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 .
DETAILED ACTION
Claims 10-13, 20-25, 28, 30-32, 34, 36, 38, 39, 41 and 47 are currently pending in the instant application, filed October 15, 2023.
Therefore, claims 10-13, 20-25, 28, 30-32, 34, 36, 38, 39, 41 and 47 are under consideration to which the following grounds of rejection are applicable.
Priority
The present application filed April 20, 2023 is a CON of PCT/US2021/057742, filed November 2, 2021, which claims the benefit of US Provisional Patent Application 63108565, filed November 2, 2020.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on April 20, 2023 has been considered. An initialed copy of the IDS accompanies this Office Action.
Claim Objections/Rejections
Claim Interpretation: based on the teachings of the as-filed Specification (paragraph [0122]), claim 34 is interpreted to refer to an RNA-Seq library that comprises a step selected from the different steps as recited in instant claim 34.
Claim Objection
Claims 10, 12, 20, 28, 30-32 and 34 are objected to because of the following informalities: Claims 10, 12, 20, 28, 30-32 and 34 recite terms such as: PCR, DNase, CviQI, NIaIII, Pmel, dNTPs, and bp, where an abbreviation should be spelled out in the first encounter of the claims.
Appropriate correction is required.
Specification
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code, such as in paragraphs [0095]-[0097]; [0141]; [0143]-[0146]; [0200]; [0202]-[0205]; [0285]; [0287]-[0289]. Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code. See MPEP § 608.01.
Claim Rejections - 35 USC § 112(b)
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.
Claims 10-13, 20-25, 28, 30-32, 34, 36, 38, 39, 41 and 47 are rejected under 35 U.S.C. 112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter which applicant regards as the invention.
Claims 10 and 12 are indefinite for the recitation of the term “the transcriptome” such as recited in claim 10, line 6. There is insufficient antecedent basis for the term “the transcriptome” in the claim. The Examiner suggests that Applicant amend claim 10, line 6 to recite, for example, “a transcriptome.”
Claim 10 is indefinite for the recitation of the term “the collected RNA” such as recited in claim 10, line 7. There is insufficient antecedent basis for the term “the collected RNA” in the claim because claim 10, line 6 recites the term “collecting cytoplasmic and nucleic RNA.” The Examiner suggests that Applicant amend claim 10 to recite, for example, “the collected cytoplasmic and nucleic RNA.”
Claim 11 is indefinite for the recitation of the terms “nuclei,” “population of cells,” “first restriction enzyme,” “isolated nuclei,” “assembled Tn5 transposome,” “Tn5 adaptors,” “proximal genomic DNA,” “crosslink,” “splint oligonucleotide,” “reverse cross-linked DNA,” “second restriction enzyme,” and “third restriction enzyme” because claim 11 depends from instant claim 10, where claim 10 does not recite nuclei, cells, restriction enzymes, splint oligonucleotides, an assembled Tn5 transposome, Tn5 adaptors, proximal genomic DNA, reverse cross-linked DNA, crosslinks, and/or crosslinking and, thus, the metes and bounds of the claim cannot be determined.
Claims 11 is indefinite for the recitation of the term “a population of cells” such as recited in claim
11, line 3. There is insufficient antecedent basis for the term “a population of cells” in the claim because claim 10, line 1 recites the term “a single population of cells.”
Claim 11 is indefinite for the recitation of the term “the digested nuclei” such as recited in claim 11, line 6. There is insufficient antecedent basis for the term “the digested nuclei” in the claim because claim 11, line 5 recites the term “digesting the isolated nuclei.”.
Claim 11 is indefinite for the recitation of the term “the Tn5 adaptors” such as recited in claim 11, line 7. There is insufficient antecedent basis for the term “the Tn5 adaptors” in the claim.
Claim 11 is indefinite for the recitation of the term “the proximal genomic DNA” such as recited in claim 11, line 7. There is insufficient antecedent basis for the term “proximal genomic DNA” in the claim.
Claims 11 and 12 are indefinite for the recitation of the term “the crosslink” such as recited in claim 11, line 8. There is insufficient antecedent basis for the term “the crosslink” in the claim.
Claims 11 and 12 are indefinite for the recitation of the term “the reverse cross-linked DNA” such as recited in claim 11, line 9. There is insufficient antecedent basis for the term “the reverse cross-linked DNA” in the claim because claim 11, line 8 recites the term “reversing the crosslink.”
Claims 11 and 12 are indefinite for the recitation of the term “the purified DNA” such as recited in claim 11, lines 9, 10 and 12. There is insufficient antecedent basis for the term “the purified DNA” in the claim because claim 11, line 9 recites the term “purifying the reverse cross-linked DNA.”
Claim 11 is indefinite for the recitation of the term “the digested DNA” such as recited in claim 11, line 11. There is insufficient antecedent basis for the term “the digested DNA” in the claim because claim 11, line 10 recites the term “digesting the purified DNA.”
Claim 11 is indefinite for the recitation of the term “the circularized DNA” such as recited in claim 11, line 11. There is insufficient antecedent basis for the term “the circularized DNA” in the claim because claim 11, line 11 recites the term “circularizing the digested DNA.”
Claim 12 is indefinite for the recitation of the term “the supernatant” such as recited in claim 12, line 3 because claim 12 depends from instant claim 10, wherein claim 10 does not recite the presence of a supernatant and, thus, the metes and bounds of the claim cannot be determined.
Claims 12, 22 and 34 are indefinite for the recitation of the term “cytoplasmic RNA” such as recited in claim 12, line 3. There is insufficient antecedent basis for the term “cytoplasmic RNA” in the
claim because claim 10, line 6 recite the term “cytoplasmic and nucleic RNA.”
Claims 12, 23 and 34 are indefinite for the recitation of the term “nucleic RNA” such as recited in claim 12, line 4. There is insufficient antecedent basis for the term “nucleic RNA” in the claim because claim 10, line 6 recite the term “cytoplasmic and nucleic RNA.”
Claim 12 is indefinite for the recitation of the term “the purified RNA” such as recited in claim 12, lines 7 and 8. There is insufficient antecedent basis for the term “the purified RNA” in the claim because claim 12, line 6 recite the term “purifying the reverse crosslinked RNA.”
Claim 12 is indefinite for the recitation of the term “creating an RNA-Seq library” such as recited in claim 12, line 9 because claim 12 depends from instant claim 10, wherein claim 10, line 7 recites analyzing the transcriptome followed by “creating an RNA-Seq library using the collected RNA.” such that it is unclear how analyzing can be carried out by skipping the analyzing step recited in claim 10. Moreover, it is unclear whether the RNA-Seq library created according to claim 12 is created from some other RNA and, thus, the metes and bounds of the claim cannot be determined.
Claim 13 is indefinite for the recitation of the term “the resulting DNA library” such as recited in claim 13, lines 1-2. There is insufficient antecedent basis for the term “the resulting DNA library” in the claim.
Claim 13 is indefinite for the recitation of the terms “the resulting DNA library,” “bioinformatics software program,” “uniquely mapped paired-end tags,” “cis-regulatory chromatin contacts,” and “anchor interaction anchor,” because claim 13 depends from instant claim 10, where claim 10 does not recite a resulting DNA library, a computer, a bioinformatics software program, uniquely mapped paired-end tags, cis-regulatory chromatin contacts, and/or anchor interaction anchors and, thus, the metes and bounds of the claim cannot be determined.
Claim 13 is indefinite for the recitation of the term “the uniquely mapped paired-end tags” such as recited in claim 13, line 3. There is insufficient antecedent basis for the term “the uniquely mapped paired-end tags” in the claim.
Claim 13 is indefinite for the recitation of the term “anchor interaction anchor” in claim 13, lines 5-6 because the as-filed Specification does not teach the term “anchor interaction anchor” such that it is unclear what the term refers to and/or how a cumulative interactive score can be calculated for each “anchor interaction anchor” and, thus, the metes and bounds of the claim cannot be determined.
Claims 21-23 are indefinite for the recitation of the term “the isolating nuclei step” such as recited in claim 21, line 2. There is insufficient antecedent basis for the term “the isolating nuclei step” in the claim. Moreover, claims 21-23 depend from instant claims 10 and 11, wherein claims 10 and 11 do not recite a “step” of isolating nuclei and, thus, the metes and bounds of the claim cannot be determined.
Claim 24 is indefinite for the recitation of the term “assembling the Tn5 transposome” such as recited in claim 24, lines 1-2 because claim 24 depends from claims 10 and 11, wherein claims 10 and 11 do not recite a Tn5 transposome such that it is unclear what is being assembled and, thus, the metes and bounds of the claim cannot be determined.
Claim 22 is indefinite for the recitation of the term “the cells” such as recited in claim 22, line 2. There is insufficient antecedent basis for the term “the cells” in the claim because claim 11, line 3 recites that term “a population of cells.”
Claim 28 is indefinite for the recitation of the term “the performing PCR step” such as recited in claim 28, line 1. There is insufficient antecedent basis for the term “the performing PCR step” in the claim. Moreover, claim 28 depends from instant claims 10 and 11, wherein claims 10 and 11 do not recite a “step” of performing PCR and, thus, the metes and bounds of the claim cannot be determined.
Claim 28 is indefinite for the recitation of the term “the digested purified DNA” such as recited in claim 28, line 2. There is insufficient antecedent basis for the term “the digested purified DNA” in the claim.
Claim 30 is indefinite for the recitation of the term “the resulting amplified DNA fragments” such as recited in claim 30, lines 1-2. There is insufficient antecedent basis for the term “the resulting amplified DNA fragments” in the claim. Moreover, claim 30 depends from claims 10, 11 and 28, where claims 10, 11 and 28 do not recite fragmenting DNA, a Tn5-tagmented open chromatin sequence, and/or different ends derived from different processes and, thus, the metes and bounds of the claim cannot be determined.
Claim 30 is indefinite for the recitation of the term “the Tn5-tagmented open chromatin sequence” such as recited in claim 30, line 3. There is insufficient antecedent basis for the term “the Tn5-tagmented open chromatin sequence” in the claim.
Claim 31 is indefinite for the recitation of the term “the end” such as recited in claim 31, lines 1-2. There is insufficient antecedent basis for the term “the end” in the claim because claim 31 depends from instant claim 30, which recites the term “one end.”
Claim 31 is indefinite for the recitation of the term “the CviQI digested genomic DNA is captured by Read 1 of each pair-end sequence…by Read 2 of each pair-end sequence” such as recited in claim 31, lines 1-3 because claim 31 depends from instant claims 10, 11, 28 and 30, where none of claims 10, 11, 28 and 30 recite capturing digested genomic DNA, Read 1, Read 2, and/or pair-end sequences and, thus, the metes and bounds of the claim cannot be determined.
Claim 32 is indefinite for the recitation of the term “those PCR products” such as recited in claim 32, line 2. There is insufficient antecedent basis for the term “those PCR products” in the claim. Moreover, claim 32 depends from claims 10, 11 and 28, wherein claims 10, 11 and 28 do not recite the presence of PCR products and, thus, the metes and bounds of the claim cannot be determined.
Claim 32 is indefinite for the recitation of the term “the gel extracted PCR products” such as recited in claim 32, lines 2-3. There is insufficient antecedent basis for the term “the gel extracted PCR products” in the claim because claim 32, line 2 recites that term “those PCR products.”
Claim 34 is indefinite for the recitation of the term “creating an RNA-Seq library comprises combining supernatant comprising cytoplasmic RNA…and creating an RNA-Seq library” such as recited in claim 34, lines 1-5 because claim 34 depends from instant claim 12, wherein claim 12 recites each of these steps; and it is completely unclear how the process of “creating an RNA-Seq library comprises” as recited in line 1 includes a step of “creating an RNA sequencing library” as recited in line 5, such that the claim is entirely unclear and, thus, the metes and bounds of the claim cannot be determined.
Claims 36 and 41 indefinite for the recitation of the term “the method” such as recited in claim 36, line 1. There is insufficient antecedent basis for the term “the method” in the claim because claim 36, line 1 recites the term “the method of claim 10.”
Claim 36 is indefinite for the recitation of the term “does not comprise antibody-mediated immunoprecipitations, adaptor ligation, or biotin pull down” such as recited in claim 36, lines 1-2 because claim 36 depends from instant claim 10, wherein claim 10 does not recite antibody-mediated immunoprecipitations, adaptor ligation, or biotin pull down and, thus, the metes and bounds of the claim cannot be determined.
Claim 38 is indefinite for the recitation of the term “the population of cells comprise cells obtained from a biosample and then subjected to a crosslinking protocol” such as recited in claim 38, lines 1-2 because claim 38 depends from instant claims 10 and 11, wherein claims 10 and 11 do not recite obtaining a population of cells, a biosample, subjecting a population of cells to a reaction, and/or a crosslinking protocol and, thus, the metes and bounds of the claim cannot be determined.
Claim 39 is indefinite for the recitation of the term “the biosample is obtained from a subject diagnosed with or is suspected of having a disease or disorder” such as recited in claim 39, lines 1-2 because claim 39 depends from instant claims 10, 11 and 38, wherein claims 10, 11 and 38 do not recite obtaining a biosample, a subject, and/or diagnosing (or having diagnosed) a disease or disorder and, thus, the metes and bounds of the claim cannot be determined.
Claim 41 is indefinite for the recitation of the term “repeating the method using a second population of cells” such as recited in claim 41, line 2 because it is unclear what method is being repeated. Moreover, claim 41 depends from claim 10, wherein claim 10 does not recite the presence of a population of cells (e.g., cells are recited only in the preamble of the claim), such that it is completely unclear where cells including a second population of cells originates and, thus, the metes and bounds of the claim cannot be determined.
Claim 47 is indefinite for the recitation of the term “one or more components and/or reagents for use in the method of claim 10” such as recited in claim 47, lines 1-2 because it is unclear what components and/or reagents are encompassed by the terms, such that they can be used in the method of claim 10 including whether the terms encompass devices, apparatus, flow cells, columns, electrodes, humans, heat, weather, solvents, water, etc. and, thus, the metes and bounds of the claim cannot be determined.
Claims 20 and 25 are indefinite insofar as they ultimately depend from instant claim 10.
Claim Rejections - 35 USC § 112(d)
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claims 11-13, 21-24, 28, 30-32, 36, 38 and 39 are rejected under 35 U.S.C. 112(d) as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 11 recites (in part): “wherein purifying and tagmenting DNA comprises one or more of the following…or any combination thereof” such as recited in claim 11, lines 1-13 because claim 11 depends from instant claim 10, where claim 10 does not recite nuclei, cells, restriction enzymes, splint oligonucleotides, an assembled Tn5 transposome, Tn5 adaptors, proximal genomic DNA, reverse cross-linked DNA, crosslinks, and/or crosslinking. Thus, claim 11 is an improper dependent claim for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 12 recites (in part): “wherein analyzing the transcriptome comprises one or more of the following…or any combination thereof” such as recited in claim 12, lines 1-10 because claim 12 depends from claim 10, wherein claim 10 does not recite crosslinked RNA and/or supernatants. Thus, claim 12 is an improper dependent claim for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 13 recites (in part): “further comprising processing the resulting DNA library…or any combination thereof” such as recited in claim 13, lines 1-6 because claim 13 depends from instant claim 10, where claim 10 does not recite a resulting DNA library, a computer, a bioinformatics software program, uniquely mapped paired-end tags, cis-regulatory chromatin contacts, and/or anchor interaction anchors. Thus, claim 13 is an improper dependent claim for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claims 21-23 recite (in part): “the isolating nuclei step” such as recited in claim 21, lines 1-2 because claims 21-23 depend from instant claims 10 and 11, where claims 10 and 11 do not recite a an isolating nuclei step. Thus, claims 21-23 are improper dependent claims for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 24 recites (in part): “assembling the Tn5 transposome” such as recited in claim 24, lines 1-2 because claim 24 depends from claims 10 and 11, wherein claims 10 and 11 do not recite the presence of a Tn5 transposome. Thus, claim 24 is an improper dependent claim for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 28 recites (in part): “wherein the performing PCR step comprises mixing the digested purified DNA with…and a polymerase” such as recited in claim 28, lines 1-3 because claim 28 depends from instant claims 10 and 11, wherein claims 10 and 11 do not recite a “step” of performing PCR. Thus, claim 28 is an improper dependent claim for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 30 recites (in part): “wherein the resulting amplified DNA fragments contain…open chromatin sequence” such as recited in claim 30, lines 1-3 because claim 30 depends from claims 10, 11 and 28, where claims 10, 11 and 28 do not recite fragmenting DNA, a Tn5-tagmented open chromatin sequence, and/or different ends derived from different processes. Thus, claim 30 is an improper dependent claim for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 31 recites (in part): “wherein the end derived from the CviQI digested genomic DNA is capture…by Read 2 of each pair-end sequence” such as recited in claim 30, lines 1-3 because claim 31 depends from instant claims 10, 11, 28 and 30, where none of claims 10, 11, 28 and 30 recite capturing digested genomic DNA, Read 1, Read 2, and/or pair-end sequences. Thus, claim 31 is an improper dependent claim for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 32 recites (in part): “using gel extraction to obtain those PCR products having a size of about 400-600 bp” such as recited in claim 32, lines 1-2 because claim 32 depends from instant claims 10, 11 and 28, wherein claims 10, 11 and 28 do not recite the presence of PCR products. Thus, claim 32 is an improper dependent claim for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 36 recites (in part): “wherein the method does not comprise…or biotin pull down” such as recited in claim 36, lines 1-2 because claim 36 depends from instant claim 10, wherein claim 10 does not recite antibody-mediated immunoprecipitations, adaptor ligation, or biotin pull down. Thus, claim 36 is an improper dependent claim for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 38 recites (in part): “wherein the population of cells…then subjected to a crosslinking protocol” such as recited in claim 38, lines 1-2 because claim 38 depends from instant claims 10 and 11, wherein claims 10 and 11 do not recite obtaining a population of cells, a biosample, subjecting a population of cells to a reaction, and/or a crosslinking protocol. Thus, claim 38 is an improper dependent claim for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 39 recites (in part): “wherein the biosample is obtained from a subject…or disorder” such as recited in claim 39, lines 1-2 because claim 39 depends from instant claims 10, 11 and 38, wherein claims 10, 11 and 38 do not recite obtaining a biosample, a subject, and/or diagnosing (or having diagnosed) a disease or disorder. Thus, claim 39 is an improper dependent claim for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Applicant may cancel the claim, amend the claim to place the claim in proper dependent form, rewrite the claim in independent form, or present a sufficient showing that the dependent claim complies with the statutory requirements.
Claim Rejections - 35 USC § 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.
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 may not be obtained though the invention is not identically disclosed or described as set forth in section 102 of this title, if the differences between the subject matter sought to be patented and the prior art are such that the subject matter as a whole would have been obvious at the time the invention was made to a person having ordinary skill in the art to which said subject matter pertains. Patentability shall not be negatived by the manner in which the invention was made.
The factual inquiries 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.
Claims 10-13, 20-25, 28, 30-32, 34, 36, 38, 39, 41 and 47 are rejected under 35 U.S.C. 103 as being unpatentable over Shendure et al. (hereinafter “Shendure”) (US Patent Application Publication No. 20220356461, published November 10, 2022; effective filing date December 19, 2019) in view of Green et. al. (hereinafter “Green”) (US Patent No. 10089437, issued October 2, 2018) as evidenced by Picelli et al. (hereinafter “Picelli”) (Genome Research, 2014, 24, 2033-2040).
Regarding claims 1, 36 and 47, Shendure teaches methods for preparing a sequencing library that includes nucleic acids from a plurality of single cells, wherein the sequencing library includes nucleic acids that represent the chromatin accessibility from the plurality of single cells including three index sequences, such that the present disclosure provides methods for characterizing rare events in isolated cells and nuclei (interpreted as generating a DNA library; a plurality of cells; isolating nuclei; and components or reagents for the method, claims 10 and 47) (Abstract). Shendure teaches that Cicero co-accessibility scores and Cicero gene activity scores for each tissue in the dataset were generated, wherein Cicero co-accessibility scores can be used to predict cis-regulatory interactions between accessible elements, wherein the elements paired by positive co-accessibility scores were combined to create a database of putative cis-regulatory interactions including 80 million unique co-accessible pairs including 4.5 million (6%) promoter-distal pairs, 76 million (94%) distal-distal pairs and 128,000 (0.2%) promoter-promoter pairs (interpreted as identifying cis-regulatory chromatin interactions, claim 10) (paragraph [0363]). Shendure teaches the development of an improved assay for single cell profiling of chromatin accessibility based on three-level combinatorial indexing (sci-ATAC-seq3) and applied it to 59 fetal samples representing 15 organs, altogether profiling on the order of one million single cells.(interpreted as characterizing chromatin accessibility; and cells as components or reagents for the method, claims 10 and 47) (paragraph [0329], lines 3-7). Shendure teaches that Figure 9 shows a schematic of sci-ATAC-seq3, wherein nuclei of 1.6 million cells from 59 fetal samples were tagmented with Tn5 transposase in bulk, such that the first two rounds of indexing were achieved by successive ligation to each end of the Tn5 transposase complex, and the third round by PCR, such that the first round of indexing was used as a sample index (interpreted as ATAC-Seq; isolating nuclei; ATAC-Seq including Tn5 adaptors; tagmenting DNA; Tn5 transposase; performing PCR; and paired-end tags, claims 10-13) (paragraph [0067]; and Figure 9). Figure 9 is shown below:
PNG
media_image1.png
752
608
media_image1.png
Greyscale
Figure 9
Shendure teaches that extracted nuclei were purified by one or more rounds of washing with a nuclei buffer (interpreted as purifying DNA; and extraction, claim 10) (paragraph [0080], lines 27-28). Shendure teaches a method for preparing a sequencing library that includes nucleic acids from a plurality of single nuclei or cells, wherein the method includes providing a plurality of nuclei or cells, where the nuclei or cells include nucleosomes and contacting the plurality of nuclei or cells with a transposome complex that includes a transposase and a universal sequence, wherein the plurality of nuclei or cells are in bulk when contacted with the transposome complex, and/or when contacted with the transposome complex the plurality of nuclei or cells are distributed in a first plurality of compartments, such that the DNA molecules in each subset of nuclei or cells are processed to generate indexed nuclei or cells, where each compartment includes a subset of nuclei or cells or represents a sample, wherein processing can include ligation, primer extension, hybridization, amplification, or a combination thereof (interpreted as generating a DNA library, purifying and tagmenting; a population of cells; incubating with a transposome; adaptors; and does not comprise immunoprecipitation, adaptor ligation, or biotin pull down, claims 10 and 36) (paragraph [0007]). Shendure teaches that a sample comprises DNA, RNA, protein, or a combination thereof, wherein a sample can include any biological, clinical, surgical, agricultural, atmospheric or aquatic-based specimen containing one or more nucleic acids and/or one or more proteins including any isolated nucleic acid from sample such a genomic DNA or a transcriptome, and any isolated protein from a sample; and/or the sample includes a collection of cells or nuclei, wherein the term “nucleic acid” includes natural and non-natural DNA, mRNA, and non-coding RNA such as RNA without poly-A at the 3’ end, and nucleic acids derived from an RNA (e.g., cDNA) (interpreted as analyzing the transcriptome by collecting cytoplasmic and nucleic RNA while performing step (i), claim 10) (paragraphs [0019]; and [0022], lines 29-32). Shendure teaches that the methods provided by the present disclosure can be easily integrated into essentially any application that includes sequencing library preparation, such as whole genome, transcriptome, epigenome, accessible (e.g., ATAC), and conformational state (e.g., HiC), wherein a multitude of sequencing library methods are known to a skilled person that can be used in the construction of whole-genome or targeted libraries (paragraph [0186]). Shendure teaches that the methods are directed to detecting rare events, and can be easily integrated into essentially any application with single-cell combinatorial indexing (sci) methods including, but not limited to, whole genome (e.g., sci-WGS-seq), epigenome (e.g., sci-MET-seq), accessible (e.g., sci-ATACseq), transcriptome (sci RNA-seq), and conformational (sci-HiC-seq) (interpreted as ATAC-Seq; and creating an RNA-Seq library, claim 10) (paragraph [0187], lines 1-8). Shendure teaches that for suspension cells, obtain between ~10-100 million cells, and pellet cells by spinning at 500xg at room temperature, aspirate supernatant and resuspend pellet in Omni-ATAC lysis buffer; and crosslink the nuclei by adding 37% formaldehyde with methanol in one shot at a final concentration (interpreted as crosslinking nuclei) (paragraph [0375], lines 1-13).
Regarding claim 11, Shendure teaches providing isolated nuclei or cells, wherein the method provided herein can include providing the cells or isolated nuclei from a plurality of cells (e.g., Figure 1A, block 10, Figure 3, block 30, Figure 4, block 40, and Figure 6, block 600) (interpreted as purifying and tagmenting DNA by isolating nucleic from a population of cells, claim 11) (paragraphs [0072]-[0073], lines 1-4; and Figures 1, 3, 4 and 6). Figures 1A & 3 are shown below:
PNG
media_image2.png
708
598
media_image2.png
Greyscale
PNG
media_image3.png
560
624
media_image3.png
Greyscale
Figure 1A Figure 3
Regarding claims 12 and 34, Shendure teaches that for suspension cells, obtain between ~10-100 million cells, and pellet cells by spinning at 500xg at room temperature, aspirate supernatant and resuspend pellet in Omni-ATAC lysis buffer; and crosslink the nuclei by adding 37% formaldehyde with methanol in one shot at a final concentration (interpreted as combining supernatant, claim 12) (paragraph [0375], lines 1-13). Shendure teaches that to reverse crosslink the nuclei, make a reverse crosslink master mix of EB buffer, Proteinase k and 1% SDS, respectively per well) were added to each well of nuclei (interpreted as reversing the crosslink, claim 12) (paragraphs [0382], lines 72-76; and [0525]).
Regarding claim 13, Shendure teaches methods include conformational sci-HiC-Seq (interpreted as anchor interactions, claim 13) (paragraph [0187]). Shendure teaches that the sequence data from the targeted sequencing of the biological feature and sequencing of the indexes is then analyzed using routine bioinformatic methods, and those combinations of index sequences that are present on the same library members as the biological feature are identified (interpreted as bioinformatics software program, claim 13) (paragraph [0180], lines 1-6). Shendure teaches Tn5 Mosaic End (ME) sequences can be used (interpreted as paired-end tags, claim 13) (paragraph [0088]). Shendure teaches that gene-level accessibility scores for the scATAC-Seq data were calculated, aggregating the number of transposition events falling within gene bodies extended by 2 kb upstream of their TSS (interpreted as calculating a cumulative interactive score, claim 13) (paragraph [0343], lines 7-10). Shendure teaches that data processing for the barnyard experiments conducted to develop sci-ATAC-seq3 was done as previously described, wherein BCL files were converted to fastq files with bcl2fastq v2.16 (Illumina), wherein each read was associated with a cell barcode made up of 4 components: on the P5 end of the molecule there was a row address for tagmentation and for PCR added and on the P7 end of the molecule there was a column address for tagmentation and PCR added; and trimmed reads were mapped to a hybrid human/mouse (hg19/mm9) genome using bowtie2 with options '-X 2000-3 1' (interpreted as processing a DNA library by mapping using a bioinformatics software program, claim 13) (paragraph [0384]). Shendure teaches integrating and comparing chromatin accessibility in cell types across all 15 organs; and randomly sampling 800 cells per cell type per organ (or in cases where fewer than 800 cells of a given cell type were represented in a given organ, all cells were taken), and performed UMAP visualization (interpreted as visualizing uniquely mapped paired-ends, claim 13) (paragraph [0346]). Shendure teaches MACS2, given a BAM file as input, will either discard one of the read pairs which using R1/R2 independently (effectively down-sampling the input data) or use the entire insert when computing coverage if explicitly specifying that the BAM file is paired-end (to compute coverage along the endpoints) (interpreted as paired end tags, claim 13) (paragraph [0389], lines 13-18).
Regarding claim 21, Shendure teaches that conditions for fixing cells or nuclei with formaldehyde was tested to allow for long-term storage (interpreted as crosslinking cells before isolating nuclei, claim 20) (paragraphs [0370]). Shendure teaches that for renal and digestive organs where RNases and proteases are abundant, paraformaldehyde-fixed cells were used rather than nuclei, which increased cell and mRNA recovery (interpreted as crosslinking cells before isolating nuclei, claim 20) (paragraphs [0413], lines 6-9).
Regarding claims 22 and 23, Shendure teaches nuclei isolation and fixation of frozen fetal tissues, wherein nuclei are isolated, lysis buffer is added, the nuclei are centrifuged and the supernatant is aspirated (interpreted as collecting nucleic and cytoplasmic RNA, claim 22 and 23) (paragraphs [0378]; [380], lines 1-3 and 10-12).
Regarding claim 24, Shendure teaches that 59 fetal samples were tagmented with Tn5 transposase in bulk, wherein the first two rounds of indexing were achieved by successive ligation to each end of the Tn5 transposase complex (interpreted as assembling the Tn5 transposome, claim 24) (paragraph [0067]; and Figure 9).
Regarding claim 28, Shendure teaches that DNA nanoballs can be captured on substrates, wherein consecutive rounds of adapter ligation, amplification and digestion are carried out prior to circularization to produce head to tail constructs having several genomic DNA fragments separated by adapter sequences, wherein amplification can include PCR (interpreted as digested, purified DNA and performing PCR, claim 28) (paragraphs [0030], last two lines; and [0140], last 5 lines). Shendure teaches that amplification conditions include dNTPs to promote extension of the primer; and PCR refers to the method of Mullis (US4683195 and 4683202) for increasing the concentration of a segment of a polynucleotide of interest in a mixture of gDNA, wherein two oligonucleotide primers are introduced to the DNA mixture, followed by a series of thermal cycling in the presence of a DNA polymerase (interpreting PCR as encompassing dNTPs, primers, and a polymerase, claim 28) (paragraphs [0031], lines 1-2 and 17-19; and [0033], lines 1-11).
Regarding claim 31 (in part), Shendure teaches that a universal primer binding site is used as a site to which a universal primer (e.g., a sequencing primer for Read 1 or Read 2) anneals for sequencing (interpreted as being captured by read 1 and read 2, claim 31) (paragraph [0024]). Shendure teaches that all libraries were sequences on one NovaSeq platform (Illumina) (Read 1, 34 cycles; and Read 2, 10 cycles) (interpreted as being captured by read 1 and read 2, claim 31) (paragraph [0467]).
Regarding claim 32, Shendure teaches that solid phase PCR covers systems such as emulsions, wherein one primer is anchored to a bead and the other is in free solution, and colony formation in solid phase gel matrices wherein one primer is anchored to the surface, and one is in free solution (interpreted as gel extraction to obtain PCR products, claim 32) (paragraph [0126], lines 11-15). Shendure teaches PCR amplification cleanup, where for fragment analysis, create a region table from 200-1000 bp in which a region molarity is calculated (interpreted as encompassing 400-600 bp, claim 32) (paragraph [0530], lines 1-2 and 17-19). Shendure teaches that the indexed nucleic acid fragments can be subjected to conditions that select for a predetermined size range, such as from 150 to 400 nucleotides in length, such as from 150 to 300 nucleotides, wherein the resulting indexed nucleic acid fragments are pooled, and optionally can be subjected to a clean-up process to enhance the purity to the DNA molecules by removing at least a portion of unincorporated universal adapters or primers, such that any suitable clean-up process can be used (interpreted as encompassing PCR cleanup by gel extraction, claim 32) (paragraph [0117]).
Regarding claim 38, Shendure teaches that a sample can include any biological, clinical, surgical, agricultural, atmospheric or aquatic-based specimen containing one or more nucleic acids and/or one or more proteins; as well as, any isolated nucleic acid from sample such a genomic DNA or a transcriptome, and any isolated protein from a sample including a collection of cells or nuclei (interpreted as a biosample, claim 38) (paragraph [0019]).
Regarding claim 39, Shendure teaches that targeted sequencing can be based on a biological feature that is typically present in a small percentage of the cells used to make the library, wherein biological features include, but are not limited to, a nucleotide sequence indicative of cell class, species type, or disease state (interpreted as a subject having a disease, claim 39) (paragraph [0013]). Shendure teaches that the terms "organism," "subject," are used interchangeably and refer to microbes ( e.g., prokaryotic or eukaryotic), animals, and plants including a mammal, such as a human (interpreted as a subject, claim 39) (paragraph [0016]). Shendure teaches that the cells can be embryonic cells, e.g., cells obtained from an embryo and/or the cells or nuclei can be from cancer or a diseased tissue (interpreted as a subject having a disease including a diagnosed disease, claim 39) (paragraph [0073], lines 8-10).
Regarding claim 41, Shendure teaches that the methods provided herein can be used to produce sequencing libraries from a plurality of single cells including essentially any single-nuclei or single-cell library preparation method or sequencing method can be used including, but not limited to, single-cell combinatorial indexing methods such as single-nuclei sequencing of transposon accessible chromatin (sci-ATAC, U.S. Pat. No. 10,059,989), whole genome sequencing of single-nuclei (U.S. Pat. Appl. Pub. No. US 2018/0023119), single-nuclei transcriptome sequencing (U.S. Prov. Pat. App. No. 62/680,259 and Gunderson et al. (WO2016/130704)), sci-HiC (Ramani et al., Nature Methods, 2017, 14:263-266), DRUG seq (Ye et al., Nature Commun., 9, article number 4307), or any combination of analytes from DNA and proteins, for example sci-CAR (Cao et al., Science, 2018, 361(6409):1380-1385) and RNA and proteins, for example CITE-seq (Stoeckius et al., 2017, Nature Methods, 14 (9): 865-868), wherein cell atlas experiments can be conducted with the readout restricted to chromatin accessible DNA, whole cell transcriptomes, a limited number of mRNAs that are highly informative, or a combination thereof (interpreted as repeating the method, claim 41) (paragraph [0071]).
Although Shendure does not specifically exemplify Tn5 transposome assembly, Shendure does teach that samples were tagmented with Tn5 transposase, indexing using the Tn5 transposase complex, the use of hyperactive Tn5 transposase including Tn5 mosaic end sequences; as well as, custom-loading of the Tn5 enzyme with barcoded adapters, where it is known that Tn5 transposomes can be assembled with pre-annealed mosaic end double-stranded oligonucleotides using A/B oligonucleotide sequences as evidenced by Picelli (pg. 2038, col 1, first full paragraph), such that one of ordinary skill in the art would clearly recognize, and be able to carry out, assembly of a Tn5 Transposome.
Shendure does not specifically exemplify the restriction enzymes CviQI, NIaIII or Pmel (claim 20); DNA fragment derived from CviQI (claims 30 and 31, both in part).
Regarding claims 20, 30 (in part) and 31 (in part), Green teaches methods to assemble genomes of eukaryotic or prokaryotic organisms (Abstract). Green teaches the importance is the use of reconstituted chromatin in forming associations among very distant, but molecularly-linked, segments of DNA, such that the disclosure enables distant segments to be brought together and covalently linked by chromatin conformation, thereby physically connecting previously distant portions of the DNA molecule, wherein subsequent processing can allow for the sequence of the associated segments to be ascertained, yielding read pairs whose separation on the genome extends up to the full length of the input DNA molecules; and since the read pairs are derived from the same molecule, these pairs also contain phase information (col 1, lines 46-57). Green teaches a plurality of read pairs can be generated by probing the physical layout of chromosomes, chromatin, or reconstituted chromatin using a Hi-C based technique comprising crosslinking chromosomes, chromatin, or reconstituted chromatin with a fixative agent, such as formaldehyde, to form DNA-protein cross links; cutting the cross-linked DNA-Protein with one or more restriction enzymes so as to generate a plurality of DNA-protein complexes comprising sticky ends; filling in the sticky ends with nucleotides containing one or more markers, such as biotin, to create blunt ends that are then ligated together; fragmenting the plurality of DNA-protein complexes into fragments; pulling down junction containing fragments by using the one or more of the markers; and sequencing the junction containing fragments using high throughput sequencing methods to generate a plurality of read pairs including those generated from data produced by probing the physical layout of reconstituted chromatin (col 2, lines 56-67; and col 3, lines 1-9). Green teaches that the restriction enzyme can have a restriction site of 1, 2, 3, 4, 5, or 6 bases long, wherein example enzymes include CviQI, NlaIII and PmeI (col 15, lines 7-8, 21,31 and 33). Green teaches that the disclosed methods can rapidly produce complete, accurate genomes at low cost, and thereby yield many highly sought capabilities in the study and treatment of human disease, including for performing a number of evolutionary and biomedical studies such as disease and trait association studies (col 10, lines 44-47; and col 12, lines 38-40).
It is prima facie obvious to combine prior art elements according to known methods to yield predictable results; the court held that, "…a conclusion that a claim would have been obvious is that all the claimed elements were known in the prior art and one skilled in the art could have combined the elements as claimed by known methods with no change in their respective functions, and the combination would have yielded nothing more than predictable results to one of ordinary skill in the art. KSR International Co. v. Teleflex Inc., 550 U.S. ___, ___, 82 USPQ2d 1385, 1395 (2007); Sakraida v. AG Pro, Inc., 425 U.S. 273, 282, 189 USPQ 449, 453 (1976); Anderson’s-Black Rock, Inc. v. Pavement Salvage Co., 396 U.S. 57, 62-63, 163 USPQ 673, 675 (1969); Great Atlantic & P. Tea Co. v. Supermarket Equipment Corp., 340 U.S. 147, 152, 87 USPQ 303, 306 (1950)”. Therefore, in view of the benefits of fragmenting chromatin as exemplified by Green, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the method of preparing sequencing libraries that include nucleic acids that represent the chromatin accessibility from a plurality of single cells as disclosed by Shendure to include the restriction enzymes including CviQI, NlaIII and PmeI that can have a restriction site of 1-6 bases long as taught by Green with a reasonable expectation of success in generating smaller genomic fragments for use in an improved assay for jointly profiling chromatin accessibility and gene expression; in ascertaining associations between very distant, but molecularly-linked, nucleic acid segments; in cataloging hundreds-of-thousands of cell type-specific DNA regulatory elements; and/or in implicating specific cell types in common human traits and/or diseases
Thus, in view of the foregoing, the claimed invention, as a whole, would have been obvious to one of ordinary skill in the art at the time the invention was made. Therefore, the claims are properly
rejected under 35 USC §103(a) as obvious over the art.
Conclusion
Claims 10-13, 20-25, 28, 30-32, 34, 36, 38, 39, 41 and 47 are rejected.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to AMY M BUNKER whose telephone number is (313) 446-4833. The examiner can normally be reached on Monday-Friday (6am-2:30pm).
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
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.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/AMY M BUNKER/Primary Examiner, Art Unit 1684