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
Claims 20-39 are under examination (11/30/2023). Applicant previously cancelled claims 1-19 (11/30/2023).
Priority
Claims 20-39 receive the priority date of 12/2/2020, the filing date of European Application No. EP20211177.9.
Information Disclosure Statement
The listing of references in the specification is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
Information Disclosure Statement from 7/12/2023 is considered.
Nucleotide and/or Amino Acid Sequence Disclosures
REQUIREMENTS FOR PATENT APPLICATIONS CONTAINING NUCLEOTIDE AND/OR AMINO ACID SEQUENCE DISCLOSURES
Items 1) and 2) provide general guidance related to requirements for sequence disclosures.
37 CFR 1.821(c) requires that patent applications which contain disclosures of nucleotide and/or amino acid sequences that fall within the definitions of 37 CFR 1.821(a) must contain a "Sequence Listing," as a separate part of the disclosure, which presents the nucleotide and/or amino acid sequences and associated information using the symbols and format in accordance with the requirements of 37 CFR 1.821 - 1.825. This "Sequence Listing" part of the disclosure may be submitted:
In accordance with 37 CFR 1.821(c)(1) via the USPTO patent electronic filing system (see Section I.1 of the Legal Framework for Patent Electronic System (https://www.uspto.gov/PatentLegalFramework), hereinafter "Legal Framework") as an ASCII text file, together with an incorporation-by-reference of the material in the ASCII text file in a separate paragraph of the specification as required by 37 CFR 1.823(b)(1) identifying:
the name of the ASCII text file;
ii) the date of creation; and
iii) the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(1) on read-only optical disc(s) as permitted by 37 CFR 1.52(e)(1)(ii), labeled according to 37 CFR 1.52(e)(5), with an incorporation-by-reference of the material in the ASCII text file according to 37 CFR 1.52(e)(8) and 37 CFR 1.823(b)(1) in a separate paragraph of the specification identifying:
the name of the ASCII text file;
the date of creation; and
the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(2) via the USPTO patent electronic filing system as a PDF file (not recommended); or
In accordance with 37 CFR 1.821(c)(3) on physical sheets of paper (not recommended).
When a “Sequence Listing” has been submitted as a PDF file as in 1(c) above (37 CFR 1.821(c)(2)) or on physical sheets of paper as in 1(d) above (37 CFR 1.821(c)(3)), 37 CFR 1.821(e)(1) requires a computer readable form (CRF) of the “Sequence Listing” in accordance with the requirements of 37 CFR 1.824.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed via the USPTO patent electronic filing system as a PDF, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the PDF copy and the CRF copy (the ASCII text file copy) are identical.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed on paper or read-only optical disc, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the paper or read-only optical disc copy and the CRF are identical.
Specific deficiencies and the required response to this Office Action are as follows:
Specific deficiency - This application fails to comply with the requirements of 37 CFR 1.821 - 1.825 because it does not contain a "Sequence Listing" as a separate part of the disclosure or a CRF of the “Sequence Listing.”.
Required response - Applicant must provide:
A "Sequence Listing" part of the disclosure; together with
An amendment specifically directing its entry into the application in accordance with 37 CFR 1.825(a)(2);
A statement that the "Sequence Listing" includes no new matter as required by 37 CFR 1.821(a)(4); and
A statement that indicates support for the amendment in the application, as filed, as required by 37 CFR 1.825(a)(3).
If the "Sequence Listing" part of the disclosure is submitted according to item 1) a) or b) above, Applicant must also provide:
A substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3) and 1.125 inserting the required incorporation-by-reference paragraph, consisting of:
A copy of the previously-submitted specification, with deletions shown with strikethrough or brackets and insertions shown with underlining (marked-up version);
A copy of the amended specification without markings (clean version); and
A statement that the substitute specification contains no new matter.
If the "Sequence Listing" part of the disclosure is submitted according to item 1) c) or d) above, applicant must also provide:
A CRF in accordance with 37 CFR 1.821(e)(1) or 1.821(e)(2) as required by 1.825(a)(5); and
A statement according to item 2) a) or b) above.
Specification
The disclosure is objected to because of the following informalities (see MPEP § 608.01):
The use of the term, “Illumina” (p. 65) is a trade name or mark used in commerce, has been noted in this application. The terms should be accompanied by the generic terminology; furthermore, the term should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the term.
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code (p. 74 in “References”). Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01.
Claim Rejections - 35 USC § 112
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 20-39 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 20 is rejected. Claim 20 recites the limitation “the range” (step i, line 2). There is insufficient antecedent basis for this limitation in the claim.
Claim 20 is further rejected. Claim 20 recites the limitation “the first barcode” (step j, part i, line 1). There is insufficient antecedent basis for this limitation in the claim.
Claim 20 is further rejected. Claim 20 recites the limitation “the frequency of gDNA fragment” (step k, line 1). There is insufficient antecedent basis for this limitation in the claim.
Claims 22, 24-25, 27-28, 30-37 are included in this rejection due to their dependency on claim 20.
Claim 21 is rejected. Claim 21 recites the limitation “the local levels” (line 1). There is insufficient antecedent basis for this limitation in the claim.
Claim 21 is further rejected. Claim 21 recites the limitation “the range” (step i, line 1). There is insufficient antecedent basis for this limitation in the claim.
Claim 21 is further rejected. Claim 21 recites the limitation “the frequency of gDNA fragment” (step k, line 1). There is insufficient antecedent basis for this limitation in the claim
Claims 26 and 29 are included in this rejection due to their dependency on claim 21.
Claim 23 is rejected. Claim 23 is rejected for indefiniteness because the metes and bounds of the claim cannot be determined. Specifically, claim 23 depends from claim 00, which does not exist. Accordingly, it is unclear what subject matter claim 23 incorporates and further limits. Therefore, claim 23 has not been further examined, and no determination has been made regarding compliance with any other statutory requirements.
Claim 35 is further rejected. Claim 35 is rejected for indefiniteness because the limitation “from each chromatin modification sub pool per” and it is unclear what “per” refers to. Accordingly, the metes and bounds of the claim cannot be determined.
Claim 38 is rejected. Claim 38 is rejected for indefiniteness because the metes and bounds of the claim cannot be determined. Specifically, claim 38 depends from claim 1, which is a previously cancelled claim. Accordingly, it is unclear what subject matter claim 38 incorporates and further limits. Therefore, claims 38 and 39 have not been further examined, and no determination has been made regarding compliance with any other statutory requirements.
Claim 39 is included in this rejection due to its dependency on claim 38.
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claims 20-22 and 24-37 are rejected under 35 U.S.C. 101 because the claimed invention is directed to an abstract idea without significantly more.
The claims recite obtaining sequencing information from tagged genomic DNA fragments, determining the number of unique tagged genomic DNA fragments comprising each barcode sequence, calculating the frequency of tagged genomic DNA fragments comprising each barcode sequence within a chromatin modification sub-pool, and determining or assessing chromatin modification levels based on the calculated frequencies. These limitations recite the evaluation, analysis, and comparison of information, followed by identifying a result based upon that information.
Such steps constitute a mental process because they involve observation, evaluation, and analysis of information that could practically be performed in the human mind or with pen and paper, albeit more effectively using laboratory equipment and computers. To the extent the claims recite determining numerical values, including frequencies, normalized read counts, ratios, or other calculated values from observed sequencing data, the claims additionally recite mathematical concepts.
The additional elements, including providing test samples, fragmenting chromatin, tagging genomic DNA fragments with ID-tags, combining tagged chromatin fragments, incubating tagged chromatin fragments with antibodies, obtaining chromatin modification sub-pools, optionally amplifying tagged genomic DNA fragments, randomly selecting tagged genomic DNA fragments, and sequencing tagged genomic DNA fragments, merely gather and generate data for use in the recited analysis and do not integrate the judicial exception into a practical application. These steps represent routine and conventional laboratory techniques for generating biological information that is subsequently analyzed according to the claimed mental process.
The integration of the judicial exception into the claims does not render them patent eligible because the claims are written at a high level of generality and merely use well-known, routine, and conventional techniques in the field.
Subject Matter Eligibility Test for Products and Processes
Step 1 - Is the Claim to a Process, Machine, Manufacture or Composition of Matter? YES.
The claims provide for a method comprising:
providing a plurality of test samples comprising chromatin from a cell population;
fragmenting the chromatin into chromatin fragments;
tagging at least a fraction of the genomic (gDNA) fragments with one or more ID-tags comprising barcode sequences and, optionally, unique molecular identifier (UMI) sequences;
combining tagged chromatin fragments into a pool;
incubating the tagged chromatin fragments with antibodies to obtain chromatin modification sub-pools;
optionally amplifying, randomly selecting, and sequencing tagged gDNA fragments; and
determining the number of unique tagged gDNA fragments comprising each barcode sequence, calculating the frequency of tagged gDNA fragments comprising each barcode sequence within each chromatin modification sub-pool, and determining or assessing chromatin modification levels based on the calculated frequencies.
Dependent claims further recite calculating normalized read counts, comparing chromatin modification levels between samples using mathematical levels between samples using mathematical relationships, utilizing second barcode sequences, selecting specified numbers of tagged gDNA fragments, and applying the method to determine the influence of test compounds on chromatin modification levels.
Thus, the claims are directed to statutory categories (i.e., processes).
Step 2A, Prong One — Does the Claim Recite an Abstract Idea, Law of Nature, or Natural Phenomenon? YES.
Abstract ideas have been identified by the courts by way of example, including fundamental economic practices, certain methods of organizing human activities, an idea ‘of itself,’ and mathematical relationships/formulas. The claims recite a judicial exception. The “mental process” of determining the number of unique tagged genomic DNA (gDNA) fragments comprising each barcode sequence, calculating the frequency of tagged gDNA fragments comprising each barcode sequence, calculating the frequency of tagged gDNA fragments comprising each barcode sequence within a chromatin modification sub-pool, and determining or assessing chromatin modification levels based on the calculated frequencies is an abstraction (an idea having no particular concrete or tangible form). The mathematical concepts involving frequencies, normalized read counts, ratios and other calculated numerical values are abstract ideas. Thus, the claimed invention describes a judicial exception, which correspond to abstractions (ideas, having no particular concrete or tangible form) and mathematical relationships.
Step 2A, Prong Two — Does the Claim Recite an Additional Elements that Integrate the Judicial Exception into a Practical Application? NO.
The Supreme Court has long distinguished between principles themselves, which are not patent eligible, and the integration of those principles into practical applications, which are patent eligible. However, absent are any additional elements recited in the claim beyond the judicial exceptions which integrate the exception into a practical application of the exception. The “integration into a practical application” requires an additional element or a combination of additional elements in the claim to apply, rely on, or use the judicial exception in a manner that imposes a meaningful limit on the judicial exception, such that it is more than a drafting effort designed to monopolize the exception.
The claim limitations are considered to be directed to the abstract idea of evaluating and analyzing information. Specifically, the claims recite determining the number of unique tagged genomic DNA (gDNA) fragments comprising each barcode sequence, calculating the frequency of tagged gDNA fragments comprising each barcode sequence within each chromatin modification sub-pool, determining or assessing chromatin modification levels based on the calculated frequencies, and, in certain claims, calculating normalized read counts, ratios, or other numerical values derived from the sequencing data. These limitations recite observation, evaluation, and analysis of information, followed by identification of a result based upon that information, which constitutes a mental process and, to the extent mathematical relationships are used to derive frequencies, normalized read counts, ratios, or other calculated values, a mathematical concept.
While the claims further recite providing test samples comprising chromatin, fragmenting chromatin into chromatin fragments, tagging genomic DNA fragments with ID-tags comprising barcode sequences and optionally unique molecular identifier (UMI) sequences, combining tagged chromatin fragments, incubating the tagged chromatin fragments with antibodies to obtain chromatin modification sub-pools, optionally amplifying tagged genomic DNA fragments, randomly selecting tagged genomic DNA fragments, and sequencing tagged genomic DNA fragments, these additional steps do not integrate the judicial exception into a practical application. Rather, these steps are recited as data gathering activities used to obtain information that is subsequently analyzed through the determining, calculating and assessing steps.
There are no additional steps which apply either of the identified judicial exceptions into a practical application. Thus, the claims do not provide for any element/step that integrates the law of nature into a practical application. Specifically, the claims do not recite and particular improvement in DNA sequencing technology, chromatin analysis technology, barcode tagging technology, antibody-based chromatin isolation technology, or other laboratory technology. Instead, these elements are merely gather biological information that is subsequently analyzed according to the recited mental process and mathematical concepts.
As a result, there are no additional steps that apply the identified judicial exception in a manner that imposes a meaningful limit on the exception. Thus, the claims do not integrate the abstract idea into a practical application.
Step 2B - Does the Claim Recite Additional Elements that Amount to Significantly More than the Judicial Exception? NO.
The Supreme Court has identified a number of considerations for determining whether a claim with additional elements amounts to “significantly more” than the judicial exception(s) itself. The claims as a whole are analyzed to determine whether any additional element/step, or combination of additional elements/steps, in addition to the identified judicial exception(s) is sufficient to ensure that the claim amounts to “significantly more” than the exception(s).
However, the additional elements of the instant application, individually and in combination, do not amount to “significantly more.” Under the Step 2B analysis, the “physical” elements/steps of, providing test samples comprising chromatin, fragmenting chromatin into chromatin fragments, tagging genomic DNA (gDNA) fragments with ID-tags comprising barcode sequences and optionally unique molecular identifier (UMI) sequences, combining tagged chromatin fragments, incubating the tagged chromatin fragments with antibodies to obtain chromatin modification sub-pools, optionally amplifying tagged gDNA fragments, randomly selecting tagged gDNA fragments, and sequencing tagged gDNA fragments are merely physical steps used to obtain information that is subsequently evaluated through the abstract determining, calculating, and assessing steps.
For example, Park (“ChIP-seq: advantages and challenges of a maturing technology, NATURE Reviews, published 2009, from IDS 7/12/2023), teaches that chromatin immunoprecipitation followed by sequencing (ChIP-seq) utilizes conventional laboratory techniques including crosslinking chromatin, fragmenting chromatin (i.e., sonication or micrococcal nuclease digestion), immunoprecipitating chromatin fragments with antibodies specific to proteins or histone modifications, constructing sequencing libraries, performing size selection, and sequencing DNA fragments to analyze chromatin modifications (Abstract; Box 1; ChIP-seq basics: Paragraphs 1-3). Thus, chromatin fragmentation, antibody-based enrichment of chromatin fragments, library preparation, size selection, and sequencing were well-understood, routine, and conventional activities in the art.
Further, van Galen et al. (“A Multiplexed System for Quantitative Comparisons of Chromatin Landscapes”, Molecular Cell: Technology, published: 2016; from IDS 7/12/2023) discloses, a multiplexed chromatin immunoprecipitation sequencing (ChIP-seq) workflow in which chromatin is fragmented, ligated to barcoded adapters unique to each sample, pooled and split for parallel ChIP assays, immunoprecipitated with antibodies, amplified, sequenced, and computationally de-multiplexed based on the barcode sequences (Summary; Figure 1; “Chromatin Isolation and Indexing”; “Pool-and-Split Multiplexing”; “Linear Amplification and Library Construction”; “In-silico De-multiplexing”). Thus, chromatin fragmentation, sample barcoding, pooling, antibody-based chromatin isolation, amplification, sequencing, and barcode-based identification of sequencing reads were well-understood, routine, and conventional activities in the art.
Further, Grosselin et al. (“High-throughput single-cell ChIP-seq identifies heterogeneity of chromatin states in breast cancer”, Nature Genetics, published 2019, from IDS 7/12/2023) discloses a high-throughput single-cell chromatin immunoprecipitation sequencing (scChIP-seq) workflow that combines droplet microfluidics with single-cell DNA barcoding technologies to profile chromatin landscapes of thousands of cells at single-cell resolution (Abstract; Results: “Droplet microfluidics workflow for scCHiP-seq”). Grosselin further teaches chromatin immunoprecipitation, DNA barcoding, sequencing, and computational analysis of chromatin profiles to characterize chromatin states and identify biologically relevant cell populations (Abstract; Results; Figures 1-2). Thus, barcoding chromatin profiling, sequencing, and computational characterization of chromatin features were well-understood, routine, and conventional activities in the art.
Therefore, obtaining chromatin fragments associated with one or more chromatin modifications, attaching barcodes or adapters to the chromatin fragments, pooling chromatin fragments, sequencing the chromatin fragments, identifying chromatin modifications associated with sequence reads, assigning sequence reads to chromatin modification sub-pools, and generating chromatin modification profiles were routine and conventional before the effective filing date of the claimed invention.
Simply appending routine and conventional activities previously known to the industry specified at a high level of generality to the judicial exception and/or generally linking the use of the judicial exception(s) to a particular technological environment or field of use, are not found to be enough to qualify as “significantly more.” Nothing is added by identifying the techniques to be used (i.e., chromatin immunoprecipitation, barcode ligation, sequencing, pooling, amplification, or chromatin profiling techniques) because those techniques were well-understood, routine, and conventional techniques that a practitioner would have thought of when instructed to obtain sequencing information associated with chromatin modifications.
In context with the other recited claim limitations, the language requiring obtaining chromatin fragments associated with one or more chromatin modifications, attaching barcodes or adapters to the chromatin fragments, pooling and sequencing the chromatin fragments, identifying chromatin modifications associated with individual sequence reads, assigning sequence reads to chromatin modification sub-pools, determining a chromatin modification profile, calculating one or more chromatin modification metrics, and classifying or identifying a chromatin state based on the calculated metrics merely indicates whether a relationship exists between the measured sequencing information and the identified chromatin state.
This information simply tells a practitioner about the relevant informational relationship between the measured fluorescence kinetic profile and the identified target nucleic acid, and does not recite any technological improvement in chromatin immunoprecipitation, sequencing technology, barcode generation, library preparation, chromatin isolation, or sequence analysis technology. Thus, when viewed both individually and as an ordered combination, the claimed elements/steps in addition to the identified judicial exception are found insufficient to supply an inventive concept because the elements/steps are considered conventional and specified at a high level of generality. The claim limitations do not transform the abstract idea that they recite into patent-eligible subject matter because “the claims simply instruct the practitioner to implement the abstract idea with routine, conventional activity.”
Accordingly, the claims do not qualify as patent-eligible subject matter.
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 (i.e., changing from AIA to pre-AIA ) 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 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 text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
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 20-22 and 24-37 are rejected under 35 U.S.C. 103 as being unpatentable over Buenrostro et al., (USPGPub 2020/0248255, published 8/6/2020) and van Galen et al. (“A Multiplexed System for Quantitative Comparisons of Chromatin Landscapes”, Molecular Cell: Technology, published: 2016; from IDS 7/12/2023).
Regarding claims 20-21, Buenrostro teaches methods and compositions for analyzing nucleic acid in individual cells and in some embodiments, the methods herein include generating, within individual cells, fragmented cellular genomic DNA and cDNA copies of cellular RNA molecules, barcoding the fragmented genomic DNA and the cDNA within each cell such that the genomic DNA and the cDNA from the same cell receive the same unique barcode sequence, isolating the barcoded genomic DNA and cDNA, and characterizing one or more features of the individual cells based, at least in part, on sequencing of the isolated barcoded genomic DNA and the cDNA (Abstract). Further, Buenrostro teaches that recent development of methods such as RNA-seq and ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) have provided the ability to analyze different types of nucleic acids in cells, however, simultaneous analysis of different types of nucleic acids in single cells remain underdeveloped due to challenges such as making the processing cross compatible between the two approaches (Paragraph 4, lines 1-5). Buenrostro also teaches that the present disclosure provides a method for single cell analysis of genomic DNA accessibility and RNA expression in a cell, the method comprising generating, within individual cells, fragmented cellular genomic DNA and cDNA copies of cellular RNA molecules; barcoding the fragmented genomic DNA and the cDNA within each cell such that the genomic DNA and the cDNA from the same cell receive the same unique cell barcode sequence; isolating the barcoded genomic DNA and the cDNA; and characterizing one or more features of the individual cells based, at least in part, on sequencing of the isolated barcoded genomic DNA and the cDNA (Paragraph 6, lines 1-5). Also, Buenrostro teaches that in some embodiments, the method further comprises fragmenting the cellular genomic DNA and/or the cDNA using an insertional enzyme and, in some embodiments, the fragmented cellular genomic DNA is generated by contacting chromatin in the individual cells with an insertional enzyme (Paragraph 7, lines 1-5).
Buenrostro further teaches that in some embodiments, the insertional enzyme is a transposase and in some embodiments, the transposase is an engineered transposase with an activity higher than a wild type counterpart and in some embodiments, the insertional enzyme comprises two or more enzymatic moieties where in some embodiments, the insertional enzyme forms a complex with a phosphorylated oligonucleotide and also where in some embodiments, the method further comprises generating cDNA from the RNA in the cell using a primer comprising i) a unique molecular identifier (UMI), ii) an affinity tag, and/or iii) a poly(T) sequence (Paragraph 7, lines 1-5). Also, Buenrostro teaches that in some embodiments, the barcoded cDNA is isolated by capturing the affinity tag on a solid support. In some embodiments, before isolation, the genomic DNA forms a complex with one or more proteins, and the genomic DNA is isolated by capturing the one or more proteins on a solid support and in some embodiments, the method further comprises amplifying the genomic DNA, the cDNA, or a combination thereof and in some embodiments, the sequencing comprises sequencing a portion of the genomic DNA fragments, a portion of the cDNA molecules, and/or a portion of the barcode attached thereof (Paragraph 7, lines 5-10). Buenrostro teaches that in some embodiments, the one or more features comprise an epigenetic feature of a genomic DNA region in the cell where in some embodiments, the epigenetic feature comprises a profile of chromatin accessibility along the genomic DNA region; a DNA binding protein occupancy for a binding site in the genomic DNA region; a nucleosome-free DNA in the genomic DNA region; a positioning of nucleosomes along the genomic DNA region; chromatin states; or a combination thereof. In some embodiments, the one or more features comprise an expression profile of the cellular RNA and also some embodiments, the genomic DNA is tagged (Paragraph 7, lines 10-15).
Buenrostro also teaches that the invention also provides a method for single cell analysis of genomic DNA accessibility and mRNA expression, comprising contacting chromatin within individual cells with a transposase to generate fragmented cellular genomic DNA; reverse transcribing the mRNA to generate cDNA; isolating the individual cells in separate individual discrete volumes, each of the individual discrete volumes further comprising a primer pair and a volume-specific barcode that hybridizes to both fragmented cellular genomic DNA and cDNA; using combinatorial split-and-pool strategies, such as ligation, to add sequential barcodes to the cellular genomic DNA fragments; amplifying the cellular genomic DNA fragments using the primer pair to generate amplicons; and sequencing the amplicons (Paragraph 11, lines 1-10). Specifically, Buenrostro teaches that in some embodiments, the mRNA is reverse transcribed using an oligonucleotide comprising a poly(dT) sequence where in some embodiments, the oligonucleotide further comprises a unique molecular identifier (UMI) and a biotin tag and in some embodiments, the hybridization is repeated three or more times (Paragraph 13, lines 1-5). Also, Buenrostro teaches that the present disclosure further provides a method of diagnosing a condition in a subject, comprising characterizing a feature of one or more cells in the subject using the method and compositions described herein; and providing a diagnosis or prognosis based on the feature (Paragraph 8, lines 1-3).
Buenrostro teaches that SHARE-seq enabled joint profiling of chromatin accessibility and transcription in tissues and (FIG. 12A) a schematic of tissues profiled with SHARE-seq, highlighting the cellular diversity within mouse skin, including (FIGS. 12B-12C) comparison of library size estimates of SHARE-seq and other single-cell or nucleic based approaches for scATAC-seq (FIG. 12B) and scRNA-seq (FIG. 12C) approaches (Paragraph 28, lines 1-5). Further, Buenrostro teaches that in certain embodiments, the fragments may be amplified using PCR primers that hybridize to the tags that have been added to the fragments, where the primer used for PCR have 5′ tails that are compatible with a particular sequencing platform and in certain cases, the primers used may contain a molecular barcode (an “index”) so that different pools can be pooled together before sequencing, and the sequence reads can be traced to a particular sample using the barcode sequence (Paragraph 139, lines 20-25). Buenrostro teaches that in some cases, the sequencing may be performed at certain “depth”, where the terms “depth” or “coverage” as used herein refers to the number of times a nucleotide is read during the sequencing process and in regards to single cell RNA sequencing, “depth” or “coverage” as used herein refers to the number of mapped reads per cell where depth in regards to genome sequencing may be calculated from the length of the original genome (G), the number of reads(N), and the average read length(L) as N×L/G (Paragraph 140, lines 1-10). Buenrostro further teaches that for example, a hypothetical genome with 2,000 base pairs reconstructed from 8 reads with an average length of 500 nucleotides will have 2× redundancy and in some cases, the sequencing herein may be low-pass sequencing, where the terms “low-pass sequencing” or “shallow sequencing” as used herein refers to a wide range of depths greater than or equal to 0.1× up to 1× (shallow sequencing may also refer to about 5000 reads per cell (e.g., 1,000 to 10,000 reads per cell)) (Paragraphs 140-141).
Buenrostro teaches in reference to organizing ChIP-seq reads, that the scatter plot showing the length of super-enhancer is not correlated with the number of associated peaks, where (FIG. 21H) a cumulative distribution function plot of peak-gene associations for each gene (FIG. 21I) and the overlapping between DORCs identified in TAC/IRS/Hair shaft and in all cells, where specifically (FIG. 21J) DORC activity for each defined cluster, values are normalized by the min and max activity (Paragraph 37, lines 10-15). For example, Buenrostro teaches that applicants then re-calculate peak-gene association by expanding the window to ±500 kb around TSSs and the DORC score was calculated by summing up all the significantly correlated peak counts per gene, and then normalized by dividing the total unique fragments in peaks (Paragraph 298, lines 1-5).
Regarding claim 22, Buenrostro teaches that in some embodiments, a barcode may be attached to sequences that allow for amplification and sequencing (for example, SBS3 and P5 elements for Illumina® sequencing) and in certain embodiments, a nucleic acid barcode can further include a hybridization site for a primer (for example, a single-stranded DNA primer) attached to the end of the barcode (Paragraph 97, lines 1-5). Buenrostro teaches that for example, an origin-specific barcode may be a nucleic acid including a barcode and a hybridization site for a specific primer and in particular embodiments, a set of origin-specific barcodes includes a unique primer specific barcode made, for example, using a randomized oligo type (SEQ ID NO:2) (Paragraph 97, lines 1-5).
Regarding claim 24, Buenrostro teaches that in certain embodiments, the fragments may be amplified using PCR primers that hybridize to the tags that have been added to the fragments, where the primer used for PCR have 5′ tails that are compatible with a particular sequencing platform and in certain cases, the primers used may contain a molecular barcode (an “index”) so that different pools can be pooled together before sequencing, and the sequence reads can be traced to a particular sample using the barcode sequence (Paragraph 139, lines 20-25). Buenrostro teaches that in some cases, the sequencing may be performed at certain “depth”, where the terms “depth” or “coverage” as used herein refers to the number of times a nucleotide is read during the sequencing process and in regards to single cell RNA sequencing, “depth” or “coverage” as used herein refers to the number of mapped reads per cell where depth in regards to genome sequencing may be calculated from the length of the original genome (G), the number of reads(N), and the average read length(L) as N×L/G (Paragraph 140, lines 1-10). Buenrostro further teaches that for example, a hypothetical genome with 2,000 base pairs reconstructed from 8 reads with an average length of 500 nucleotides will have 2× redundancy and in some cases, the sequencing herein may be low-pass sequencing, where the terms “low-pass sequencing” or “shallow sequencing” as used herein refers to a wide range of depths greater than or equal to 0.1× up to 1× (shallow sequencing may also refer to about 5000 reads per cell (e.g., 1,000 to 10,000 reads per cell)) (Paragraphs 140-141).
Regarding claims 25-27, Buenrostro teaches that the method may further comprise attaching one or more barcodes to the fragmented DNA and the cDNA and in some examples, the fragmented DNA and the cDNA from or derived from the same cell may receive a unique barcode sequence, which may comprise one or more barcodes and when the nucleic acids are sequenced, the unique barcode sequence may be used to identify sequence reads identifying a single cell, and further where in some examples, a barcode may be a unique cell barcode, e.g., molecules from the same cell comprises the same unique cell barcode (Paragraph 90, lines 1-5). Buenrostro teaches that such cases, molecules from different cells may be distinguished and/or identified based on the unique cell barcodes where in some examples, a barcode may be a unique molecular identifier (UMI), e.g., two different molecules comprise different UMIs and can be distinguished based on the UMIs (Paragraph 90, lines 5-10).
Regarding claims 28-30, Buenrostro teaches that in certain embodiments, a barcode may identify the type of nucleic acids molecules and for example, all DNA molecules may comprise a first common barcode sequence and all RNA molecules or cDNA molecules generated from RNA molecules may comprise a second common barcode sequence, which is different from the first common barcode sequence, where in some cases, a barcode may identify the individual discrete volume (Paragraph 93, lines 1-5). Buenrostro teaches that in some cases, the sequencing may be performed at certain “depth”, where the terms “depth” or “coverage” as used herein refers to the number of times a nucleotide is read during the sequencing process and in regards to single cell RNA sequencing, “depth” or “coverage” as used herein refers to the number of mapped reads per cell where depth in regards to genome sequencing may be calculated from the length of the original genome (G), the number of reads(N), and the average read length(L) as N×L/G (Paragraph 140, lines 1-10). Buenrostro further teaches that for example, a hypothetical genome with 2,000 base pairs reconstructed from 8 reads with an average length of 500 nucleotides will have 2× redundancy and in some cases, the sequencing herein may be low-pass sequencing, where the terms “low-pass sequencing” or “shallow sequencing” as used herein refers to a wide range of depths greater than or equal to 0.1× up to 1× (shallow sequencing may also refer to about 5000 reads per cell (e.g., 1,000 to 10,000 reads per cell)) (Paragraphs 140-141).
Regarding claims 31-32, Buenrostro teaches that the splitting and pooling steps may be repeated for at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, or at least 500 times and in some cases, the splitting and pooling steps may be repeated once, twice, three times, or four times, where in some cases, the pooled sample may be used for further processing and analysis (Paragraph 112, lines 10-15). Further, Buenrostro teaches that in certain cases, the split samples in partitions may be used for further processing and analysis and in some cases, the split-pooling (one or multiple rounds) may be performed for barcode ligation, where multiple rounds of split-pooling may create barcode possibilities to identify cells, thus increase the throughput of analysis methods (Paragraph 112, lines 15-20).
Regarding claims 33-34, Buenrostro teaches that when powered by the massive scalability of this approach, SHARE-seq can be adapted for identifying RNA barcodes, particularly useful for CRISPR-based perturbation screens and SHARE-seq can be further extended by replacing ATAC-seq with whole-genome transposition enabling methods for DNA methylation and chromatin conformation and in these efforts, scRNA-seq data may be used as a common scaffold for integration, providing a unique opportunity to comprehensively map between multiple layers of gene regulation, as well as to train algorithms that learn to map between different data modalities in a cell (Paragraph 254, lines 5-10). Further, Buenrostro teaches that the tags attached to the DNA during tagmentation may be any barcode described herein and in some examples, the tags may comprise sequencing adaptors, locked nucleic acids (LNAs), zip nucleic acids (ZNAs), RNAs, affinity reactive molecules (e.g. biotin, dig), self-complementary molecules, phosphorothioate modifications, azide or alkyne groups, where in some cases, the sequencing adaptors further comprise a barcode label and further, the barcode labels may comprise a unique sequence where the unique sequences can be used to identify the individual insertion events and any of the tags can further comprise fluorescence tags (e.g. fluorescein, rhodamine, Cy3, Cy5, thiazole orange, etc.) (Paragraph 80, lines 1-5).
Regarding claims 35-37, Buenrostro teaches that the splitting and pooling steps may be repeated for at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, or at least 500 times and in some cases, the splitting and pooling steps may be repeated once, twice, three times, or four times, where in some cases, the pooled sample may be used for further processing and analysis (Paragraph 112, lines 10-15). Buenrostro further teaches that for example, a hypothetical genome with 2,000 base pairs reconstructed from 8 reads with an average length of 500 nucleotides will have 2× redundancy and in some cases, the sequencing herein may be low-pass sequencing, where the terms “low-pass sequencing” or “shallow sequencing” as used herein refers to a wide range of depths greater than or equal to 0.1× up to 1× (shallow sequencing may also refer to about 5000 reads per cell (e.g., 1,000 to 10,000 reads per cell)) (Paragraphs 140-141).
Buenrostro does not teach or suggest multiplexed quantitative assessment of multiple chromatin modification sub-pools based on barcode frequencies associated with tagged genomic DNA fragments using antibodies.
Van Galen teaches a multiplexed chromatin immunoprecipitation sequencing (ChIP-seq) workflow in which chromatin from multiple samples is fragmented, ligated to barcoded adapters unique to each sample, pooled and split for parallel ChIP assays, immunoprecipitated with antibodies, amplified, sequenced, and computationally de-multiplexed based on the barcode sequences followed by quantitatively comparing chromatin modification states across samples (Summary; Figure 1; “Chromatin Isolation and Indexing”; “Pool-and-Split Multiplexing”; “Linear Amplification and Library Construction”; “In-silico De-multiplexing”).
It would have been obvious to one of ordinary skill in the art to modify the multiplexed sequencing workflow of Buenrostro by incorporating the multiplexed chromatin modification profiling of van Galen in order to simultaneously quantify multiple chromatin modifications while maintaining sample identity through barcode-based de-multiplexing, thereby increasing throughput, reducing experimental variability, and enabling quantitative comparison of chromatin modification levels across multiple samples using a common sequencing workflow. Because both references employ barcoded sequencing libraries, pooled sequencing, amplification, and computational de-multiplexing to characterize chromatin-derived nucleic acids, a person of ordinary skill in the art would have had a reasonable expectation of successfully combining the teachings using known molecular biology techniques.
Although van Galen nor Buenrostro does not expressly disclose selecting n x m x 100-100,000 tagged genomic DNA fragments or using the identical numerical relationship recited in the claims, selection of sequencing depths, read count, and sampling level constitutes a result-effective variable that was routinely optimized by those of ordinary skill in the art depending on the desired sequencing coverage, throughput, sample number, and quantitative accuracy. Likewise, although van Galen performs immunoprecipitation using multiple antibodies directed to different chromatin modifications rather than expressly referring to chromatin modification sub-pools, each antibody-specific immunoprecipitation inherently produces a distinct subpopulation (sub-pool) of tagged chromatin fragments corresponding to the association chromatin modification, which are separately identified following barcode de-multiplexing. Optimizing the number of reads obtained from each sub-pool would have been an obvious matter of routine experimentation to achieve the desired sequencing coverage and quantitative confidence.
Conclusion
No claim is allowed.
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/ELIZABETH ROSE LAFAVE/Examiner, Art Unit 1684
/HEATHER CALAMITA/Supervisory Patent Examiner, Art Unit 1684
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