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 .
Claim Status and Action Summary
This action is in response to the papers filed on May 27, 2026.
Claims 2-3 were canceled in the response. Currently, claims 1, 5-6, 12-16, 18, 20-22, 25-27, and 36-38 are pending and under examination.
Any objections and rejections not reiterated below are hereby withdrawn.
The objections to the specification over the use of trade names in the specification have been withdrawn in view of the amendments to the specification.
The rejections under 35 U.S.C. 112(b) and (d) have been withdrawn in view of the amendments to the claims.
The rejection of record of claims 1-3, 5-6, 12-16, 18, 20-22, 26-27, and 36-38 under 35 U.S.C. 103 as being obvious over Belgrader et al., US 2020/0002764 A1 (published January 2, 2020) in view of Pratt et al., US 2020/0377937 A1 (published December 3, 2020) has been withdrawn because the response, signed by the applicant’s representative, includes the following clear and conspicuous statement that the claimed invention of the application under examination and the subject matter disclosed in the Pratt et al. reference were owned by the same person or subject to an obligation of assignment to the same person not later than the effective filing date of the claimed invention (see Remarks page 14, dated 05/27/2026):
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Priority
The present application, filed on September 13, 2023, is a 371 of PCT/US2022/071272, filed on March 22, 2022 and claims priority to U.S. Provisional Patent Application No: 63/164,958, filed on March 23, 2021.
Drawings
The drawings filed on September 13, 2023 are acceptable.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1, 5-6, 12-16, 18, 20-22, 25-27, and 36-38 are rejected under 35 U.S.C. 103 as being unpatentable over Belgrader et al., US 2020/0002764 A1 (published January 2, 2020) in view of Rigatti et al., US 2013/0281306 A1 (published October 24, 2013).
This rejection has been updated as necessitated by the amendments to the claims.
Regarding claim 1, Belgrader et al. teach methods of sequencing polynucleotides comprising two or more barcode regions on the same end of a polynucleotide relative to a region of interest (Figures 9A, 51A, and paragraph 0673).
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Belgrader et al. further teach hybridizing a sequencing primer to the polynucleotide, wherein the polynucleotide comprises an intervening (i.e. spacer) sequence between the first and second barcode sequences (Belgrader et al., paragraph 0604-0605), and sequencing the barcode sequences (Belgrader et al., paragraph 0052-0054 and paragraphs 0604-0605) by known sequencing techniques such as “Illumina sequencing” (Belgrader et al., paragraph 0004). (i.e. combining the hybrid with labeled nucleotides and detecting the presence of incorporated nucleotides in a plurality of sequencing flow steps).
Belgrader et al. further teach that the barcode and intervening sequences are known (Belgrader et al., paragraphs 0551-0557, 0608, and Figure 51A).
Belgrader et al. does not teach extending the sequencing primer through the intervening sequence using a set of one or more dark sequencing flow steps wherein nucleotides are incorporated into the polynucleotide without detecting the presence or absence of the incorporated nucleotide(s).
However, Rigatti et al. teach methods of sequencing polynucleotides comprising “fast forward” “limited dark steps” (Rigatti et al., figure 5 and paragraphs 0325-0328) (i.e. extending a sequencing primer through an intervening region without detecting the presence or absence of incorporated nucleotide(s)).
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Rigatti et al. further teach that “a barcode sequence can include an ordered series of determined regions and at least one dark region” and “at least a portion of the sequence of the dark region may be undetermined” (Rigatti et al., paragraphs 0306-0307) (i.e. also reading on the implicit alternative wherein the sequence of the dark region is not undetermined). Rigatti et al. further teach “Sequence information obtained using iterations of at least one limited dark extension step and at least one limited read extension step can produce a molecular signature for a target nucleic acid that is predictable and informative (Rigatti et al., paragraph 0165). Rigatti et al. also teaches the “fast forward” sequence synthesized by the limited dark extension steps is known (Figure 5 and paragraphs 0362-0364). Rigatti et al. teach the dark sequencing flow steps extend the primer during each dark sequencing flow step (Rigatti et al., Figure 2A and paragraph 0353).
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 have modified the method taught by Belgrader et al. comprising identifying uniquely barcoded nucleic acids from single cells wherein two or more barcode sequences are separated by a spacer (i.e. intervening) sequence with the teachings of Rigatti et al. that limited dark sequencing flow steps can be implemented to “fast forward” through non-informative sequence between barcode sequences that “produce a molecular signature for a target nucleic acid that is predictable and informative” (Rigatti et al., paragraph 0165). The ordinary artisan would have been motivated to modify the sequencing methods taught by Belgrader et al. with the limited dark sequencing steps taught by Rigatti et al. by the suggestion of Rigatti et al. that target nucleic acids can be identified by their molecular signatures comprising iterations of read extension steps and dark extension steps. Additionally, the ordinary artisan would have recognized the predictable advantages of reducing the amount of sequencing data required to identify particular target sequences in sequencing methods wherein the total number of read cycles is limited per flow cell/sequencing run and by extension, determines the total cost and time required for obtaining said sequencing data. As made explicit by Rigatti in paragraph 0171, “the sequence of a target nucleic acid may be predicted, determined concurrently in real-time or previously known. Additionally, in performing a series of limited dark extension steps, it may be desirable to minimize the number of repeated limited dark extension steps in a homopolymer sequence (e.g. poly-A). In this example, a sequencing reagent containing at least one type of nucleotide monomer including `T` could be utilized.” (Rigatti et al., paragraph 0171).
Regarding claim 5, Rigatti et al. teach sequencing the region of interest (Rigatti et al., paragraphs 0073 and 0288). Belgrader et al. also teach sequencing the region of interest (Belgrader et al., paragraph 0274)
Regarding claim 6, Belgrader et al. teach sequencing the two barcodes (i.e. “BC” and “UMI” from the “Read 1 primer” prior to sequencing the region of interest (i.e. V D J C) (Belgrader et al., figure 84B).
Regarding claim 12, Belgrader et al. teach associating each sequencing read (i.e. region of interest) with its barcode sequences (Belgrader et al., paragraph 0064).
Regarding claims 13-14, Rigatti et al. teach the nucleotides used in the sequencing flow steps are non-terminating nucleotides (Rigatti et al., paragraph 0166-0171).
Regarding claim 15, Rigatti et al. teach that the nucleotides used in the sequencing by synthesis steps can be labeled or unlabeled (Rigatti et al., paragraph 0242).
Regarding claim 16, Rigatti et al. teach that dark extensions (i.e. dark sequencing flow steps) include incorporating nucleotide monomer(s) without detecting the incorporation (Rigatti et al., paragraph 0162). Furthermore, Rigatti et al. teaches an example wherein a dark extension step is performed with nucleotide monomers only and two read extension steps are performed with nucleotide monomers comprising labels (Rigatti et al., paragraph 0325) (i.e. the nucleotides in the read extension, but not the dark extension step comprise labels).
Regarding claim 18, Belgrader et al. teach that the polynucleotide may further comprise a unique molecular identifier (Belgrader et al., paragraph 0011 and 0029).
Regarding claim 20, Belgrader et al. teach the polynucleotide can be a cDNA molecule (Belgrader et al., paragraph 0031).
Regarding claim 21, Rigatti et al. teach that each of first or second plurality of sequencing flow steps comprise a single type of nucleotide base (either mixtures of combinations of labeled nucleotides, or sequential addition of a single type of each of the four labeled nucleotides) (i.e. consist of a single type of nucleotide base) (Rigatti et al., paragraph 0168).
Regarding claim 22, Rigatti et al. teach the nucleotides used in the first and second plurality of sequencing flow steps can comprise limited read extension steps including “doublet” and “triplet” deliveries of nucleotide monomers (i.e. comprising two or three different types of nucleotide bases) (Rigatti et al., paragraph 0170).
Regarding claims 25-26, Rigatti et al. teach examples of “limited dark extension” wherein “no more than one type of nucleotide monomer is incorporated without being detected” or “two or more types of nucleotide monomers are incorporated without being detected” or “three or more types of nucleotide monomers are incorporated without being detected” (Rigatti et al., paragraph 0162).
Regarding claim 27, Belgrader et al. teach the barcode regions, in combination, uniquely identify a cell of origin for the polynucleotide (Belgrader et al., paragraph 0317).
Regarding claim 36, Belgrader et al. teach the sequence of each of a plurality of polynucleotides each having different sequences are determined in parallel (Belgrader et al., paragraph 0317 and 0406).
Regarding claim 37, Belgrader et al. teach that cDNAs (i.e. polynucleotides) derived from the same cell will include common barcode sequences as well as a unique molecular identifier enabling quantitation of the different mRNA molecules present in that particular single cell (Belgrader et al., paragraph 0317).
Regarding claim 38, Belgrader et al. teach associating polynucleotides having the same barcode sequences to the same cell of origin (Belgrader et al., paragraph 0317).
Response to arguments
The response asserts that the claims as amended now recite limitations not taught by the prior art, namely, “the intervening region has a known sequence and that the set of one or more dark sequencing flow step” is configured to extend the primer during each dark sequencing step. Because each dark sequencing flow step in amended claim 1 incorporates one or more nucleotides, the total number of dark sequencing flow steps needed to extend the primer through the intervening region is minimized” (Response, page 11-12).
These assertions regarding the teachings of the prior art have been thoroughly reviewed and are not persuasive. As is addressed in the rejection above, Belgrader et al. in view of Rigatti et al. teach embodiments wherein the sequence of an intervening region between two barcode sequences is known (Belgrader et al., paragraphs 0551-0557, 0608, and Figure 51A); see also, as discussed at length above, (Rigatti et al., paragraphs 0165, 0306-0307, and 0362-0364).
The response asserts that Rigatti does not “teach the dark sequencing flow steps extend the primer during each dark sequencing flow step”… For instance…” and references several working examples taught by Rigatti to assert that Rigatti teaches that “limited dark extension step cycles may extend a primer by an average, not predetermined number of nucleotides… and contemplates that some dark extension steps fail to extend” (with reference to Examples 1 and 2).
These arguments have been thoroughly reviewed and are not persuasive. The teachings of Rigatti et al. are not limited to the particular examples cited by the response. As is discussed above, Rigatti et al. teach embodiments wherein the sequence of the intervening template nucleotides are known, and that it is desirable to minimize the number of repeated limited dark extension steps. Even more, Rigatti et al. demonstrate specific implementations of such “fast forward” sequencing wherein a computer system is used to determine an appropriate composition of sequencing reagents (i.e. at least one of A/C/T/G) to be delivered for a particular extension step wherein the template sequence being synthesized is known (Rigatti et al., paragraph 0282-0283).
The response further asserts that the technical problems addressed by Rigatti et al. and are “entirely different from that of Belgrader et al. and the subject application such that one skilled in the art would not have been motivated to [combine the references]”. The response asserts that: a) Belgrader et al. describes accurately sequencing barcodes requiring accuracy of sequencing results at each position in the barcode to correctly identify the nucleic acid molecules in question and b) The methods of Rigatti: do not guarantee primer extension at every step, would risk misaligning the resulting sequence reads, are not positioned a priori relative to high resolution read regions, and have an “objective… to obtain… a low resolution representation of a target nucleic acid”.
These assertions have been thoroughly reviewed and are not persuasive. As addressed above, the teachings of Rigatti et al. are not limited to examples wherein the sequence of the intervening (i.e. dark) sequence between two sequences that are read, but clearly contemplate embodiments wherein the entire sequence is known and motivate the minimization of the number of dark flow steps required to “fast-forward” through the uninformative intervening sequence.
For all of the reasons set forth above and those already of record, the claims remain rejected as unpatentable under 35 U.S.C. 103 over Belgrader et al. in view of Rigatti et al.
Conclusion
No claim is allowed.
THIS ACTION IS MADE FINAL. 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.
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/Z.M.T./Examiner, Art Unit 1682
/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682
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