unique labels that are defined by different combination of the random transposon locations and the random UMIs (i.e., random N-mers).
It would have been obvious to one having ordinary skill in the art to have modified the single-cell RNA sequencing method taught by Regev with the emulsification method taught by Hatori. The ordinary artisan would have been motivated to make this combination in order to prepare single cell droplets comprising a single template particle without the need for specialized microfluidic chips and control systems (pg. 9818, column 2, ¶ 2). It would have additionally been obvious to one having ordinary skill in the art to further modify the combined method of Regev and Hatori with the RNA/cDNA polynucleotide tagmentation taught by Di and the incorporation of UMIs into the transposon complex as taught by Zhu, to arrive at the instantly claimed invention with a reasonable expectation of success. It is noted that while Regev teaches the presence of UMI sequences on the RNA capture beads ([0118]), however these UMIs only label the ends of each captured RNA molecule. The addition of UMIs into the transposome complex provides the additional advantage of labeling each fragment with a UMI for PCR deduplication. The ordinary artisan would have been motivated to make these modifications in order to add specific adapter sequences to the RNA/cDNA duplex polynucleotides (as taught by Di) and to incorporate UMIs into each of the tagmented polynucleotides in order to distinguish unique polynucleotide fragments from PCR duplicates (as taught by Zhu). In addition, one having ordinary skill in the art would have recognized that the know techniques of the cited references could have been combined with predictable results because the known techniques of the cited references predictably result in the design, formation, or preparation of sequencing libraries.
Neither Regev, Di, Zhu, or Hatori teach a UMI or barcode (i.e., random N-mers) that provide a total number of sequence combinations that are substantially less than the amount of distinct RNA species in the sample. This combination also does not teach that some of the cDNA polynucleotides comprise identical oligos.
However, Wu teaches a method wherein a “less unique” UMI (i.e., a random N-mer) is used in conjunction with other identification techniques (i.e., sequence alignment location) to create unique DNA molecules comprising a random nucleic acid fragment and a random UMI. Wu teaches that multiple fragments comprise the same UMI ([0064]).
It would have been obvious to a person having ordinary skill in the art to have simply substituted the barcodes/UMIs taught by the combination of Regev, Di, Zhu and Hatori with the less unique UMI taught by Wu 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 removing the need for having a unique UMI sequence for every labeled RNA fragment provides the advantage of being able to use shorter UMI sequences and freeing up sequence space in short-read sequencers. In addition, it would have been obvious to one having ordinary skill in the art 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 methods for the identification of nucleic acid sequences using UMIs.
The combination of Regev, Di, Zhu and Hatori teaches the method of library preparation and sequencing, but they do not teach the method of analyzing sequencing reads to identify PCR duplicates.
However, Smith teaches that identifying duplicate sequences during sequencing comprises aligning the sequencing reads to a reference genome to determine genomic coordinates corresponding to random fragments of DNA (pg. 494, column 2, ¶ 1).
It would have been obvious to a person having ordinary skill in the art to have modified the sequencing methods taught by the combination of Regev, Di, Zhu, Hatori and Wu with the processing methods taught by Smith to arrive at the instantly claimed invention with a reasonable expectation of success. The ordinary artisan would have been motivated to make this modification because said modification would provide superior estimations of unique molecules sequences as taught by Smith (pg. 496, column 2, ¶ 2).
Regarding claim 9, Hatori teaches that the template particles introduce reagents for cell lysis (pg. 9813, column 2, ¶ 2).
Regarding claim 10, Regev teaches that the particles comprise an oligo-dT (i.e., a poly-T) sequence ([0069]).
Regarding claim 11, Regev teaches that, after hybridization, the RNA is reverse transcribed into complementary DNA ([0069]).
Regarding claim 17, Smith teaches that two sequencing reads with the same genomic coordinates are identified as potential PCR duplicates (pg. 494, column 2, ¶ 1).
Regarding claim 18, Smith teaches that once potential PCR duplicates are identified via the method of claim 17, PCR duplicates are removed using the UMI sequence (i.e., the random N-mers; pg. 494, column 2, ¶ 1).
Regarding claims 19 and 20, Hatori teaches that single cells and hydrogel particles are captured in droplet emulsions via emulsification by vortexing (i.e., shaken with a homogenizer; FIG 1, FIG 6, and pg. 9818 column 2 ¶ 2). Droplet emulsification by vortexing inherently comprises shearing forces between two immiscible layers.
Regarding claim 21, Regev teaches that the RNA captured by beads is mRNA ([0119]).
Regarding claim 22, Di teaches that the method of reverse transcribing RNA using a polyT primer and fragmenting the RNA/cDNA polynucleotides biases the RNA-seq signal (i.e., the sequencing library) towards the 3' end of the mRNA (pg. 2892, column 1, ¶ 4).
Regarding claim 23, Regev teaches amplifying the barcoded polynucleotides prior to transposase-based fragmentation ([0201]).
Response to Arguments
5. Applicant’s arguments, see Remarks, filed 26 May 2026, with respect to the rejections of claims 1, 4, 9-11 and 17-23 under U.S.C. § 103 have been fully considered and are persuasive. Therefore, the rejection has been withdrawn. However, upon further consideration, a new ground(s) of rejection is made in view of Regev et al (US Patent Application No. US 20180030515 A1, published 2018-02-01) in view of Di et al (RNA sequencing by direct tagmentation of RNA/DNA hybrids, PNAS, 117(6), 2886-2893, published 11 February 2020), Zhu et al (ATAC-seq with unique molecular identifiers improves quantification and footprinting, BioRxiv, doi: https://doi.org/10.1101/2020.10.22.351478, published 23 October 2020), Hatori et al (Particle-templated emulsification for microfluidics-free digital biology, Analytical Chemistry, 90, 9813-9820, published 23 July 2018), Wu et al (International Patent Application No. WO 2018136248 A1, published 2018-07-26), and Smith et al (UMI-tools: modeling sequencing errors in unique molecular identifiers to improve quantification accuracy, Genome Research, 27, 491-499, published 2017-01-18).
6. 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.
Conclusion
7. No claims are allowed.
8. 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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/BRIAN ELLIS YOUNG/Examiner, Art Unit 1684
/JULIET C SWITZER/Primary Examiner, Art Unit 1682
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