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 . Applicant’s amendment filed 6/26/2026 is acknowledged. Claims 1-58 have been canceled. Claim 59 has been amended. Claims 59-78 are pending.
All of the amendment and arguments have been thoroughly reviewed and considered. Any rejection not reiterated in this action has been withdrawn as being obviated by the amendment of the claims.
This action is made Final.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Previous Rejections
The objection to the specification is withdrawn in view of Applicant’s amendment of the claims. The prior art rejection under 35 USC 103 directed to the claims 59-78 is withdrawn in view of the new grounds of rejections necessitated by applicant’s amendment of the claims:
New Ground(s) of Rejections
The new ground(s) of rejections were necessitated by applicant’s amendment of the claims:
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
4. Claim(s) 59-78 is/are rejected under 35 U.S.C. 103 as being unpatentable over Kennedy et al (WO 2018119452, June 28, 2018) in view of Osborne et al (WO 2017037657, March 2017) and further in view of Kim et al (C-2921620, citations based on WO 2015026853 version, February 2015).
Regarding claims 59-78, Kennedy et al teach at paragraphs [0023] – [0025] a method of fractionating DNA molecules from a human sample to generate two or more partitions; applying differential molecular tags and NGS-enabling adapters to each of the two or more partitions to generate molecular tagged partitions (see also [00211] which discuss nuclei acid partitioned based on methylation differences); assaying the molecular tagged partitions on an NGS instrument to generate sequence data for deconvoluting the sample into molecules that were differentially partitioned. In one embodiment the method further comprises analyzing the sequence data by deconvoluting the sample into molecules that were differentially partitioned. In another embodiment the DNA molecules are from extracted blood plasma. In another embodiment physical fractionating comprises fractionating molecules based on various degrees of methylation (see also [000224] and [00234] which further teach fragmentome signatures based on fragmentomic data). In another embodiment various degrees of methylation comprise hypermethylation and hypomethylation.
In another embodiment physically fractionating comprises fractionating with methyl-binding domain protein ("MBD")-beads to stratify into various degrees of methylation. In another embodiment the differential molecular tags are different sets of molecular tags corresponding to a MBD-partition. In another embodiment the physical fractionation comprises separating DNA molecules using immunoprecipitation. In another embodiment the method further comprises re-combining two or more molecular tagged fractions of the generated molecular tagged fractions. In another embodiment the method further comprises enriching the re-combined molecular tagged fractions or groups. In another embodiment the one or more characteristics is methylation. In another embodiment the fractionation comprises separating methylated nucleic acids from non-methylated nucleic acids using proteins comprising a methyl-binding domain to generate groups of nucleic acid molecules comprising varying degrees of methylation. In another embodiment one of the groups comprises hypermethylated DNA. In another embodiment at least one group is characterized by a degree of methylation. In another embodiment fractionation comprises isolating protein-bound nucleic acids. In another embodiment the isolating comprises immunoprecipitation.
[00024] In another aspect provided herein is a method for molecular tag identification of MBD-bead fractionated libraries through NGS, comprising: physical fractionation of an extracted DNA sample using a methyl-binding domain protein-bead purification kit, saving all elutions for downstream processing; parallel application of differential molecular tags and NGS- enabling adapter sequences to each fraction or group; re-combining all molecular tagged fractions or groups, and subsequent amplification (see also paragraphs [00160], [00176], [00178] which teaches amplification by PCR) using adapter-sequence specific DNA primer sequences; (d) enrichment/hybridization of re-combined and amplified total library, targeting genomic regions of interest; re-amplification of the enriched total DNA library, appending a sample tag; and pooling different samples, and assaying them in multiplex on an NGS instrument; wherein NGS sequence data provides sequence of the molecular tags being used to identify unique molecules, and sequence data for deconvolution of the sample into molecules that were differentially MBD-partitioned. In one embodiment the method comprises performing analysis of NGS data, with the molecular tags being used to identify unique molecules, as well deconvolution of the sample into molecules that were differentially MBD- partitioned. In another embodiment the fractionation comprises physical fractionation. In another embodiment the population of nucleic acid molecules is partitioned based on one or more characteristics selected from the group consisting of: methylation status, glycosylation status, histone modification, length and start/stop position. In another embodiment the method further comprises pooling the nucleic acid molecules. In another embodiment fractionation comprises fractionating based on a difference in a mono-nucleosomal profile. In another embodiment fractionation is capable of generating different mono-nucleosomal profiles for at least one group of nucleic acid molecules when compared to a normal. In another embodiment the method further comprises fractionating at least one group of nucleic acid molecules based on a different characteristic. In another embodiment analyzing comprises, at one or more loci, comparing a first characteristic corresponding to a first group of nucleic acid molecules to a second characteristic corresponding to a second group of nucleic acid molecules. In another embodiment the nucleic acid molecules are circulating tumor DNA. In another embodiment the nucleic acid molecules are cell-free DNA ("cfDNA"). In another embodiment the tags are used to distinguish different molecules in the same sample. In another embodiment the one or more characteristic is a cancer marker.
[00025] In another aspect provided herein is a method, comprising: providing a population of nucleic acid molecules obtained from a bodily sample of a subject; fractionating the population of nucleic acid molecules based on one or more characteristics to generate a plurality of groups of nucleic acid molecules, differentially tagging nucleic acid molecules in the plurality of groups to distinguish the nucleic acid molecules in each of the plurality of groups from one another based on the one or more characteristics; sequencing the plurality of groups of nucleic acid molecules to generate sequence reads; containing sufficient data to generate relative information about nucleosome positioning, nucleosome modification, or binding DNA-protein interaction for each of the plurality of groups of nucleic acid molecules. In one embodiment the method further comprises analyzing the sequence reads to generate relative information about nucleosome positioning, nucleosome modification, or binding DNA-protein interaction for each of the plurality of groups of nucleic acid molecules. In another embodiment the method further comprises using a trained classifier to classify the subject based on the one or more characteristics. In another embodiment the one or more characteristics comprise a quantitative characteristic of the mapped reads. In another embodiment the fractionation comprises physical fractionation. In another embodiment the method further comprises pooling the nucleic acid molecules. In another embodiment fractionation comprises fractionating based on a difference in a mono-nucleosomal profile. In another embodiment fractionation is capable of generating different mononucleosomal profiles for at least one group of nucleic acid molecules when compared to a normal. In another embodiment the method further comprises fractionating at least one group of nucleic acid molecules based on a different characteristic. In another embodiment analyzing comprises, at one or more loci, comparing a first characteristic corresponding to a first group of nucleic acid molecules to a second characteristic corresponding to a second group of nucleic acid molecules. In another embodiment analyzing comprises analyzing a characteristic of the one or more characteristics in a group relative to a normal sample at one or more loci. In another embodiment the one or more characteristics are selected from the group consisting of: a base-call frequency at a base position on the reference sequence, a number of molecules mapping to one base or sequence on the reference sequence, a number of molecules having a start site mapping to a base position on the reference sequence, and a number of molecules having a stop site mapping to a base position on the reference sequence, and a length of a molecule mapping to a locus on the reference sequence. In another embodiment the method further comprises using a trained classifier to classify the subject based on the one or more characteristics. In another embodiment the trained classifier classifies the one or more characteristics as associated with a tissue in the subject. In another embodiment the trained classifier classifies the one or more characteristics as associated with a type of cancer in the subject. In another embodiment the one or more characteristics are indicative of gene expression or status of a disease. In another embodiment the nucleic acid molecules are circulating tumor DNA. In another embodiment the nucleic acid molecules are cell-free DNA ("cfDNA"). In another embodiment the tags are used to distinguish different molecules in the same sample. In another embodiment the one or more characteristic is a cancer marker.
Kennedy teaches at paragraph [0026], in another aspect provided herein is a method, comprising: providing a population of nucleic acid molecules obtained from a bodily sample of a subject; fractionating the population of nucleic acid molecules based on methylation status to generate a plurality of groups of nucleic acid molecules; differentially tagging nucleic acid molecules in the plurality of groups to distinguish the nucleic acid molecules in each of the plurality of groups from one another based on the one or more characteristics; sequencing the plurality of groups of nucleic acid molecules to generate sequence reads; and analyzing the sequence reads to detect one or more characteristics in one of the plurality of groups of nucleic acid molecules, wherein the one or more characteristics is indicative of nucleosome positioning, nucleosome modification, or a DNA-protein interaction. In another embodiment the method further comprises using a trained classifier to classify the subject based on the one or more characteristics. In another embodiment the one or more characteristics comprise a quantitative characteristic of the mapped reads. In another embodiment the fractionation comprises physical fractionation. In another embodiment the method further comprises pooling the nucleic acid molecules. In another embodiment fractionation comprises fractionating based on a difference in a mono-nucleosomal profile. In another embodiment fractionation is capable of generating different mononucleosomal profiles for at least one group of nucleic acid molecules when compared to a normal. In another embodiment the method further comprises fractionating at least one group of nucleic acid molecules based on a different characteristic. In another embodiment analyzing comprises, at one or more loci, comparing a first characteristic corresponding to a first group of nucleic acid molecules to a second characteristic corresponding to a second group of nucleic acid molecules. In another embodiment analyzing comprises analyzing a characteristic of the one or more characteristics in a group relative to a normal sample at one or more loci. In another embodiment the one or more characteristics are selected from the group consisting of: a base-call frequency at a base position on the reference sequence, a number of molecules mapping to one base or sequence on the reference sequence, a number of molecules having a start site mapping to a base position on the reference sequence, and a number of molecules having a stop site mapping to a base position on the reference sequence, and a length of a molecule mapping to a locus on the reference sequence. In another embodiment the method further comprises using a trained classifier to classify the subject based on the one or more characteristics. In another embodiment the trained classifier classifies the one or more characteristics as associated with a tissue in the subject. In another embodiment the trained classifier classifies the one or more characteristics as associated with a type of cancer in the subject. In another embodiment the one or more characteristics are indicative of gene expression or status of a disease. In another embodiment the nucleic acid molecules are circulating tumor DNA. In another embodiment the nucleic acid molecules are cell-free DNA ("cfDNA") (see also [00171] which further discuss the cell free nucleic acid samples used in the method). In another embodiment the tags are used to distinguish different molecules in the same sample. In another embodiment the one or more characteristic is a cancer marker.
Kennedy teaches the use of differential fragmentation pattern to partition ctDNA for differential labelling/tagging ([0048]). The reference teaches that the adapters may be Y-shaped adapters or hairpin adapters ([0109]) (see also [00160] which further teaches Y-shaped adapters/forked with 5-methylcytosine replacing) used in BIS-seq with a label on one of the two adapter strands. At paragraph [00134] –[00135], Kennedy teaches wherein each partition differentially tagged. Tags can be molecules, such as nucleic acids, containing information that indicates a feature of the molecule with which the tag is associated. For example, molecules can bear a sample tag (which distinguishes molecules in one sample from those in a different sample), a partition tag (which distinguishes molecules in one partition from those in a different partition) or a molecular tag (which distinguishes different molecules from one another (in both unique and non-unique tagging scenarios). In certain embodiments, a tag can comprise one or a combination of barcodes. A barcode can have, for example, between 10 and 100 nucleotides. A collection of barcodes can have degenerate sequences or can have sequences having a certain hamming distance, as desired for the specific purpose. So, for example, a sample index, partition index or molecular index sequence can be comprised of one barcode or a combination of two barcodes, each attached to different ends of a molecule.
Kennedy differs from the instant invention in that Kennedy does not teach wherein the method comprised concatenating polynucleotides, wherein the concatenating comprises denaturing, annealing, and/or extension seps to concatenate the tagged polynucleotide.
Osborne et al teaches a method of sequencing comprising concatenating a plurality of fragments of genomic DNA to produce concatenated DNA; sequencing the concatenated DNA to produce a plurality of sequence reads, wherein at least some of the sequence reads comprise: at least the sequence of the 3' and/or 5' ends of a fragment that corresponds to the locus of interest and sequence of one or both of the fragments that flank the fragment in the concatenated DNA; and grouping the sequence reads that corresponds to the locus of interest using, for each of the grouped sequence reads: the 3' and/or 5' end sequences; and/or the flanking sequence (abstract). Osborne teaches that the concatenated DNA may comprise at least a thousand or at least ten thousand concatenated DNA molecules, and each molecule may contain at least 3, at least 5, at least 10, at least 50, at least 100, at least 500, at least 1000 fragments that are joined one another in a random order and orientation (page 17). The reference teaches that the concatenated DNA may be fragmented to a desired length and ligated to adapter sequences, wherein an amplification step may occur prior to sequencing (page 18). Osborne teaches that in some embodiment fragments are attached to a generic asymmetric adapter before amplification allowing the identification sequencing reads that derived either the top or bottom strand of a double stranded fragment. The reference teaches that this approach can be used in error correction thereby increasing the confidence that a sequence variation/mutation is genuine (page 18 and 20).
With regards to the limitation, wherein the fragmented polynucleotides comprise fragments less than 50 base pairs in length, the specification teaches at paragraph [0020] that “the polynucleotide sample is fragmented to a size of 200-500 base pair prior to the partitioning”. The specification teaches at paragraph [0013] that “the method, further comprises, prior to tagging, selecting amplified polynucleotides of size less than 50 base pairs.”
Kennedy teaches that populations of nucleic acids may be partitioned based on nucleic acid lengths (e.g., molecules of up to 160 bp and greater than 160 bp ([000127]).
Osborne teaches that the amplification products or sequenced fragments may have a median length below 1 kb, such as in the range from 50 base pairs to 500 base pairs, although fragments having a median size outside of this range may be used (page 18, lines 26-29). The limitation median is interpreted as being “a midpoint.” and therefore encompasses values higher and lower than what is recited as the median. MPEP 2144.05 states that if the ranges do not overlap but are close enough that one skilled in the art would expect similar properties, obviousness may still be established. In this case, neither the specification nor clams provide any evidence of the criticality of fragments sizes less than 50 base pairs as instantly claimed and therefore, the fragment sizes are deemed prima facie obvious in view of the teachings of Kennedy and Osborne.
Still further, Kim et al teach a method of sequencing nucleic acid, wherein the method encompasses steps of extracting sequence data comprising concatenating sequence data files, identifying and extracting target sequence and aligning target sequences (page 32, Figure 5). Kim teaches fragmentation of nucleic acids and sequencing of fragmented sequences less than 50 base pairs in length and greater than 50bps which further provides evidence supporting a prima facie case of obvious of the teachings of Kennedy and Osborne.
It would have been further obvious to one of ordinary skill in the art at the time of the effective filing date of the claimed invention to have been motivated to have modified the sequence analysis method of Kennedy to encompass a concatenating step as taught by Osborne and Kim for the obvious further benefit of increasing efficiency of accurately analyzing a polynucleotide sample for the presence of cancer(s) as Kennedy and Osborne teaches that their method are used in the detection mutations implicated in cancers or diagnosis and development of treatment for cancers as taught by Kim.
Conclusion
5. No claims are allowed. Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
6. Any inquiry concerning this communication or earlier communications from the examiner should be directed to CYNTHIA B WILDER whose telephone number is (571)272-0791. The examiner can normally be reached Flexible.
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/CYNTHIA B WILDER/ Primary Examiner, Art Unit 1681
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