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 and arguments have been thoroughly reviewed and considered. Claims 11-14 have been canceled. Claims 1-6, 8-10, and 15-22 are pending and are examined on the merits herein.
Response to Applicant’s Amendments and Arguments
Regarding the 35 USC 103 Rejections, Applicant argues that none of the claimed references teach the newly amended portions of the claims, namely that the claims detect a low frequency allele in a subject, where the alternate allele frequency is less than 0.1%. Of the references, Applicant states that Reid only teaches detecting rare variant frequencies of <1% in very large samples, which is not encompassed by the instant claims, as they entail using a single sample from a single subject. Thus, modifying Reid to arrive at the claimed invention would allegedly render Reid inoperable for its intended purpose (Remarks, pages 7-8).
Applicant’s response is almost entirely focused on the teachings of Reid, when the rejections in the Non-Final Rejection mailed 8/18/2025 were based on a combination of Reid in view of Head. In response to Applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). In combining Reid in view of Head, Head is specifically cited because its overlapping amplification methods allow for reduced sequencing computational burden and the ability for low frequency variants (such as an SNP or other sequence novelty) to still be detected (see para. 20 of the Non-Final Rejection). This final benefit is directly related to the alleged deficiencies of Reid argued by Applicant, as the overlapping amplicons of Head would allow for more copies of a particular low frequency mutation, and thus allow for greater detection. Applicant does not directly address the motivation or reasonable expectation of success given by the Examiner for this combination, and so it is considered proper.
In claim 1, the language of the claim has been amended to focus on a “low frequency somatic mosaic variant,” where the variant has an alternate allele fraction. In the instant specification, “low frequency somatic mosaic variant” or “low frequency variant” does not have a particular definition. “Low frequency” refers to the frequency of a variant within a population (pages 12-13, joining para.), and “alternative allele” is any allele other than a reference allele (page 4, para. 5), where the reference allele can differ between populations. It is noted that page 5, para. 1 defines “alternative allele fraction” as the frequency of an allele in a population of cells within a single individual, but the instant claims do not utilize this specific phrase. “Sequence variants” can also be variants in relation to a reference/consensus sequence that may utilize data from multiple individuals or a population (page 12, para. 5). “Somatic mosaicism” refers to two or more cells or populations of cells within a single individual (page 8, para. 3).
Taken together, the phrase “low frequency somatic mosaic variant” will be considered to be referring to a variant with low frequency occurrence within a single subject, where the alternative allele frequency represents the more uncommon allele for a particular loci either within a single individual or on a population level. It is noted that this term (“low frequency somatic mosaic variant”) was not used in previous versions of the claim.
Applicant cites paras. 114 and 326-327 to support their conclusions about Reid. Para. 114 states that “statistically significant associations for rare variants may only be detected in very large samples,” but this is in a discussion of statistical power. In para. 113, the reference explicitly teaches that mutant allele frequencies of 0.01% can be detected with their method. Additionally, when discussing sample size, Reid specifically mentions that an increased sample size is an increase in the number of sequenced subjects and states, “The sample size required for detecting a variant is influenced by both the frequency of the variant, for example the minor allele frequency (MAF), and the effect size of the variant,” (para. 112). In para. 327, it is stated, “Very large genomic variant databases are needed to identify rare variants with large effects on clinical traits of interest; such variants can be exceedingly rare due to purifying selection, but can be extremely informative in revealing novel biological mechanisms and identifying therapeutic targets. Each individual in the cohort harbored ~20 rare predicted LoF variants in multiple genes.” Generally, Reid also teaches assigning one or more of a plurality of variants to an individual (para. 193 and 201). Thus, with the methods of Reid, low frequency genetic variants within individuals can still be detected, even if multiple subjects must be sequenced to do so. In instant claim 1, the method is comprising the listed steps. Thus, while the method is focused on a single subject, the method also does not prohibit performing the method on multiple subjects, and so the methods of Reid can still apply.
Though the method of Reid outlined in the Non-Final Rejection (Example 1 of the reference) measures multiple subjects, said method is specifically designed to generate clinically actionable data on genetic variants in individuals (para. 263). Para. 272 notes that sequencing reads were compared to a reference genome, and paras. 273-274 note that differences between individuals’ reads and the reference were used. Genotyping was conducted for each sample to determine read counts for reference and alternate alleles. For each individual who went through each step of the method, both low frequency and more common variants were detected (para. 286 and Table 4), and para. 287 notes variants that were only present in single individuals. Thus, this example shows determining the presence of low frequency variants within individuals, even if multiple samples are used. The comparison of an individual’s sequencing reads to a reference would allow for quantitative notation of sequence differences from said reference for each individual (e.g. an alternate allele frequency calculation), and when combined with the amplification teachings of Head, low frequency variants would be more easily detected due to increased amplification coverage. MPEP 2141.03 I states, “"A person of ordinary skill in the art is also a person of ordinary creativity, not an automaton." KSR Int'l Co. v. Teleflex Inc., 550 U.S. 398, 421, 82 USPQ2d 1385, 1397 (2007). "[I]n many cases a person of ordinary skill will be able to fit the teachings of multiple patents together like pieces of a puzzle." Id. at 420, 82 USPQ2d 1397. Office personnel may also take into account "the inferences and creative steps that a person of ordinary skill in the art would employ." Id. at 418, 82 USPQ2d at 1396.” Thus, the Examiner argues that the ordinary artisan would be capable of combining the teachings of Reid and Head, along with ordinary skill, creativity, and knowledge in the art, to arrive at the claimed alternative allele frequency calculations.
Applicant also argues that neither Reid nor Head specifically teach determining allele frequency for somatic mosaic variants. As noted above, “somatic mosaicism” in the instant specification refers to two or more cells or populations of cells within a single individual (page 8, para. 3). In the Non-Final Rejection, teachings of Reid regarding cancer were described to read on mosaic variants generally (see para. 24 of the Non-Final Rejection). In Example 1 of Reid, samples from cancer patients are used (see Table 2), and para. 344 notes that cancer samples can contain somatic variants. Figure 16F of Reid notes that cancer gene classes were examined in Example 1. While a cancer/tumor cell specific mutation would be considered a somatic mosaic variant according to the definition provided by the instant specification, it is unclear if any such mutations were found with the claimed alternate allele frequency in Reid. Head also generally teaches that variants are associated with cancer (para. 285), as well as that their methods can be used on somatic mutations related to cancer (para. 295). The reference has general clinical applications for determining allelic/gene variants (paras. 281-282), teaches testing for known disease-associated alleles (para. 352), and notes how to distinguish true genomic variants from sequencing or amplification errors (para. 366). However, this reference also does not specifically note detection of a somatic mosaic variant with the claimed alternate allele frequency.
Thus, the previous 35 USC 103 Rejections have been withdrawn for all currently pending claims. Claims 11-14 have been canceled, and so these rejections have been rendered moot. However, see new grounds of rejection below. It is noted that the relevant portions of both Reid and Head presented in the Non-Final Rejection have been reiterated in the rejections below in accordance with the arguments provided by the Examiner above, and additional teachings have been included to clarify the Examiner’s position.
Claim Objections
Claim 3 is objected to because of the following informalities: in line 3, “bin” should read “bins,” as multiple bins are used in claims 1 and 2. Appropriate correction is required.
Claim 15 is objected to because of the following informalities: in lines 2-3, it is recommended to end the claim with “a disease or a predisposition to a disease in the subject,” as the amendments to claim 1 now already recite a subject from whom the sample is derived. Appropriate correction is required.
Claim Interpretation
For clarity, as noted in the “Response to Applicant’s Arguments” section, though claim 1 requires the analysis of a single sample from a single subject, as the method comprises the listed steps, the analysis of multiple samples from multiple subjects is not prohibited. See MPEP 2111.03.
Additionally, as noted in the Final Rejection, claim 1 requires the performance of three or more "separate" amplification reactions on a "single sample." In the instant specification, the term "separate" is not defined. On page 8, para. 6, "separate" is used to refer to simultaneous sequencing reactions, which the ordinary artisan would recognize could be performed on a single array. On page 31, para. 4, "separate nucleic acid amplification reactions" can be prepared for each pair of primers, but the number of mixtures or containers used is not specified. Therefore, amplification reactions will be considered separate if they have distinct primer pairs, even if a single reaction is performed in the same container as another occurring reaction. There is no requirement that the amplification reactions must be physically separate, and so any prior art which teaches performing at least three successful amplification reactions as described in instant claim 1 will be considered to meet this limitation (see para. 7 of the Non-Final Rejection).
Claim Rejections - 35 USC § 112(b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1-6, 8-10, and 15-22 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 1 is rejected because the amended phrase “a known low frequency somatic mosaic variant in a subject” in unclear in the context of the claimed method. Specifically, it is unclear if the known variant is known as occurring with low frequency generally (e.g. within a population) or if it is known to occur with low frequency within the subject to be examined specifically. In the latter case, the utility of detecting a variant already known to be present in a subject would be unclear. Thus, the “known low frequency somatic mosaic variant” will be interpreted to be a variant that is known within a population to occur at low frequency, and the overall method will be interpreted as detecting that more generally known low frequency variant in a particular individual subject.
Claims 2-6, 8-10, and 15-22 are rejected based on their dependence on rejected claim 1.
Claim Rejections - 35 USC § 112(d)
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claim 18 is 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 18 requires that the genetic variant of claim 1 originate from a somatic event. However, claim 1 has been amended to require that the variant be a somatic variant. Thus, claim 18 does not further limit the subject matter of the claim upon which it depends.
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (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 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 1-5, 15-16, 17-19, and 22 are rejected under 35 U.S.C. 103 as being unpatentable over Reid et al. (US 2017/0286594 A1), in view of Head et al. (US 2015/0265995 A1), and in view of Mortimer et al. (WO 2017/181146 A1).
Reid discloses methods for determining alternate allele frequency (paras. 9 and 215). One such method, in Example 1 of the reference, involves performing PCR and qRT-PCR on samples during library preparation (para. 270). Samples in a study population where patients had diseases associated with somatic mutations (e.g. lung and breast cancer, and see para. 344, which notes that cancer samples can contain somatic variants) were used (paras. 264 and Table 2). Samples were given unique barcodes, and the process utilized Illumina paired-end sequencing (para. 270). Raw sequence data was then sorted via the specific barcodes (para. 272). Variant calling and genotyping was then performed, which involved computing a variant quality score and collecting read counts for alternate alleles (paras. 274 - 275). Reid also explicitly notes that at least some of the produced variants had overlapping sites (para. 281).
Reid shows that the alternate allele fractions for their study range from just above 0% to 1% (para. 287 and Figure 16B). In para. 113, the reference explicitly teaches that mutant allele frequencies of 0.01% can be detected with their method. Additionally, when discussing sample size, Reid specifically mentions that an increased sample size is an increase in the number of sequenced subjects and states, “The sample size required for detecting a variant is influenced by both the frequency of the variant, for example the minor allele frequency (MAF), and the effect size of the variant,” (para. 112). In para. 327, it is stated, “Very large genomic variant databases are needed to identify rare variants with large effects on clinical traits of interest; such variants can be exceedingly rare due to purifying selection, but can be extremely informative in revealing novel biological mechanisms and identifying therapeutic targets. Each individual in the cohort harbored ~20 rare predicted LoF variants in multiple genes.” Generally, Reid also teaches assigning one or more of a plurality of variants to an individual (para. 193 and 201). Thus, with the methods of Reid, low frequency genetic variants within individuals can still be detected, even if multiple subjects must be sequenced to do so. As noted above in the “Claim Interpretation” section, the claimed method is comprising the listed steps. Thus, while the method is focused on a single subject, the method also does not prohibit performing the method on multiple subjects, and so the methods of Reid remain relevant
However, it is unclear if the overlapping sites in Reid include overlapping sites from single samples, or simply from all the samples overall. Reid also does not make clear the specific primers used, and whether index or adapter sequences are present on those primers. Additionally, regarding the claimed alternate allele frequency for a somatic mosaic variant, Example 1 of Reid does teach analysis of DNA from cancer patients, as noted above (and additionally see Figure 16F which quantifies that cancer gene classes were examined in Example 1). However, while a cancer/tumor cell specific mutation would be considered a somatic mosaic variant according to the definition provided by the instant specification, it is unclear if any such mutations were found with the claimed alternate allele frequency (<0.1%) in Reid, as Reid does not provide specific information on the alternate allele frequencies for particular disease-associated genes.
Head teaches methods for nucleic acid analysis and library labeling (Abstract). These methods can involve primer tiling. Tiled primers can comprise a plurality of primer pairs that overlap one another (para. 112). They can be designed to cover the target loci multiple times (including at least three times; para. 112). Head also specifically teaches that primers can have both adapters and barcode sequences, particularly at their 5’ ends (para. 120). Primers can contain sequences used for capture on various sequencing platforms, such as Illumina (para. 219). Adapters in particular can be used for sequencing, and can comprise regions complementary to sequencing primers (paras. 121, 136, 193, 267, 271). Adapters can comprise multiple types of sequences for anchoring, amplification, and sequencing (para. 271), meaning they could differ between forward and reverse primers, as different amplification sequences are required for each. Head also teaches that primers can have unique identifiers (para. 10) and that this unique identifier can be a molecular barcode (paras. 91, 189, and 191-192). Head teaches that primer sets can later be binned for particular targets, which can be helpful for assembly of sequence reads (para. 139), and that the binning can occur via the molecular barcodes (para. 137 and 229). When this occurs, duplicate reads can be removed (para. 137). Head also teaches the pooling of multiple samples using the barcodes to identify the source of each sample (para. 70), and the reference specifically mentions the pooling of amplicons (e.g. paras. 124, 141, and 183). Head specifically teaches that their methods can be used for identifying multiple alleles, particularly in medical contexts such as disease diagnosis, monitoring, and/or treatment (para. 282).
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to combine the teachings of Reid and Head. Reid teaches methods for preparing multiple samples via isolation of targets, amplification, and sequencing, but does not provide the details for many of these aspects. Head provides extensive details on primer design and amplification, and teaches that these methods can provide more targeted sequencing. Regarding tiled primers specifically, these are taught to allow redundant sampling and consensus read lengths. This can “avoid target drop out because of SNPs or novel biology under one primer site,” (para. 139). Adding additional sequences (such as the adaptors and barcodes) “can allow for dramatically reduced computational burden,” (para. 139). Thus, the ordinary artisan would be motivated to use the methods of Head in a computationally heavy sequencing and analysis method such as the one described by Reid. In the specific combination of these references, tiled primers can be used for the PCR described by Reid – this would allow for amplification producing at least three (and potentially more) overlapping amplicons, where the particular genes of Reid are targeted. Because the primers overlap a particular target, and because these primers would all be unique, as the overlapping portions are not identical, the target would be at a different position within each unique amplicon produced by each unique primer pair in each amplification reaction, and each target would have redundant coverage. This tiling would also mean that the overlapping amplicons would each occur for each single sample, as well as over all the samples. The primers would contain 5’ adapters that can hybridize to sequencing primers and unique molecular barcode sequences. Head teaches that these barcodes on the primers can be used for binning amplicons, thus identifying particular amplicons, and it would be prima facie obvious to substitute the binning described by Reid for the binning described by Head, as this barcoding occurs during PCR and not before, so sequence degeneration of the barcodes would be less likely before sequencing occurred. The results of this substitution would also remain the same, namely in producing binned sequence reads. Thus, after PCR, samples would be pooled and sequenced, binned according to their unique molecular barcodes (where duplicates would be removed), and then allele frequency could be calculated. There would be a reasonable expectation of success in combining these references as designing tiling primers with adapters and barcodes is well-known in the art, as described by Head, and the results of this combination would be predictable, as sequence reads would still be produced, binned, and analyzed.
Though the method of Reid outlined in Example 1 of the reference measures multiple subjects, said method is specifically designed to generate clinically actionable data on genetic variants in individuals (para. 263). Para. 272 notes that sequencing reads were compared to a reference genome, and paras. 273-274 note that differences between individuals’ reads and the reference were used. Genotyping was conducted for each sample to determine read counts for reference and alternate alleles. For each individual who went through each step of the method, both low frequency and more common variants were detected (para. 286 and Table 4), and para. 287 notes variants that were only present in single individuals. Thus, this example shows determining the presence of low frequency variants within individuals, even if multiple samples are used. The comparison of an individual’s sequencing reads to a reference would allow for quantitative notation of sequence differences from said reference for each individual (e.g. an alternate allele frequency calculation), and when combined with the amplification teachings of Head, low frequency variants would be more easily detected due to increased amplification coverage. MPEP 2141.03 I states, “"A person of ordinary skill in the art is also a person of ordinary creativity, not an automaton." KSR Int'l Co. v. Teleflex Inc., 550 U.S. 398, 421, 82 USPQ2d 1385, 1397 (2007). "[I]n many cases a person of ordinary skill will be able to fit the teachings of multiple patents together like pieces of a puzzle." Id. at 420, 82 USPQ2d 1397. Office personnel may also take into account "the inferences and creative steps that a person of ordinary skill in the art would employ." Id. at 418, 82 USPQ2d at 1396.” Thus, the ordinary artisan would be capable of combining the teachings of Reid and Head, along with ordinary skill, creativity, and knowledge in the art, to arrive at the claimed alternative allele frequency calculations for an individual sample within a group of samples.
It is noted that under the interpretation of “separate nucleic acid amplification reactions” described in the “Claim Interpretation” section above, Applicant’s limitation in the final lines of step (a) of instant claim 1, “and wherein each nucleic acid amplification reaction comprises only one of the unique pairs of forward and reverse primers,” would be inherently met by Reid in view of Head, as the use of unique primer pairs is the clear distinction between the amplification reactions in this combination of references.
Head also generally teaches that variants are associated with cancer (para. 285), as well as that their methods can be used on somatic mutations related to cancer (para. 295). The reference has general clinical applications for determining allelic/gene variants (paras. 281-282), teaches testing for known disease-associated alleles (para. 352), and notes how to distinguish true genomic variants from sequencing or amplification errors (para. 366). However, this reference also does not specifically note detection of a somatic mosaic variant with the claimed alternate allele frequency (<0.1%).
Mortimer teaches methods for early cancer detection that include sequencing cell-free nucleic acid molecules and detecting one or more tumor markers (Abstract). cfDNA can be used, where the cfDNA is from a single subject that does not detectably exhibit a cancer (para. 5). Tumor markers are then detected, where the markers can be detected at a frequency as low as 0.01%. Para. 9 lists panels of genes that may be sequenced, and para. 10 lists the various mutations that a tumor marker may consist of. Para. 45 states that cancer driver mutations may be a somatic variant detected at a mutant allele frequency of no more than 0.05%-0.1%. The cfDNA may be amplified prior to sequencing (para. 37). Para. 269 also notes that the sequencing analysis methods can involve aligning sequence data with a reference genome and filtering/binning sequence reads. In applying their method, Mortimer notes that mutant allele frequency can be tracked over time (see Figure 2 and paras. 284-286). Similar teachings are shown in para. 294 and Figure 14. Tables 1 and 2 also show various MAF calculations for particular mutations associated with colorectal cancer (explained in para. 290). These tables show MAFs for individual patients, though population was also calculated (see Table 4 for example). Mortimer teaches that their methods are capable of being used with many types of cancers (see para. 149 for example), and para. 147 notes that their methods can be used on subjects that are suspected of having cancer but have no symptoms, or on subjects who have cancer that is not detectable with other screening methods. Mortimer also notes that their methods can be accomplished with readily available and well-known sequencing methods (para. 153).
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to utilize the teachings of Mortimer to inform the method of Reid in view of Head to arrive at the inventions of instant claims 1-5, 15-16, and 18. Specifically, Reid in view of Head teaches performing amplification and sequencing reactions on samples to find alternate allele frequencies, where the samples may be from cancer patients. Mortimer teaches analysis of subjects likely to have cancer utilizing amplification and sequencing as well, and utilizing these methods to detect alternate allele frequencies for cancer-driving genes, where the alternate allele frequency for said genes can be below 0.1%. Thus, the methods of Reid in view of Head could be used to focus on the specific genes disclosed by Mortimer to be associated with cancer, particularly in subjects being evaluated for cancer. As Mortimer teaches that their gene panels can be used on subjects that may have cancer but show no symptoms, or may be used to detect cancer when other methods have failed, the ordinary artisan would be motivated to evaluate these genes in subjects to provide early diagnostic testing that can increase a subject’s chance of survival and also inform treatment plans. Mortimer also teaches measuring allele frequencies over time for cancer patients given particular interventions, which would also motivate the ordinary artisan, as this method can be used to determine if treatments are effective in a particular subject, or if alternative approaches are needed. There would be a reasonable expectation of success in utilizing the teachings of Mortimer with those of Reid in view of Head because it would simply involving using the teachings of Reid in view of Head on particular genes, and Mortimer already teaches successful sequencing of these genes, as well as the use of amplification methods.
Thus, claims 1-5, 15-16, and 18 are prima facie obvious over Reid in view of Head.
Regarding claim 17, Reid teaches that subjects from their study had blood and DNA samples taken (para. 264), but does not specify that cell-free DNA is used. Mortimer teaches the use of cell-free DNA throughout their methods (e.g. paras. 5-6, 9, 12, 32 and Examples 1-5). Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use cfDNA in addition to the genomic DNA present in the method of Reid, in view of Head, and in view of Mortimer. cfDNA is already fragmented, and so fragments around the average fragment length used by Reid (150 bp, para. 270) could be utilized along with the fragmented genomic DNA sequences. This would provide additional sequencing reads and more potential data, increasing the accuracy of results. This could be important for subjects who may be at risk for particular diseases, such as cancer. As Mortimer teaches that cfDNA can be used to analyze allele frequency, there would be a reasonable expectation of success in incorporating this additional analysis into the method of Reid, in view of Head, and in view of Mortimer. Additionally, no steps of the method of Reid, in view of Head, and in view of Mortimer would need to be altered in terms of PCR amplification and sequencing, there would simply be more DNA to use.
Regarding claim 19, Reid, in view of Head, and in view of Mortimer teaches the method of claim 15, as described above. Reid also teaches that “genetic variant data” refers to “data obtained by identifying allelic variants in a subject's nucleic acid, relative to a reference nucleic acid sequence,” (para. 102). Reid also teaches displaying information about reference alleles (para. 215) and matching individuals to reference allele frequencies (para. 292). In para. 116, Reid describes how the incidence of a variant of interest can be determined in case and control groups, and later teaches that statistical tests can be done on case versus control phenotypes (para. 153). In para. 412, Reid discusses comparing allele frequencies of mutations associated with diseases with those of control groups. Reid contemplates mutations associated with particular diseases (see Figures 30 and 33) and notes that their methods can uncover genetic factors that influence disease (para. 92). In Example 3 of their method, Reid correlates heterozygosity for a particular allele with risk of COPD, emphysema, and liver disease (paras. 145-149), and in Example 4, Reid demonstrates how a particular mutation can be a marker for early-onset inflammatory bowel disease (paras. 420-424). Head teaches that by using the data provided by their methods, disease diagnosis can occur and disease progression can be monitored (paras. 283-285), and that their methods can detect sequences associated with particular diseases (para. 195). Head also teaches tracking of patient disease over time to gather actual data about disease progression (para. 286). Mortimer also teaches the use of a reference genome to which a sequence can be compared (para. 102), and para. 264 notes the use of frequencies from reference samples.
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use the teachings of Reid, Head, and Mortimer to arrive at the invention of instant claim 19. Reid, in view of Head, and in view of Mortimer already teaches the calculation and analysis of alternate allele frequencies, and Reid provides many teaching and examples of comparing patients to controls, particularly with regard to incidents of genetic variants. Reid and Mortimer also provide the genetic basis and/or genetic contributing factors for many diseases (with Mortimer in particular detailing cancer), showing that these factors would be known and could be compared across case and control groups. Head also specifically teaches the use of genetic data to diagnose and predict progression of disease. Thus, the ordinary artisan would be capable of measuring alternate allele frequencies in controls (thus acting as reference samples) and comparing these values to those of patients. The presence of particular disease-associated alleles in patients that are not present in controls could then determine if a disease is present in a patient. Head’s teachings about monitoring a disease over time could then be used to determine if patient alternate allele frequencies are the same, increased, or decreased compared to previous measurements and those of controls. These indications of disease progression can aid in treatment decisions and potentially improve patient outcomes. There would be a reasonable expectation of success incorporating this aspect into the method of Reid, in view of Head, and in view of Mortimer because it simply involves calculating additional alternate allele frequencies, which is shown by Reid to be possible for the ordinary artisan.
Regarding claim 22, Head teaches that nucleic acid fragments can be spatially separated from one another (para. 4). These separations can be separate compartments of wells (para. 56 and Figure 13), or separate chambers, plates, or droplets (para. 108). Head notes that when amplification reactions are performed separately, they may be pooled prior to sequencing (para. 124). In particular methods, spatial separation can occur before amplification (paras. 249 and 264), and particular primers may be associated with particular partitions on a well plate (paras. 257 and 261). Reid also notes that for each patient examined, at least 100ng of sample was used (para. 270), and so each sequence of interest appears more than once in a given sample.
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to spatially separate the targets of Reid, in view of Head, and in view of Mortimer before amplification occurred via the use of physically separated partitions as taught in Head. This is because with the tiling method of Head, primers that are very similar in sequence to one another are acting on similar portions of sequences for a given sample. By separating amplification for each of these portions, this can ensure that each portion is properly amplified, and that the primers do not inhibit one another. This would provide additional amplification product for each sequence of interest, and would likely lead to more accurate results. There would be a reasonable expectation of success because Head teaches separating sequences before amplification with means that would be available to the ordinary artisan, such as with a well array.
Thus, claim 22 is prima facie obvious over Reid, in view of Head, and in view of Mortimer.
Claims 6 and 8 are rejected under 35 U.S.C. 103 as being unpatentable over Reid et al. (US 2017/0286594 A1), in view of Head et al. (US 2015/0265995 A1), and in view of Mortimer et al. (WO 2017/181146 A1), as evidenced by Roche Diagnostics (2010).
Regarding claims 6 and 8, Reid, in view of Head, and in view of Mortimer teaches the method of claims 1-5, 15-16, 17-19, and 22, as described above. Reid also teaches that the DNA exome fragments are captured by streptavidin beads using Roche NimbleGen technology (para. 270). Roche Diagnostics teaches the design of their probes for facilitating exome capture. These probes contain biotin (page 9, “SeqCap EZ probe pool”). Reid teaches wash steps after capture and before PCR of the pooled samples (para. 270), and Roche Diagnostics notes that there is no need to remove the DNA from the beads, as the captured DNA can be used as a PCR template (page 31). After the amplification of Reid, in view of Head, and in view of Mortimer, the amplicon pool would thus contain biotin labels that are captured by streptavidin beads.
Thus, claims 6 and 8 are prima facie obvious over Reid, in view of Head, and in view of Mortimer, as evidenced by Roche Diagnostics.
Claim 9 is rejected under 35 U.S.C. 103 as being unpatentable over Reid et al. (US 2017/0286594 A1), in view of Head et al. (US 2015/0265995 A1), in view of Mortimer et al. (WO 2017/181146 A1), and further in view of Sutherland et al. (US 5,985,619).
Regarding claim 9, Reid, in view of Head, and in view of Mortimer teaches the method of claims 1-5, 15-16, 17-19, and 22, as described above. However, none of these references teach that the primers used may be enzymatically digested during the method.
Sutherland teaches mixtures for PCR amplification including an exonuclease in the PCR reaction mix (Abstract). This reference teaches that particular exonucleases, such as Exonuclease III, are capable of digesting primer-dimer intermediates (column 3, para. 2). Example 1 (columns 11-14) shows PCR with the addition of Exonuclease III and an anti-Taq antibody, and Sutherland concludes that Exonuclease III has a positive effect on PCR amplification efficiency by controlling primer-dimer formation, and can perform well under even “challenging” PCR conditions (column 14, paras. 1-2).
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use the teachings of Sutherland in the method of Reid, in view of Head, and in view of Mortimer. Specifically, the ordinary artisan would be motivated to include Exonuclease III and an anti-Taq antibody in the method of Reid, in view of Head, and in view of Mortimer to reduce primer-dimers and improve amplification efficiency. Because the primers of Reid, in view of Head, and in view of Mortimer are tiled, and so more primers may have regions capable of hybridizing to each other compared to primers of traditional PCR, digesting primer-dimers can improve detection efficiency and accuracy for target sequences. Because the primers of Reid, in view of Head, and in view of Mortimer also contain adapters and barcodes, digesting primer-dimers would prevent including primer-dimers in binning protocols, which could lead to sequencing or downstream analysis inaccuracies. There would be a reasonable expectation of success in using the methods of Sutherland in the method of Reid, in view of Head, and in view of Mortimer because Sutherland provides evidence that Exonuclease III and anti-Taq antibodies are well-known and can be successfully added to PCR reactions, and Reid, Head, and Mortimer provide teachings related to PCR methods involving Taq (e.g. Read paras. 365 and 403, Head paras. 83 and 96, Mortimer paras. 222 and 234), showing that these are also well-known in the art and could easily be used by the ordinary artisan in the method of Reid, in view of Head, and in view of Mortimer.
Thus, claim 9 is prima facie obvious over Reid, in view of Head, in view of Mortimer, and further in view of Sutherland.
Claim 10 is rejected under 35 U.S.C. 103 as being unpatentable over Reid et al. (US 2017/0286594 A1), in view of Head et al. (US 2015/0265995 A1), and in view of Mortimer et al. (WO 2017/181146 A1), as evidenced by Illumina (2017).
Regarding claim 10, Reid, in view of Head, and in view of Mortimer teaches the method of claims 1-5, 15-16, 17-19, and 22, as described above. Reid also teaches that sequencing was done via Illumina methods (para. 270). As evidenced by Illumina, this type of sequencing (including sequencing done on the HiSeq 2500) involves use of a bridge amplification step before the actual sequencing begins (Figure 3 and page 7, para. 4). Thus, the amplicons would be amplified before they are sequenced.
Thus, claim 10 is prima facie obvious over Reid, in view of Head, and in view of Mortimer, as evidenced by Illumina.
Claims 20-21 are rejected under 35 U.S.C. 103 as being unpatentable over Reid et al. (US 2017/0286594- A1), in view of in view of Head et al. (US 2015/0265995 A1), in view of Mortimer et al. (WO 2017/181146 A1), and further in view of Lim et al. (Nature Genetics, 2014).
Regarding claims 20-21, Reid, in view of Head, and in view of Mortimer teaches the method of claims 1-5, 15-16, 17-19, and 22, as described above. However, none of these references discuss measuring mean alternate allele frequencies for sequencing bins. Mortimer discusses sequencing error rates generally, but not necessarily for sequences surrounding an alternate allele (e.g. paras. 258, 260, and 264).
Lim teaches detection of somatic mutations associated with breast cancer fibroadenomas (Abstract). During amplification and sequencing protocols, mean alternate allele frequencies, standard deviations, and estimated background error rates were calculated (Online Methods, “Ultra-deep targeted amplicon sequencing of MED12 exon 2,” specifically the final para.) for positive control samples. The error rate in particular is a measurement of the likelihood of error from sequencing and alignment artifacts. This was done in order to gauge the sensitivity of the assay used.
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use the teachings of Lim in the method of Reid, in view of Head, and in view of Mortimer. Specifically, this would involve calculating average alternate allele frequencies and error rates for the binned samples in the method of Reid, in view of Head, and in view of Mortimer. In the method of Reid, in view of Head, and in view of Mortimer, samples are given barcodes and then separated into bins based on these barcodes before sequencing, Thus, the alternate allele frequency calculations are calculated based on these bins, and could easily be averaged across multiple bins, as this requires only simple calculations that would be possible for the ordinary artisan. Lim provides evidence that such calculations would have a reasonable expectation of success. Lim also notes that calculations of error rates are possible for the ordinary artisan, and that these calculations can indicate how accurate sequencing and alignment methods are, which can provide information about the accuracy of overall results. These methods would not change any preceding aspects of the method of Reid, in view of Head, and in view of Mortimer, and would simply add subsequent analysis steps.
Thus, claims 20-21 are prima facie obvious over Reid, in view of Head, in view of Mortimer, and further in view of Lim.
Conclusion
No claims are currently allowable.
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.
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/FRANCESCA FILIPPA GIAMMONA/Examiner, Art Unit 1681
/ANGELA M. BERTAGNA/Primary Examiner, Art Unit 1681