DETAILED ACTION
Status of the Claims
Claims 1-20 are currently pending.
Claims 15-20 have been withdrawn as being drawn to non-elected subject matter (see below).
Claims 1-14 are examined herein.
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Response to Restriction Requirement
Applicant’s election without traverse of Group I (claims 1-14) in the reply filed on 03/10/2026 is acknowledged.
Claims 15-20 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to nonelected groups, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 03/10/2026.
Claim Objections
Claim 1 is objected to because of the following informalities: line 36 recites the term “sequencesand”, which appears to be a typo for “sequences and”. Appropriate correction is required.
Claim Rejections - 35 USC § 112(b) -- Indefiniteness
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.
Claim 14 is 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 pre-AIA the applicant regards as the invention.
Claim 14 recites a Markush-type group “consisting of genomic DNA or cDNA”, which should instead be “consisting of genomic DNA and cDNA”. See MPEP § 2173.05(h).
Since the two options are currently in the alternative, the metes and bounds of the claim are unascertainable.
As per MPEP 2173: It is of utmost importance that patents issue with definite claims that clearly and precisely inform persons skilled in the art of the boundaries of protected subject matter. Therefore, claims that do not meet this standard must be rejected under 35 U.S.C. 112(b) or pre-AIA 35 U.S.C. 112, second paragraph as indefinite. Further, as per MPEP 2173.02: If the language of the claim is such that a person of ordinary skill in the art could not interpret the metes and bounds of the claim so as to understand how to avoid infringement, a rejection of the claim under 35 U.S.C. 112, second paragraph, would be appropriate. As currently written, the metes and bounds of the rejected claims are unascertainable for the reasons set forth above, thus the above claim(s) and all dependent claims are rejected under 35 USC 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph.
Claim Rejections – 35 U.S.C. 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
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.
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.
The factual inquiries set forth in Graham v. John Deere Co., 383 U.S. 1, 148 USPQ 459 (1966), that are applied for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
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.
Lebofsky et al. and Chen et al.
Claims 1-14 are rejected under 35 U.S.C. 103 as being unpatentable over Lebofsky et al. (U.S. PGPub 2020/0056231 A1, cited in IDS of 06/01/2023) in view of Chen et al. (ACS Applied Materials & Interfaces, 2018, 10(14):11539-11545, cited in IDS of 07/01/2025).
Regarding claim 1, Lebofsky discloses a method of deconvoluting sequencing reads from partitions, the method comprising:
performing tagmentation of nucleic acids in permeabilized cells (e.g., permeabilized as per para [0089]) in a mixture, thereby forming at least one cleavage site in a target nucleic acid from one of the cells to form a first nucleic acid fragment and a second nucleic acid fragment, wherein the first and second nucleic acid fragments have at the cleavage site a single-stranded 9 nucleotide sequence, which are complementary to each other, linked to a transposase oligonucleotide delivered by a tagmentation transposase, wherein the transposase oligonucleotide has a double-stranded portion, and a single-stranded 5' portion comprising a universal sequence (e.g., tagmenting DNA using adaptor-loaded tagmentase on beads as per para [0084]-[0094]);
forming a plurality of partitions from the mixture and a plurality of beads and the permeabilized cells (e.g., forming partitions as per para [0084] and/or [0091]), wherein one of the partitions comprise the first nucleic acid fragment and second nucleic acid fragment and at least two beads, wherein the beads are linked to 5' ends of a plurality of clonal barcoding oligonucleotides, the barcoding oligonucleotides comprising a 5' PCR handle sequence, a 3' capture sequence and a barcode sequence unique to the bead to which the barcode oligonucleotide is linked, wherein the 3' capture sequence comprises a copy of said universal sequence (e.g., beads with capture sequences linked to barcodes as per para [0096]);
gap-filling the single-stranded 5' portion of the transposase oligonucleotide to form a reverse complement of the 5' portion and gap-filling the 9 nucleotide sequences, wherein the gap filling comprises using a polymerase to insert nucleotides using the single stranded sequences as a template (e.g., during template-based polymerase extension as per para [0093]-[0097]);
hybridizing the 3' capture sequence of different barcoding oligonucleotides from different beads to the reverse complement of the 5' portion on the first and second nucleic acid fragments and extending the 3' capture sequence of the different barcoding oligonucleotides in a template-dependent manner with a polymerase to form barcoded first and second nucleic acid fragments (e.g., template-based polymerase extension as per para [0093]-[0097]);
amplifying the barcoded first and second nucleic acid fragments with primers that hybridize to the PCR handle sequences (e.g., amplifying as per para [0101]); and
generating sequencing reads from the amplified barcoded first and second nucleic acid fragments, wherein the sequencing reads include the barcode sequence, the 9 nucleotide sequence and at least a portion of the nucleic acid fragment from the cell (e.g., sequencing as per para [0102]-[0115]).
However, Lebofsky is silent to the limitations of identifying in the sequence reads the genomic location relative to the nucleic acid fragment and sequence identity of the 9 nucleotide sequence and determining sequencing reads having barcodes from the amplified barcoded first and second barcoding oligonucleotides were from the same partition if the 9 nucleotide sequences in the sequencing reads are reverse complementary sequences and the 9 nucleotide sequences in the sequencing reads are 5' to adjacent genomic positions, as set forth in claim 1. Rather, Lebofsky uses bead-specific barcoding of exogenous DNAs randomly added to the partitions to determine if a partition contained more than one bead-specific barcode (e.g., as per para [0093]).
Chen discloses a similar method of tagmentation on microbeads (e.g., as per the Abstract), but instead uses the complimentary 9-nucleotide sequences generated from Tn5 cleavage to link two reads as originating from the same cleavage event from the same bead and thus spatially near (e.g., as per Fig. 1).
It would have been prima facie obvious to a person of ordinary skill in the art prior to the effective filing date of the application to utilize the complimentary 9-nucleotide sequences to identify spatially-linked Tn5 cleavage events as per Chen in the partitioned tagmentation reactions of Lebofsky. One of ordinary skill in the art would have been motivated to do so since this would avoid the need to add exogenous DNA (as per Lebofsky) therefore reducing the complexity, time, and cost of the data collection and analysis.
One of ordinary skill in the art would have had a reasonable expectation of success as of the application’s effective filing date in combining the teachings of the prior art references to arrive at the invention as presently claimed since Lebofsky already sequences the 9-nucleotide complimentary sequences as per para [0115], which states (in part): “Identical fragments can be determined for example, by comparing start and stop sequences (i.e., end portions) of the DNA template fragment portion of different sequencing reads from all fragments that are contained within a partition, wherein identical start sequences and stop sequences of two DNA template fragment sequencing reads indicates the two origin DNA fragments are identical.”
Regarding claim 2, Lebofsky discloses the above, wherein the nucleic acids in the permeabilized cells are chromosomal DNA and different chromosomal sequences differ in how accessible the different chromosomal sequences are to the transposase (e.g., accessible chromatin as per para [0089]).
Regarding claim 3, Lebofsky discloses the above, wherein the nucleic acids in the permeabilized cells have been stripped of histones (e.g., DNA binding proteins are removed as per [0090]).
Regarding claim 4, Lebofsky discloses the above, wherein the single-stranded 5' portion of the transposase oligonucleotide comprises (ii) a unique molecular identifier barcode sequence (e.g., as per para [0056]).
Regarding claim 5, Lebofsky discloses the above, wherein the unique molecular barcode sequence is 4-10 bp long (e.g., as per para [0056]).
Regarding claim 6, Lebofsky discloses the above, wherein the single-stranded 5' portion of the transposase oligonucleotide comprises a multiplexing identifier sequence that distinguishes different samples (e.g., as per para [0056]).
Regarding claim 7, Lebofsky discloses the above, wherein the multiplexing identifier sequence is 4-10 bp long (e.g., as per para [0056]).
Regarding claim 8, Lebofsky discloses the above, wherein the nucleic acids in permeabilized cells are DNA (e.g., DNA as per [0089]).
Regarding claim 9, Lebofsky discloses the above, wherein the method comprises forming first strand cDNAs or double-stranded cDNAs in the permeabilized cells and the nucleic acids comprise cDNA (e.g., as per para [0095]-[0097]).
Regarding claim 10, Lebofsky discloses the above, wherein the DNA is cellular genomic DNA (e.g., as per para [0089]).
Regarding claim 11, Lebofsky discloses the above, wherein the partitions are droplets in a water-in-oil emulsion (e.g., water and oil droplets as per para [0055]).
Regarding claim 12, Lebofsky discloses the above, wherein the partitions are microwells (e.g., multi-well microtiter dish as per para [0055]).
Regarding claim 13, Lebofsky discloses the above, wherein the tagging further comprises tagging nucleic acids in the cells such that two or more types of nucleic acids are tagged and subsequently sequenced (e.g., as per para [0085] and/or [0097]).
Regarding claim 14, Lebofsky discloses the above, wherein the two types of nucleic acids are selected from the group consisting of genomic DNA and cDNA (e.g., as per para [0085] and/or [0097]).
Conclusion
No claims are allowed.
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/JEREMY C FLINDERS/
Primary Examiner, Art Unit 1684
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