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 .
Election/Restrictions
Applicant’s election of the coverage of an abundant sequence of the abundant sequences is the number of sequence reads aligned to the abundant sequence 2. One, at least one, or each abundant sequence of the abundant sequences comprises a plurality of consecutive subsequences of a reference nucleotide sequence of the plurality of reference nucleotide sequences, wherein the number of sequence reads aligned to each of the plurality of consecutive subsequences is above the coverage threshold. 3. Determining the abundant sequences of the reference nucleotide sequences. comprises determining the number of sequence reads aligned to subsequences of a plurality of subsequences of a reference nucleotide sequence of the plurality of reference nucleotide sequences, and determining an abundant sequence comprising a plurality of consecutive subsequences of the reference nucleotide sequence, wherein the number of sequence reads aligned to each of the plurality of consecutive subsequences is above the coverage threshold. 4. The sample comprises an organism of a species that is not predetermined. 5. The one or more abundant RNA transcripts are ribosomal RNA transcripts.in the reply filed on 6/18/2026 is acknowledged. Because applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.01(a)).
Claims 83-85 withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected species there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 6/18/2026.
Priority
The instant application was filed 11/13/2023 and is a divisional of 17125378 , filed 12/17/2020, which claims priority from provisional application 62950891 , filed 12/19/2019.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 2/23/2024 is being considered by the examiner.
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 79-82, 86-98 are rejected under 35 U.S.C. 101 because the claimed invention is directed to a mental step or abstract idea (instructions) without significantly more. The claim(s) recite(s) the abstract ideas or mental step of receiving, aligning, determining, and designing. This judicial exception is not integrated into a practical application because if there are no additional steps which integrate the mental step or abstract ideas. The claim(s) does/do not include additional elements that are sufficient to amount to significantly more than the judicial exception because the claims provide no steps which are not mental steps or abstract ideas.
Claim analysis
The instant claim 79 is directed towards a method for depleting abundant sequences of ribonucleic acid transcripts comprising: under control of a hardware processor: receiving a plurality of sequence reads of ribonucleic acid (RNA) transcripts, or products thereof, in a sample; aligning each of the plurality of sequence reads to a reference nucleotide sequence, or a subsequence thereof, of a plurality of reference nucleotide sequences; determining abundant sequences of reference nucleotide sequences, or subsequences thereof, of the plurality of reference nucleotide sequences, wherein each of the abundant sequences has a coverage, related to a number of the sequence reads aligned to the abundant sequence, above a coverage threshold; determining top abundant sequences, of the abundant sequences of the reference nucleotide sequences with coverages above the coverage threshold, with highest numbers of coverages; designing one or more nucleic acid probes for depleting each of the top abundant sequences of the reference nucleotide sequences with the highest numbers of coverages based on a sequence of the top abundant sequence, a probe length, and a tiling gap; generating the one or more nucleic acid probes for depleting each of the top abundant sequences of the reference nucleotide sequences with the highest numbers of coverages; and depleting abundant RNA transcripts in a sample using the generated one or more nucleic acid probes and one or more nucleases to generate a plurality of remaining RNA transcripts in the sample.
The receiving, aligning, determining, and designing steps are a mental steps or abstract idea ( or instructions).
While the claim recites “under control of a hardware processor” This appears to be a general use computer. There is no indication the instructions of the claims affects the operation of the hardware processor.
Dependent claims set forth further limitations to other mental steps or abstract ideas..
According to the 2019 Patent Eligibility Guidance an initial two step analysis is required for determining statutory eligibility.
Step 1. Is the claim directed to a process, machine, manufacture, or composition of matter? In the instant case the Step 1 requirement is satisfied as the claims are directed towards a process using a hardware processor in a general use computer.
Step 2A Prong one. Does the claim recite a law of nature, a natural phenomenon or an abstract idea? Yes, abstract idea and law of nature or mental steps.
The claim recites, “receiving a plurality of sequence reads of ribonucleic acid (RNA) transcripts, or products thereof, in a sample; aligning each of the plurality of sequence reads to a reference nucleotide sequence, or a subsequence thereof, of a plurality of reference nucleotide sequences; determining abundant sequences of reference nucleotide sequences, or subsequences thereof, of the plurality of reference nucleotide sequences, wherein each of the abundant sequences has a coverage, related to a number of the sequence reads aligned to the abundant sequence, above a coverage threshold; determining top abundant sequences, of the abundant sequences of the reference nucleotide sequences with coverages above the coverage threshold, with highest numbers of coverages; and designing one or more nucleic acid probes for depleting each of the top abundant sequences of the reference nucleotide sequences with the highest numbers of coverages based on a sequence of the top abundant sequence, a probe length, and a tiling gap.” This is an abstract idea or mental step..
Step 2A prong two. Does the claim recite additional elements that integrate the judicial exception into a practical application? The answer is no as the claim requires the method is performed under control of hardware processor, thus it appears to be in silico or in the mind. Alternatively, the depleting abundant RNA is considered merely applying the judicial exception.
Step 2B. Does the claim recite additional elements that are significantly more than the judicial exceptions? The answer is no as the claim provides no additional steps which integrate of otherwise provide a practical application of the mental steps..
Gu (Genome Biology (2016) 17:41),.Li (PLoS ONE 8(9): e74286. doi:10.1371/journal.pone.0074286) and Kissopoullou (PLoS ONE 8(12): e81809. doi:10.1371/journal.pone.0081809) suggest obtaining reads of sequence data and designing probes to deplete them were known and conventional at the time of the invention.
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 79-82, 86-98 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 79 recites, “under control of a hardware processor.” The claim later recites, “generating the one or more nucleic acid probes for depleting each of the top abundant sequences of the reference nucleotide sequences with the highest numbers of coverages; and depleting abundant RNA transcripts in a sample using the generated one or more nucleic acid probes and one or more nucleases to generate a plurality of remaining RNA transcripts in the sample.” The metes and bounds are unclear how nucleic acid probes are generated and abundant RNA depleted in a sample under the control of a hardware processor.
Claim 79 recites, “receiving a plurality of sequence reads of ribonucleic acid (RNA) transcripts, or products thereof, in a sample.” The metes and bounds are unclear what is required of products thereof. It is unclear how what is required or encompassed of products thereof. It is unclear if this is proteins and if so how the method is performed.
Claim 79 recites, “determining abundant sequences of reference nucleotide sequences, or subsequences thereof, of the plurality of reference nucleotide sequences, wherein each of the abundant sequences has a coverage, related to a number of the sequence reads aligned to the abundant sequence, above a coverage threshold; determining top abundant sequences, of the abundant sequences of the reference nucleotide sequences with coverages above the coverage threshold, with highest numbers of coverages; designing one or more nucleic acid probes for depleting each of the top abundant sequences of the reference nucleotide sequences with the highest numbers of coverages based on a sequence of the top abundant sequence, a probe length, and a tiling gap.” The recitation of abundant sequence or top abundant sequence suggests there is less than abundant, less than top abundant, or non-abundant sequences. Review and searching of the specification did not reveal a standard to differentiate abundant from non-abundant. Thus the metes and bounds are unclear what is required of this relative term. Further the recitation of “coverage” appears to be a relative term which is not defined by the specification or claims. Thus the recitation of “coverage threshold,” highest numbers of coverages” are vague and unclear.
Claim 79 recites, “tiling gap.” “tiling gap” is not an art accepted term. The claims and specification do not provide a definition or standard for tiling gap. The metes and bounds are unclear what is required.
Claim 88 recites, “determining a similarity score between a pair of the top abundant sequences; and iteratively removing each top abundant sequence having the similarity score, with respect to any other top abundant sequence of the plurality of top abundant sequences remaining, that is above a similarity threshold from the top abundant sequences remaining.” The metes and bounds are unclear as the claims and specific do not provide how to determine a “similarity score.” Thus the claim is vague unclear and incomplete.
Claim 89 recites, “iteratively, determining a similarity score between a pair of the top abundant sequences remaining to be above a similarity threshold; and removing one of the pair of top abundant sequences from the top abundant sequences remaining..” The metes and bounds are unclear as the claims and specific do not provide how to determine a “similarity score.” Thus the claim is vague unclear and incomplete.
Claim 90 is unclear as it recites, “top abundant,” “highest number of coveragesm “tiling top abundant sequence” and “tiling gap,” which are idnefinte for the reasons of record.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent 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.
Claim(s) 79-82, 86-98 is/are rejected under 35 U.S.C. 103 as being unpatentable over Gu (Genome Biology (2016) 17:41) and Chowdhury (.IEEE/ACM TRANSACTIONS ON COMPUTATIONAL BIOLOGY AND BIOINFORMATICS, VOL. 17, NO. 2, MARCH/APRIL 2020, pages 566-586 (published Date of publication 1 Oct. 2018).
The claims have numerous 112 issues as detailed above.
While the claim recites, “under control of a hardware processor,” based on the examples and teachings of the specification this appears to be limited to the receiving, aligning, determining, determining, designing steps of the claims. The rejection is to the interpretation the sequences of the probes are made and used to deplete RNA in a sample.
Gu teaches, “ The challenge of extracting faint signals from abundant noise in molecular diagnostics is a recurring theme across a broad range of applications. In the case of RNA sequencing (RNA-Seq) experiments specifically, there may be several orders of magnitude difference between the most abundant species and the least. This is especially true for metagenomic analyses of clinical samples like cerebrospinal fluid (CSF), whose source material is inherently limited [1], making enrichment or depletion strategies impractical or impossible to employ prior to library construction. The presence of unwanted high abundance species, such as transcripts for the 12S and 16S mitochondrial ribosomal RNAs (rRNAs), effectively increases the cost and decreases the sensitivity of counting-based methodologies.”(page 1, 1st column).
Gu teaches, “In this paper, we have exploited the unique properties of Cas9 to selectively deplete unwanted high-abundance sequences from existing RNA-Seq libraries. We refer to this approach as Depletion of Abundant Sequences by Hybridization (DASH). Employing DASH after transposon-mediated fragmentation but prior to the following amplification step (which relies on the presence of adaptor sequences on both ends of the fragment) prevents amplification of the targeted sequences, thus ensuring they are not represented in the final sequencing library (Fig. 1b). We show that this technique preserves the representational integrity of the non-targeted sequences while increasing overall sensitivity in cell line samples and human metagenomic patient samples. Further, we demonstrate the utility of this system in the context of cancer detection, in which depletion of wild-type sequences increases the detection limit for oncogenic mutant sequences. The DASH technique may be used to deplete specific unwanted sequences from existing Illumina sequencing libraries, PCR amplicon libraries, plasmid collections, phage libraries, and virtually any other existing collection of DNA species.” (page 2 first column middle)
Gu teaches, “ Existing specific sequence enrichment techniques -such as pull-down methods [5, 12–14], amplicon-based methods [3, 15], molecular inversion methods [16–18], COLD-PCR [19], competitive allele-specific TaqMan PCR (castPCR) [20], and the classic method of using restriction enzyme digestion on mutant sites [21] — can effectively enrich for targets in sequencing libraries, but these are not useful for discovery of unknown or unpredicted sequences. Brute force counting methods also exist, such as dPCR [3, 7], but they are not easy to multiplex across a large panel. While high-throughput sequencing of select regions can be highly multiplexed to detect rare and novel mutations, and barcoded unique identifiers can overcome sequencing error noise [22], it is costly since the vast majority of the sequencing reads map to non-informative wild-type sequences. A number of sequence-specific RNA depletion methods also currently exist. Illumina’s Ribo-Zero rRNA Removal Kit and Ambion’s GLOBINclear Kit pull rRNAs and globin mRNAs, respectively, out of total RNA samples using sequence-specific oligos conjugated to magnetic beads. RNAse H-based methods, such as New England BioLab’s NEBNext rRNA Depletion Kit similarly mark abundant RNA species with sequence-specific DNA oligos, and then subject them to degradation by RNAse H, which digests RNA/DNA hybrid molecules [23]. These methods are all employed prior to the start of library prep, and are limited to samples containing at least 10 ng to 1 μg of RNA. DASH, in contrast, depletes abundant species after complementary DNA (cDNA) amplification, and thus can be utilized for essentially any amount of input sample.” (page 2 first column bottom-2nd column)
Thus Gu specifically teaches the problem of depleting samples of highly abundant RNA from samples from sequencing were known.
Gu continues by teaching, “We demonstrate deletion of unwanted mitochondrial rRNA using DASH first on HeLa cell line RNA (Fig. 2) and then on CSF RNA from patients with pathogens in their CSF (Fig. 3), in order to increase sequencing bandwidth of useful data. Selection of rRNA sgRNA targets was based on examining coverage plots for standard RNA-Seq experiments on HeLa cells as well as on several patient CSF samples. Coverage of the 12S and 16S mitochondrial rRNA genes was consistently several orders of magnitude higher than the rest of the mitochondrial and non-mitochondrial genes (Figs. 2c and 3). We chose 54 sgRNA target sites within this high-coverage region of the mitochondrial chromosome, situated approximately every 50 bp over a 2.5 kb region (sequences listed in Additional file 1). sgRNA sites are indicated by red arrowheads in Fig. 2b. sgRNAs for these sites were generated as described in the "Materials and methods" section. ” (page 2, 2nd column, 1st full paragraph).
Gu teaches, “Depletion of Abundant Sequences by Hybridization (DASH) targeting abundant mitochondrial ribosomal RNA in HeLa RNA extractions. a Normalized coverage plots showing alignment to the full-length human mitochondrial chromosome. Before treatment, three distinct peaks representing the 12S and 16S ribosomal subunits characteristically account for a large majority of the coverage (>60 % of total mapped reads). After treatment, the peaks are virtually eliminated —with 12S and 16S signatures reduced 1000-fold to 0.055 % of mapped reads. b Coverage plot of Cas9-targeted region with 12S and 16S gene boundaries across the top. Each red arrowhead represents one sgRNA target site. We chose 54 target sites, spaced approximately 50 bp apart. c Scatterplot of the log of fragments per kilobase of transcript per million mapped reads (log-fpkm) values per human gene in the control versus treated samples illustrate the significant reduction in reads mapping to the targeted 12S and 16S genes. DASH treatment results in 82- and 105-fold reductions in coverage for the 12S and 16S subunits, respectively. The slope of the regression line (red) fit to the untargeted genes indicates a 2.38-fold enrichment in reads mapped to untargeted transcripts. R-squared (R2) value of the regression line (0.979) indicates minimal off-target depletion. Between replicates, the R2 coefficient between fpkm values across all genes is 0.994, indicating high reproducibility (three replicates). Notably, one gene, MT-RNR2-L12 (MT-RNR2-like pseudogene), shows significant depletion in the DASHed samples compared with the control.” (figure 2, legend.) Gu teaches, “We demonstrate deletion of unwanted mitochondrial rRNA using DASH first on HeLa cell line RNA (Fig. 2) and then on CSF RNA from patients with pathogens in their CSF (Fig. 3), in order to increase sequencing bandwidth of useful data. Selection of rRNA sgRNA targets was based on examining coverage plots for standard RNA-Seq experiments on HeLa cells as well as on several patient CSF samples. Coverage of the 12S and 16S mitochondrial rRNA genes was consistently several orders of magnitude higher than the rest of the mitochondrial and non-mitochondrial genes (Figs. 2c and 3).”
Gu does not specifically teach the use of a hardware processor or recite the specific steps of the claims.
However, Gu teaches high throughput sequencing (page 10, 2nd column). Gu further teaches (figure 2).
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Gu teaches, “DASH can be adapted to target any sequence containing a PAM site; construction of new sgRNAs is facile and inexpensive (see "Materials and methods" section). Because it is employed after sequencing adapter addition, DASH’s utility is not limited to RNA-Seq; it can be applied to any library type. Examples include ATAC-Seq libraries, in which desired nuclear DNA is contaminated with a significant amount of mitochondrial DNA sequences, and microbiome sequencing, where it may be desirable to eliminate a particularly abundant species in order to better sample the underlying diversity. Since Ribo-Zero, RiboMinus and NEBNext are all proprietary kits, they cannot easily be re-programmed by the user to target other sites.” (page 5, 2nd column, programmability).
Therefore it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claims to obtain sequencing data using a hardware processor, align the sequencing data to a reference sequence, determine the abundant sequences and top abundant sequences above a threshold and design one or more probes to the sequences for depleting the top abundant sequences, synthesizing the probes and testing probes. The artisan would be motivated to design probes to deplete sequences to allow detection of less abundant sequences such as pathogen or cancer associated sequences (Gu discussion). The artisan would have a reasonable expectation of success as Gu demonstrates the method works.
With regards to claim 80-82, Gu in figure 2 teaches alignment of abundant rRNA sequence reads or subsequences to the mitochondrial rRNA.
With regards to claim 86-89, Gu teaches peaks and valleys in the reads of mitochondrial rRNA and read length is 135 bp(high throughput sequencing page 10, 2nd column). Thus it would have been prima facie obvious to one of skill in the art prior to the effective filing date the peaks of figure 2 contain sequences and subsequences interspersed across the mitochondrial rRNA sequences can be merged and is above a coverage threshold. The artisan would be motivated as Gu teaches the reads are 135 bp and the mitochondrial rRNA is over 15 kb. The artisan would have a reasonable expectation of success as the artisan is merely recognizing what is taught by Gu.
With regards to claim 90-92, Gu teaches, “DASH can be adapted to target any sequence containing a PAM site; construction of new sgRNAs is facile and inexpensive (see "Materials and methods" section). Because it is employed after sequencing adapter addition, DASH’s utility is not limited to RNA-Seq; it can be applied to any library type. Examples include ATAC-Seq libraries, in which desired nuclear DNA is contaminated with a significant amount of mitochondrial DNA sequences, and microbiome sequencing, where it may be desirable to eliminate a particularly abundant species in order to better sample the underlying diversity. Since Ribo-Zero, RiboMinus and NEBNext are all proprietary kits, they cannot easily be re-programmed by the user to target other sites.” (page 5, 2nd column, programmability). Gu teaches figure 2.
Thus it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claims to sort the sequence reads from the most abundant to the least abundant and design tiling probes. The artisan would be motivated determine the sequences most abundant to target them first for depletion probe design. The artisan would have a reasonable expectation of success as the artisan is merely sorting sequences based on the number of reads.
With regards to claims 88-89, Gu teaches reduction of library size by clustering which is an iterative process of removing sequences (page 10, 2nd column, bottom) . Thus rendering the claims obvious.
With regards to claim 93, Gu teaches sequencing of the microbiome and thus is sequencing unknown or not predetermined sequences (page 10, 2nd column, programmability).
With regards to claim 94, Gu teaches sequencing of the microbiome and thus is sequencing at least two species, which were not predetermined ( page 10, 2nd column, programmability)..
The courts have haled that rearrangement of steps is obvious in the absence of unexpected results. Ex parte Rubin, 128 USPQ 440 (Bd. App. 1959) (Prior art reference disclosing a process of making a laminated sheet wherein a base sheet is first coated with a metallic film and thereafter impregnated with a thermosetting material was held to render prima facie obvious claims directed to a process of making a laminated sheet by reversing the order of the prior art process steps.). See also In re Burhans, 154 F.2d 690, 69 USPQ 330 (CCPA 1946) (selection of any order of performing process steps is prima facie obvious in the absence of new or unexpected results); In re Gibson, 39 F.2d 975, 5 USPQ 230 (CCPA 1930) (Selection of any order of mixing ingredients is prima facie obvious.).
Gu teaches:
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Gu teaches, “We envision that DASH will be immediately useful for the development of non-invasive diagnostic tools, with applications to low input samples or cell-free DNA, RNA, or methylation targets in body fluids.” (page 9, 1st column, 1st full paragraph).
Gu suggest a method use Cas9 to remove overexpressed cDNA. Gu does not specifically teach the use of probes designed by his method to deplete RNA prior to cDNA synthesis. However, Gu teaches, “A number of sequence-specific RNA depletion methods also currently exist. Illumina’s Ribo-Zero rRNA Removal Kit and Ambion’s GLOBINclear Kit pull rRNAs and globin mRNAs, respectively, out of total RNA samples using sequence-specific oligos conjugated to magnetic beads. RNAse H-based methods, such as New England BioLab’s NEBNext rRNA Depletion Kit similarly mark abundant RNA species with sequence-specific DNA oligos, and then subject them to degradation by RNAse H, which digests RNA/DNA hybrid molecules [23]. These methods are all employed prior to the start of library prep, and are limited to samples containing at least 10 ng to 1 μg of RNA. DASH, in contrast, depletes abundant species after complementary DNA (cDNA) amplification, and thus can be utilized for essentially any amount of input sample.” (page 2, bottom column 1- column 2).
Therefore it would have been prima facie obvious to one of skill in the art prior to the effective filing date of the claims to design probes for depletion of rRNA or globin RNA prior or following cDNA synthesis. The artisan would be motivated to determine if decreasing over abundant RNA prior to cDNA synthesis improved the sequencing of the library. The artisan would have a reasonable expectation of success as the art demonstrates method of depleting RNA before cDNA synthesis was known.(claims 95-96)
With regards to claim 97, Gu teaches
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No rRNA was depleted from the top sample of figure 2A.
With regards to claim 98, Gu teaches, “Samples were processed for high-throughput sequencing as previously described.”
Summary
No claims are allowed.
US Patents 11,421216, 11299774, 11390912, 9255265 and 10047359 appear to be owned by the assignee in this case and use probes to deplete RNA from sample. However they do not specifically teach the steps of the claims.
Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to STEVEN C POHNERT PhD whose telephone number is (571)272-3803. The examiner can normally be reached Monday- Friday about 6:00 AM-5:00 PM, every second Friday off.
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/Steven Pohnert/Primary Examiner, Art Unit 1683
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