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 .
Status of Claims
Claims 1, 4-5, 7-20, 23-24 and 27 pending and examined on the merits.
Claims 2-3, 6, 21-22, 25-26, and 28-53 cancelled.
Priority
The instant application filed on 3/14/2023 claims the benefit of priority to U.S. Provisional Patent Application No. 63/320,110 filed on 3/15/2022. Thus, the effective filing date of the claims is 3/15/2022.
The applicant is reminded that amendments to the claims and specification must comply with 35 U.S.C. § 120 and 37 C.F.R. § 1.121 to maintain priority to an earlier-filed application. Claim amendments may impact the effective filing date if new subject matter is introduced that lacks support in the originally filed disclosure. If an amendment adds limitations that were not adequately described in the parent application, the claim may no longer be entitled to the priority date of the earlier filing.
Information Disclosure Statement
The information disclosure statement (IDS) filed on 4/13/2023 has been entered and considered. A signed copy of the corresponding 1449 form has been included with this Office action.
The information disclosure statement (IDS) filed on 10/31/2024 has been entered and considered. A signed copy of the corresponding 1449 form has been included with this Office action.
Claim Objections
Claims 1, 5, 11-13, 15, 18-20, and 27 objected to because of the following informalities:
Claims 1, 5, 11-13, 15, 18-20, and 27 recite the alternative phrase "and/or" one or more times. Applicant should specify "and" or "or" at each usage of the phrase "and/or".
Appropriate correction is required.
Claim Interpretation
The claims in this application are given their broadest reasonable interpretation (BRI) using the plain meaning of the claim language in light of the specification as it would be understood by one of ordinary skill in the art.
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 1, 4-5, 7-20, 23-24 and 27 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.
Claims 1, 5, 11-13, 15, 18-20, and 27 recite the alternative phrase "and/or" one or more times. The phrase renders the claim indefinite because it may be interpreted in multiple ways, resulting in unclear metes and bounds of the claims. To further prosecution, all instances of the phrase "and/or" are interpreted as "or".
Claims 1 and 27 recite "the converted sequence" in steps (e) and (c), respectively, and claims 7, 10-13, 15, and 19 recite "the converted sequences" throughout the claims. There is insufficient antecedent basis for “the converted sequence(s)” because there are both "converted reference sequences" and "converted sequence reads". The phrase "converted sequences" is ambiguous as to which is being referred to (granted there is more context in claims 1 and 27 for one to discern there are meant to refer to the "converted sequence reads", however there is no context in claim 7). To further prosecution, "the converted sequence(s)" is interpreted as "the converted sequence read(s)".
Claim 8 recites "wherein the C-to-T converted sequence read, the reverse compliment of the C-to-T converted sequence read, the G-to-A converted sequence read, or the reverse compliment of the G-to-A converted sequence read resulting in the best alignment is the converted sequence read" in lines 9-12. There is insufficient antecedent basis for “the converted sequence read”, as there are four different types of "converted sequence read" identified in claim 8. Therefore it is not clear which type is being referred to for the limitation of "resulting in the best alignment". To further prosecution, the limitation is interpreted as not further limiting claim 8.
Claim 16 recites "wherein steps (d1) and (d2) are performed by an aligner, or wherein steps (d1), (d2), and (d3) are performed by an aligner". There is insufficient antecedent basis for step "(d3)", as this step is only introduced in dependent claim 15. To further prosecution, the claim is interpreted as “wherein steps (d1) and (d2) are performed by an aligner".
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 10, 12-13, and 15 rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, 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 10 rejected as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends. The claim recites "the number of the converted sequences that support methylated Cs at that position is the number of Cs, if any, of the converted sequences that are mapped to that position, and wherein the number of the converted sequences that support unmethylated Cs at that position is the number of Ts, if any, of the converted sequences that are mapped to that position", which does not further limit claim 1 because these limitations are effectively identical to step (e) of claim 1.
Claim 12 rejected as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends. The claim recites "determining, for each of the plurality of positions with Cs in the reference genome sequence, the number of the converted sequences that support methylated Cs or the number of the converted sequences that support unmethylated Cs at that position comprises: determining, for each of one or more positions of the plurality of positions with Cs in the reference genome sequence, a number of the converted sequences that support C-to-T mutations based on a number of As of the converted sequences that are mapped to the complementary strand of the reference genome sequence at that position", which does not further limit claim 1 because these limitations are effectively identical to step (e) of claim 1.
Claim 13 rejected as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends. The claim recites "the number of the converted sequences that support C-to-T mutations at that position is a number of As of the converted sequences that are mapped to the complementary strand of the reference genome sequence at that position, or wherein determining, for each of one or more positions of the plurality of positions with Cs in the reference genome sequence, the number of the converted sequences that support C-to-T mutations comprises: determining, for each of one or more positions of the plurality of positions with Cs in the reference genome sequence, a percentage of the converted sequences that support C-to-T mutations at that position using a percentage of the converted sequences that are mapped to the complementary strand of the reference genome sequence with As at that position, optionally wherein the percentage of the converted sequences that support C-to-T mutations at that position indicate the percentage of DNA with C-to-T mutations at that position in the sample", which does not further limit claim 1 because these limitations are effectively identical to step (e) of claim 1.
Claim 15 rejected as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends. The claim recites "for each of one or more positions of the plurality of positions with Cs in the reference genome sequence to which a C or a T of the converted sequence read is mapped to, (i) increasing a counter of C in that position if the converted sequence read comprises a C mapped to that position; and (ii) increasing a counter of T in that position if the converted sequence comprises a T mapped to that position, wherein determining, for each of the plurality of positions with Cs in the reference genome sequence, the number of the converted sequences that support methylated Cs or the number of the converted sequences that support unmethylated Cs at that position comprises: determining, for each of the plurality of positions with Cs in the reference genome sequence, the number of the converted sequences that support methylated Cs or the number of the converted sequences that support unmethylated Cs at that position based on (i) a value of the counter for C at that position and (ii) a value of the counter for T at that position", which does not further limit claim 1 because these limitations are effectively identical to step (e) of claim 1.
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
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 1, 4-5, 7-20, 23-24 and 27 are rejected under 35 U.S.C. 101 because the claimed invention is directed to an abstract idea of a mental process, a mathematical concept, organizing human activity, or a law of nature or natural phenomenon without significantly more. In accordance with MPEP § 2106, claims found to recite statutory subject matter (Step 1: YES) are then analyzed to determine if the claims recite any concepts that equate to an abstract idea, law of nature or natural phenomenon (Step 2A, Prong 1). In the instant application, the claims recite the following limitations that equate to an abstract idea:
Claim 1 and 27: “determining, for each of a plurality of positions with Cs in the reference genome sequence, a number of the converted sequences that support methylated Cs or a number of the converted sequences that support unmethylated Cs at that position based on a number of Cs, if any, or a number of Ts, if any, of the converted sequences that are mapped to that position” provides an evaluation (determining a number of reads that support methylated positions involves evaluating alignment data) that may be performed in the human mind and is therefore considered a mental process, which is an abstract idea.
Claim 9: “determining the converted sequence read is a top strand, a reverse complement of the top strand, a bottom strand, or a reverse complement of the bottom strand based on alignment types” provides an evaluation (determining strandedness and complementarity of reads involves evaluating alignment data) that may be performed in the human mind and is therefore considered a mental process, which is an abstract idea.
Claim 11: “determining [. . .] a percentage of the converted sequences that support methylated Cs [etc]” provides an evaluation (determining percentages of reads that support methylated Cs involves evaluating alignment data) that may be performed in the human mind and is therefore considered a mental process, which is an abstract idea.
These recitations are similar to the concepts of collecting information, analyzing it, and displaying certain results of the collection and analysis in Electric Power Group, LLC, v. Alstom (830 F.3d 1350, 119 USPQ2d 1739 (Fed. Cir. 2016)), organizing and manipulating information through mathematical correlations in Digitech Image Techs., LLC v Electronics for Imaging, Inc. (758 F.3d 1344, 111 U.S.P.Q.2d 1717 (Fed. Cir. 2014)) and comparing information regarding a sample or test to a control or target data in Univ. of Utah Research Found. v. Ambry Genetics Corp. (774 F.3d 755, 113 U.S.P.Q.2d 1241 (Fed. Cir. 2014)) and Association for Molecular Pathology v. USPTO (689 F.3d 1303, 103 U.S.P.Q.2d 1681 (Fed. Cir. 2012)) that the courts have identified as concepts that can be practically performed in the human mind or are mathematical relationships. Therefore, these limitations fall under the “Mental process” and “Mathematical concepts” groupings of abstract ideas. Additionally, while claim 27 recites performing some aspects of the analysis on “A system for methylation calling comprising: non-transitory memory configured to store executable instructions, [. . .] and a hardware processor in communication with the non-transitory memory, the hardware processor programmed by the executable instructions”, there are no additional limitations that indicate that this requires anything other than carrying out the recited mental processes or mathematical concepts in a generic computer environment. Merely reciting that a mental process is being performed in a generic computer environment does not preclude the steps from being performed practically in the human mind or with pen and paper as claimed. If a claim limitation, under its broadest reasonable interpretation, covers performance of the limitation in the mind but for the recitation of generic computer components, then it falls within the “Mental processes” grouping of abstract ideas. As such, claims 1, 4-5, 7-20, 23-24 and 27 recite an abstract idea (Step 2A, Prong 1: YES).
Claims found to recite a judicial exception under Step 2A, Prong 1 are then further analyzed to determine if the claims as a whole integrate the recited judicial exception into a practical application or not (Step 2A, Prong 2). The judicial exceptions listed above are not integrated into a practical application because the claims do not recite an additional element or elements that reflects an improvement to technology. Specifically, the claims recite the following additional elements:
Claim 1 and 27: “receiving a reference genome sequence” and “receiving a plurality of sequence reads generated from a sample subjected to a methylation assay” provides insignificant extra-solution activities (receiving data is a pre-solution activity involving data gathering steps) that do not serve to integrate the judicial exceptions into a practical application.
“generating a mapping reference sequence comprising a cytosine (C)-to-thymine (T) converted reference sequence and a guanine (G)-to-adenine (A) converted reference sequence from the reference genome sequence” and “generating a C-to-T converted sequence read and a G-to-A converted sequence read” provides insignificant extra-solution activities (converting C-to-T and G-to-A in both reference and sequence data is a pre-solution activity involving data manipulation steps) that do not serve to integrate the judicial exceptions into a practical application.
“mapping the C-to-T converted sequence read and the G-to-A converted sequence read to the mapping reference sequence to determine a converted sequence read mapped to the C-to-T converted reference sequence or the G-to-A converted reference sequence of the mapping reference sequence” provides insignificant extra-solution activities (mapping sequence data is a pre-solution activity involving data manipulation steps) that do not serve to integrate the judicial exceptions into a practical application.
Claim 4: “indexing the C-to-T converted reference sequence and the G-to-A converted reference sequence as two separate reference sequences together, optionally wherein the mapping reference comprises the C-to-T converted reference sequence and the G-to-A converted reference sequence with different reference identifiers” provides insignificant extra-solution activities (indexing sequence data or mapping to a combined reference sequence is a pre-solution activity involving data manipulation steps) that do not serve to integrate the judicial exceptions into a practical application.
Claim 8: “mapping the C-to-T converted sequence read and the G-to-A converted sequence read to the mapping reference sequence to determine the best alignment amongst (i) the C-to-T converted sequence read and the C-to-T converted reference sequence, (ii) a reverse complement of the C-to-T converted sequence read and the C-to-T converted reference sequence, (iii) the G-to-A converted sequence read and the G-to-A converted reference sequence, and (iv) a reverse complement of the G-to-A converted sequence read and the G-to-A converted reference sequence” provides insignificant extra-solution activities (mapping sequence data is a pre-solution activity involving data manipulation steps) that do not serve to integrate the judicial exceptions into a practical application. Examiner also notes that this is part of the standard Bismark workflow addressed below.
Claim 19: “creating a file or a report or generating a user interface (UI) comprising a UI element representing or comprising, for each of positions of the plurality of positions with Cs in the reference genome sequence, the number or percentage of the converted sequences that support methylated Cs or the number or percentage of the converted sequences that support unmethylated Cs at that position” provides insignificant extra-solution activities (outputting a report with associated metrics is a post-solution activity involving data gathering and manipulation steps) that do not serve to integrate the judicial exceptions into a practical application.
Claim 27: “A system for methylation calling comprising: non-transitory memory configured to store executable instructions, [. . .] and a hardware processor in communication with the non-transitory memory, the hardware processor programmed by the executable instructions” provides insignificant extra-solution activities (running instructions on generic computer components) that do not serve to integrate the judicial exceptions into a practical application.
The steps for receiving, indexing, converting, mapping, and outputting data are insignificant extra-solution activities that do not serve to integrate the recited judicial exceptions into a practical application because they are pre- and post-solution activities involving data gathering, data manipulation, and sample manipulation steps (see MPEP 2106.04(d)(2)). Furthermore, the limitations regarding implementing program instructions do not indicate that they require anything other than mere instructions to implement the abstract idea in a generic way or in a generic computing environment. As such, this limitation equates to mere instructions to implement the abstract idea on a generic computer that the courts have stated does not render an abstract idea eligible in Alice Corp., 573 U.S. at 223, 110 USPQ2d at 1983. See also 573 U.S. at 224, 110 USPQ2d at 1984. Therefore, claims 1, 4-5, 7-20, 23-24 and 27 are directed to an abstract idea (Step 2A, Prong 2: NO).
Claims found to be directed to a judicial exception are then further evaluated to determine if the claims recite an inventive concept that provides significantly more than the judicial exception itself (Step 2B). The claims do not include additional elements that are sufficient to amount to significantly more than the judicial exception because the claims recite additional elements that are insignificant extra-solution activities that do not serve to integrate the recited judicial exceptions into a practical application, or equate to mere instructions to apply the recited exception in a generic way or in a generic computing environment.
As discussed above, there are no additional elements to indicate that the claimed “system for methylation calling comprising: non-transitory memory configured to store executable instructions, [. . .] and a hardware processor in communication with the non-transitory memory, the hardware processor programmed by the executable instructions” requires anything other than generic computer components in order to carry out the recited abstract idea in the claims. Claims that amount to nothing more than an instruction to apply the abstract idea using a generic computer do not render an abstract idea eligible. MPEP 2106.05(f) discloses that mere instructions to apply the judicial exception cannot provide an inventive concept to the claims. Additionally, the limitations for receiving, indexing, converting, mapping, and outputting data are insignificant extra-solution activities that do not serve to integrate the recited judicial exceptions into a practical application. Furthermore, no inventive concept is claimed by these limitations as they are demonstrated to be well-understood, routine, and conventional:
Page 7 col 1 paragraph 1 "We use the popular Bismark package (50) for multiple steps of our pipeline, including genome conversion, which must be completed once before alignment" (Singer, Benjamin D. "A practical guide to the measurement and analysis of DNA methylation." American journal of respiratory cell and molecular biology 61.4 (2019): 417-428).
Page 6 last paragraph "Two mapping approaches were developed for bisulfite sequencing alignment: three-letter and wild card. The three-letter approach simplifies the four-letter-based genome to a three letter-based genome by converting all Cs to Ts in the reference genome and sequence reads [29]. Thereafter, the reads are processed with standard aligners, such as Bowtie1 or 2 [39–41], BWA mem [42, 43], and GEM3 [44, 45], mainly employing the Burrows Wheeler Transform (BWT) backtracking algorithm" (Gong et al. "Analysis and performance assessment of the whole genome bisulfite sequencing data workflow: currently available tools and a practical guide to advance DNA methylation studies." Small Methods 6.3 (2022): 2101251).
The additional elements do not comprise an inventive concept when considered individually or as an ordered combination that transforms the claimed judicial exception into a patent-eligible application of the judicial exception. Therefore, the claims do not amount to significantly more than the judicial exception itself (Step 2B: No). As such, claims 1, 4-5, 7-20, 23-24 and 27 are not patent eligible.
Claim Rejections - 35 USC § 102
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 the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claims 1, 4-5, 7-20, 23-24 and 27 rejected under 35 U.S.C. 102(a)(1), based upon a public use or sale or other public availability of the invention, as being anticipated by Krueger (Felix Krueger, Babraham Institute. June 16, 2011. "Bismark ‐ Quick Reference" v0.5.1. Babraham Bioinformatics. bioinformatics.babraham.ac.uk/projects/bismark/Bismark_User_Guide_v0.5.1.pdf).
Regarding Claims 1, 10, 12-15, and 27, Krueger teaches receiving a reference genome sequence (Page 1 paragraph 2 "First you need to download a reference genome and place it in a genome folder") and receiving a plurality of sequence reads generated from a sample subjected to a methylation assay (Page 1 paragraph 2 "Both single‐entry or multiple‐entry FastA files are supported").
Krueger also teaches: generating a mapping reference sequence comprising a cytosine (C)-to-thymine (T) converted reference sequence and a guanine (G)-to-adenine (A) converted reference sequence from the reference genome sequence; and for each of the plurality of sequence reads: (d1) generating a C-to-T converted sequence read and a G-to-A converted sequence read; and (d2) mapping the C-to-T converted sequence read and the G-to-A converted sequence read to the mapping reference sequence to determine a converted sequence read mapped to the C-to-T converted reference sequence or the G-to-A converted reference sequence of the mapping reference sequence; and (claim 14) the plurality of positions with Cs in the reference genome sequence comprises at least 10,000 positions, substantially all positions, or all positions with Cs in the reference genome sequence (Page 4 last paragraph "Sequence reads are first transformed into fully bisulfite‐converted forward (C‐>T) and reverse read (G‐>A conversion of the forward strand) versions, before they are aligned to similarly converted versions of the genome (also C‐>T and G‐>A converted)").
Krueger also teaches determining, for each of a plurality of positions with Cs in the reference genome sequence, a number of the converted sequences that support methylated Cs or a number of the converted sequences that support unmethylated Cs at that position based on a number of Cs, if any, or a number of Ts, if any, of the converted sequences that are mapped to that position [which is effectively identical to claims 10, 12-13, and 15] (Page 5 Bismark alignment and methylation call report describes several outputs including the "Number of methylated and unmethylated cytosines" and "Percentage of methylation of cytosines in either CpG, CHG or CHH context (where H can be either A, T or C)").
Regarding Claims 4 and 17, Krueger teaches generating the mapping reference sequence comprises: indexing the C-to-T converted reference sequence and the G-to-A converted reference sequence as two separate reference sequences together, optionally wherein the mapping reference comprises the C-to-T converted reference sequence and the G-to-A converted reference sequence with different reference identifiers (Page 15 Options include "--single_fasta" which will "[i]nstruct the Bowtie Indexer to write the converted genomes into single‐entry FastA files instead of making one multi‐FastA file (MFA) per chromosome", implies that the default is to combine references together into a single FASTA with different reference identifiers). With respect to Claim 17, Krueger also teaches the method is at least 2x faster than a multi-pass methylation calling method, because figure 8 (which shows the difference in approach between the claimed "single-pass" method and a "multi-pass" method) encompasses the option on page 15 in Krueger above which implicitly covers both methods and therefore would also encompass the same claimed speed improvements.
Regarding Claims 5 and 7, Krueger teaches: the C-to-T converted reference sequence comprises all Cs in the reference genome sequence converted to Ts, and wherein the G-to-A converted reference sequence comprises all Gs in the reference genome sequence converted to As; or wherein the C-to-T converted sequence read comprises all Cs in the sequence converted to Ts, and wherein the G-to-A converted sequence read comprises all Gs in the sequence converted to As; and the converted sequence is the C-to-T converted sequence, a reverse complement of the C-to-T converted sequence, the G-to-A converted sequence, or a reverse complement of the G-to-A converted sequence (Page 4 last paragraph "Sequence reads are first transformed into fully bisulfite‐converted forward (C‐>T) and reverse read (G‐>A conversion of the forward strand) versions, before they are aligned to similarly converted versions of the genome (also C‐>T and G‐>A converted)").
Regarding Claim 8, Krueger teaches mapping the C-to-T converted sequence read and the G-to-A converted sequence read to the mapping reference sequence comprises: mapping the C-to-T converted sequence read and the G-to-A converted sequence read to the mapping reference sequence to determine the best alignment amongst (i) the C-to-T converted sequence read and the C-to-T converted reference sequence, (ii) a reverse complement of the C-to-T converted sequence read and the C-to-T converted reference sequence, (iii) the G-to-A converted sequence read and the G-to-A converted reference sequence, and (iv) a reverse complement of the G-to-A converted sequence read and the G-to-A converted reference sequence (Page 4 last paragraph "Sequence reads are first transformed into fully bisulfite‐converted forward (C‐>T) and reverse read (G‐>A conversion of the forward strand) versions, before they are aligned to similarly converted versions of the genome (also C‐>T and G‐>A converted)").
Krueger also teaches wherein the C-to-T converted sequence read, the reverse compliment of the C-to-T converted sequence read, the G-to-A converted sequence read, or the reverse compliment of the G-to-A converted sequence read resulting in the best alignment is the converted sequence read (Page 4 last paragraph "Sequence reads that produce a unique best alignment from the four alignment processes against the bisulfite genomes (which are running in parallel) are then compared to the normal genomic sequence and the methylation state of all cytosine positions in the read is inferred").
Regarding Claim 9, Krueger teaches mapping the C-to-T converted sequence read and the G-to-A converted sequence read to the mapping reference sequence comprises determining the converted sequence read is a top strand, a reverse complement of the top strand, a bottom strand, or a reverse complement of the bottom strand based on alignment types (Page 6 numeral 3 "Bismark methylation extractor. This step is optional and will extract the methylation information from the Bismark alignment output. Running this additional step allows splitting the methylation information up into the different contexts, getting strand‐specific methylation information and offers some filtering options" and page 8 paragraph 4 "To summarise this again: alignments to the original top strand or to the strand complementary to the original top strand (OT and CTOT) will both yield methylation information for cytosines on the top strand. Alignments to the original bottom strand or to the strand complementary to the original bottom strand (OB and CTOB) will both yield methylation information for cytosines on the bottom strand, i.e. they will appear to yield methylation information for G positions on the top strand of the reference genome").
Regarding Claim 11, Krueger teaches determining, for each of the plurality of positions with Cs in the reference genome sequence, a number of the converted sequences that support methylated Cs or a number of the converted sequences that support unmethylated Cs at that position comprises: determining (i) a percentage of the converted sequences that support methylated Cs or (ii) a percentage of the converted sequences that support methylated Cs using (i) a percentage of the converted sequences with Cs, if any, or (ii) a percentage of the converted sequences with Ts, if any, mapped to that position, or wherein the percentage of the converted sequences that support methylated Cs at that position indicate the percentage of the DNA with methylated Cs at that position in the sample, and wherein the percentage of the converted sequences that support unmethylated Cs at that position indicate the percentage of the DNA with unmethylated Cs at that position in the sample (Page 5 Bismark alignment and methylation call report describes several outputs including the "Number of methylated and unmethylated cytosines" and "Percentage of methylation of cytosines in either CpG, CHG or CHH context (where H can be either A, T or C)").
Regarding Claim 16, Krueger teaches wherein steps (d1) and (d2) are performed by an aligner (as interpreted above) (Page 4 paragraph 1 "Bismark holds the reference genome in memory and in addition to that runs four parallel instances of Bowtie").
Regarding Claim 18, Krueger teaches the methylation assay comprises bisulfite sequencing, whole genome-bisulfite sequencing (WGBS), Enzymatic Methyl-seq (EM-seq), or TET-assisted pyridine borane sequencing (TAPS), or wherein the methylation assay comprises a directional methylation protocol, a non-directional methylation protocol, or post-bisulfite adapter tagging (PBAT); and wherein the plurality of sequence reads is generated by whole genome sequencing (WGS), optionally wherein the WGS is clinical WGS (cWGS) (Page 8 describes directional BS-Seq (bisulfite treatment sequencing libraries) used to generate the data for methylation calling).
Regarding Claim 19, Krueger teaches creating a file or a report or generating a user interface (UI) comprising a UI element representing or comprising, for each of positions of the plurality of positions with Cs in the reference genome sequence, the number or percentage of the converted sequences that support methylated Cs or the number or percentage of the converted sequences that support unmethylated Cs at that position (Page 5 Bismark alignment and methylation call report describes several outputs including the "Number of methylated and unmethylated cytosines" and "Percentage of methylation of cytosines in either CpG, CHG or CHH context (where H can be either A, T or C)").
Regarding Claim 20, Krueger teaches the plurality of sequence reads comprises sequence reads that are about 100 base pairs to about 1000 base pairs in length each, or wherein the plurality of sequence reads comprises paired-end sequence reads or single-end sequence reads (Page 9 paragraph 1 "Bismark produces a comprehensive alignment and methylation call output file for each input file or set of paired‐end input files").
Regarding Claims 23 and 24, Krueger teaches: the sample comprises cells, cell-free DNA, cell-free fetal DNA, amniotic fluid, a blood sample, a biopsy sample, or a combination thereof; and the sample is a sample of a subject, optionally wherein the sample is obtained directly from a subject (Page 4 describes a test data set that meets the requirements of claims 23 and 24 "A test BS‐Seq data set is available for download from the Bismark homepage. It contains 10,000 single‐end shotgun BS reads from human ES cells in FastQ format (from SRR020138, Lister et al, 2009; trimmed to 50 bp; base call qualities are Sanger encoded Phred values (Phred33))").
Conclusion
No claims are allowed.
Inquiries
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Robert A. Player whose telephone number is 571-272-6350. The examiner can normally be reached Mon-Fri, 8am-5pm.
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/R.A.P./Examiner, Art Unit 1686
/Karlheinz R. Skowronek/ Supervisory Patent Examiner, Art Unit 1687
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