Notice of Pre-AIA or AIA Status
The present application is being examined under the pre-AIA first to invent provisions.
DETAILED ACTION
Status of the Claims
Claims 21-53 are pending and the subject of this NON-FINAL Office Action. This is the first action on the merits.
Priority
The earliest priority date for the claims is 09/12/2023 because the claims filed in this application on that date are the first priority document that contains explicit support for circulating nucleic acids used in the duplex sequencing technique claimed herein.
It is noted that the first document filed in any of the related cases assigned to University of Washington, or with the inventors Salk, Loeb and Schmidt, and with explicit support for circulating nucleic acids, is the claim set filed in US 16/411045 on 05/24/2019. However, this Application does not claim priority to that application, or any other applications that include a claim to circulating nucleic acids.
Claim Rejection - 35 USC § 112 – Written Description
The following is a quotation of 35 U.S.C. 112 (AIA and pre-AIA ):
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention
Claims 21-53 are rejected under 35 U.S.C. 112(a) (AIA ) or 112, first paragraph (pre-AIA ) as failing to comply with the written description requirement. The claims contain subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, at the time the application was filed, had possession of the full scope of the claimed invention.
The specification does not demonstrate possession of circulating DNA for the claimed duplex sequencing using ligated adaptor tags.
“[T]he purpose of the written description requirement is to ‘ensure that the scope of the right to exclude, as set forth in the claims, does not overreach the scope of the inventor’s contribution to the field of art as described in the patent specification.”’ Ariad Pharms., Inc. v. Eli Lilly & Co., 598 F.3d 1336, 1353–54 (Fed. Cir. 2010) (en banc). Whether the disclosure of a patent satisfies the written description requirement is a question of fact. See id. at 1351. The test for sufficiency of the written description support is “whether the disclosure of the application relied upon reasonably conveys to those skilled in the art that the inventor had possession of the claimed subject matter as of the filing date sought.” Id. This “possession” test “requires an objective inquiry into the four corners of the specification from the perspective of a person of ordinary skill in the art.” Id. Possession shown by evidence “outside of the specification is not enough,” and “a description that merely renders the invention obvious does not satisfy the requirement.” Id. at 1352. Instead, it is the specification itself that must demonstrate possession. Id.
Here, the specification never uses the phrase “circulating DNA.” Nor does the specification describe any such DNA. At best, it mentions, with no further explanation or description, “quantification of circulating microRNAs [and] . . . quantification of circulating neoplastic cells” (para. 0087). However, this is in the context of discussing prior art “[s]ingle-molecule counting . . . applications” using SMI tags (para. 0087). The specification never discloses that the claimed duplex sequencing adaptor-ligation technique can or is used with circulating nucleic acids of any sort. Moreover, even if quantification of circulating microRNAs somehow provide written description support for a species of circulating nucleic acid, yet “the specification must demonstrate that the applicant has made a generic invention that achieves the claimed result and do so by showing that the applicant has invented species sufficient to support a claim to the functionally-defined genus.” Id. at 1349; AbbVie Deutschland GmbH v. Janssen Biotech, Inc., 759 F.3d 1285, 1299 (Fed. Cir. 2014). A “sufficient description of a genus instead requires the disclosure of either a representative number of species falling within the scope of the genus or structural features common to the members of the genus so that one of skill in the art can ‘visualize or recognize’ the members of the genus.” Ariad Pharms., 598 F.3d at 1350. One species of circulating nucleic acids, much less miRNA which has no relation to cfDNA such as fetal nucleic acids or ctDNA, for example, does not make a genus. Here, the claims encompass any and all “circulating DNA”; yet, at best, only one is disclosed. See Centocor Ortho Biotech, Inc. v. Abbott Labs., 636 F.3d 1341, 1351 (Fed. Cir. 2011) (“For generic claims,” the Federal Circuit has “set forth a number of factors for evaluating the adequacy of the disclosure, including ‘the existing knowledge in the particular field, the extent and content of the prior art, the maturity of the science or technology, [and] the predictability of the aspect at issue.’”) (quoting Capon v. Eshhar, 418 F.3d 1349, 1359 (Fed. Cir. 2005)). Given the scope of the claimed genus of circulating DNA, the Examiner finds that the Specification provides at most a wish or research plan, “leaving it to others to explore the unknown contours of the claimed genus.” See AbbVie Deutschland GmbH v. Janssen Biotech, Inc., 759 F.3d 1285, 1300 (Fed. Cir. 2014); see also Centocor, 636 F.3d at 1351. Thus, like in Centocor, the Specification here “at best describes a plan for [using circulating DNA] and then identifying those that satisfy the claim limitations,” but such a “‘mere wish or plan’ for obtaining the claimed invention is not sufficient.”
For further evidence that circulating DNA was never contemplated by the specification, the Examiner points out that the specification never discloses samples that have circulating DNA. For example, the specification never discloses blood samples, plasma samples, urine samples or other samples that are known to have circulating DNA such as ctDNA, fetal DNA, etc.
Finally, even if paragraph 0087 somehow supports the full scope of circulating DNA, yet it is not in the context of the claimed adaptor-ligation technique for duplex sequencing, as explained above. In other words, the specification never clearly integrates circulating DNA in the particular claimed combination of elements. For example, in Hyatt v. Dudas, 492 F.3d 1365, 1371 (Fed. Cir. 2007), the examiner made a prima facie case by clearly and specifically explaining why applicant’s specification did not support the particular claimed combination of elements, even though applicant’s specification listed each and every element in the claimed combination. The court found the "examiner was explicit that while each element may be individually described in the specification, the deficiency was lack of adequate description of their combination" and, thus, "[t]he burden was then properly shifted to [inventor] to cite to the examiner where adequate written description could be found or to make an amendment to address the deficiency." Id.; see also Novozymes A/S v. DuPont Nutrition Biosciences APS, 723 F.3d 1336, 1349 (Fed. Cir. 2013) (explaining that each claim must be taken “as an integrated whole rather than as a collection of independent limitations” and the specification must be viewed prospectively, based on PHOSITA knowledge at the time, not in hindsight); Flash-Control, LLC v. Intel Corp., -- Fed. Appx. --, 2021 WL 2944592, *4 (Fed. Cir. July 14, 2021) (“A patent owner cannot show written description support by picking and choosing claim elements from different embodiments that are never linked together in the specification.”); Stored Value Solutions, Inc. v. Card Activation Techs., 499 Fed.App’x 5, 13-14 (Fed. Cir. 2012) (non-precedential) (Finding inadequate written support for claims drawn to a method of processing debit purchase transactions requiring three separate authorization codes because "the written description [did] not contain a method that include[d] all three codes" and "[e]ach authorization code is an important claim limitation, and the presence of multiple authorization codes in [the claim] was essential".) Just as in Hyatt, Applicant has not demonstrated sufficiently how the Specification supports the particular combination of limitations identified by the Examiner recited in the selected claims.
Claim Rejection - 35 USC § 112 – Enablement
The following is a quotation of 35 U.S.C. 112 (AIA and pre-AIA ):
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 21-53 are rejected under 35 U.S.C. 112(a) or 112, first paragraph, because the specification does not reasonably provide enablement for ligating cfDNA with adaptors such that enough cfDNA would be available to be able to compare both strands of DNA to determine erroneous nucleotides in the strands. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to use the invention commensurate in scope with these claims.
The claims are directed to ligating partially double-stranded adaptors to circulating (AKA cell-free) DNA to generate tags (DNA end sequences together with associated identifier sequences on the adaptor), which are used to generate sequencing reads to compare both strands of the cfDNA and determine erroneous nucleotides in the strands. This involves very rare events (Spec., para. 0007, describing mutation frequencies generally ranging from 10-9 or 10-11). In other words, the amount of DNA required is very high. Samples of cfDNA, at the earliest time of the invention (03/20/2012), do not meet this requirement.
There are many factors to be considered when determining whether there is sufficient evidence to support a determination that a disclosure does not satisfy the enablement requirement and whether any necessary experimentation is “undue.” These factors include, but are not limited to:
(A) The breadth of the claims;
(B) The nature of the invention;
(C) The state of the prior art;
(D) The level of one of ordinary skill;
(E) The level of predictability in the art;
(F) The amount of direction provided by the inventor;
(G) The existence of working examples; and
(H) The quantity of experimentation needed to make or use the invention based on the content of the disclosure.
In re Wands, 858 F.2d 731, 737, 8 USPQ2d 1400, 1404 (Fed. Cir. 1988).
A. Breadth of Claims: Broad
The claims are broad. The claims encompass any double-stranded adaptors, any ligation technique, any “identifier sequences,” any “end sequences” and any cfDNA fragments. The claims are also directed to any cfDNA sample from any source with any cfDNA isolation. As is shown below, specific details of these missing factors are critical to sequencing cfDNA. Thus this factor weighs heavily against enablement of the full scope of the claims.
In contrast to this breadth, the Office presents evidence that sequencing of cfDNA is notoriously difficult, especially using ligated adaptors, while Applicants fail to overcome these massive hurdles with working examples.
B. The Nature Of The Invention
The instant invention relates to sequencing cfDNA for duplex (or paired-end) sequencing to determine very rare error-corrected sequences using adaptor ligation. As is shown below, sequencing cfDNA using ligated adaptors requires, at best, extensive optimization and experimentation; at worst, was impossible. Yet, no such optimization and experimentation is described in the instant specification. Even more, cfDNA samples require additional extensive optimization to enrich enough quality cfDNA for downstream nucleic acid experiments such as sequencing. Again, this is never disclosed. Thus, this factor weighs against enablement.
C. The State Of The Prior Art
Beginning in 2012, numerous publications (NPL or patent) disclose sequencing of cfDNA; yet, these publications explicitly make clear that before 2012 (at the earliest) nobody had successfully performed sequencing on cfDNA. This is near to Applicants’ earliest possible effective filing date (03/20/2012). For example, the Office is unaware of any successful attempt to use sequencing on ctDNA1 (species of cfDNA) until 11/28/2012, after Applicants’ earliest possible priority date (see Leary et al, Detection of chromosomal alterations in the circulation of cancer patients with whole-genome sequencing, Sci Transl Med. 2012 Nov 28;4(162):162ra154. doi: 10.1126/scitranslmed.3004742; Perakis et al, Advances in Circulating Tumor DNA Analysis, Adv Clin Chem. 2017;80:73-153. doi: 10.1016/bs.acc.2016.11.005. Epub 2017 Jan 3 (“The first whole-genome analyses of cfDNA were conducted by Leary et al., who demonstrated the feasibility of directly detecting chromosomal alterations in the plasma of cancer patients (Fig. 1) [Leary et al.].”)). This is why Leary states that “we wondered whether we could directly identify chromosomal alterations in the circulation of cancer patients” because “[s]equencing analyses of chromosome content in the maternal circulation are now being used for detection of fetal aneuploidy, although such approaches have not been evaluated for detection of chromosomal alterations in cancer patients” (pg. 1, cols. 1-2; emphasis added). In fact, their technique required five specific chromosome arm SNP alterations to achieve the ability to determine copy number variations (pg. 6, col. 2, pg. 10, col. 1, Fig. 5). Leary had to use specialized bioinformatics to distinguish the relatively few somatic rearrangements present in ctDNA from the much larger number of structural variants resulting from copy number variations in the germline of all individuals (pg. 4, col. 1). Finally, Leary does not explain how the ctDNA was isolated, but does explain the extensive ctDNA preparation required. This included the following specific steps:
Plasma DNA libraries were prepared following Illumina’s suggested protocol with the following modifications: (i) circulating DNA isolated from plasma was mixed with 10 μl of End Repair Reaction Buffer and 5 μl of End Repair Enzyme Mix (catalog no. E6050, New England Biolabs) in a final volume of 100 μl. The end-repair mixture was incubated at 20°C for 30 min, purified by a PCR purification kit (catalog no. 28104, Qiagen), and eluted with 45 μl of elution buffer (EB) prewarmed to 70°C. If starting DNA volumes were >85 μl, multiple 100 μl end-repair reactions were used per sample and purified over the same Qiagen column as indicated above. (ii) To A-tail, all 42 μl of end-repaired DNA was mixed with 5 μl of 10× dA Tailing Reaction Buffer and 3 μl of Klenow (exo-) (catalog no. E6053, New England Biolabs). The 50-μl mixture was incubated at 37°C for 30 min before DNA was purified with a MinElute PCR purification kit (catalog no. 28004, Qiagen). Purified DNA was eluted with 27 μl of EB. (iii) For adaptor ligation, 25 μl of A-tailed DNA was mixed with 10 μl of PE-adaptor (Illumina), 10 μl of 5× Ligation buffer, and 5 μl of Quick T4 DNA ligase (catalog no. E6056, New England Biolabs). The ligation mixture was incubated at 20°C for 15 min. (iv) To purify adaptor-ligated DNA, 50 μl of ligation mixture from step (iv) was mixed with 200 μl of NT buffer and cleaned up by NucleoSpin column (catalog no. 636972, Clontech). DNA was eluted in 50 μl of EB
(pg. 8, cols. 1-2). Thus, this reference demonstrates not only that cfDNA techniques do not necessarily apply to ctDNA, further ctDNA was not successfully sequenced until after Applicants’ earliest possible priority date, using specific bioinformatics approach and specific isolation and preparation techniques.
Further, ctDNA, as all cfDNAs, is a notoriously-difficult sample type. In blood samples, ctDNA is known to be as low as 1%. Such low amounts in samples were a large hurdle to successful nucleic acid assays, such as sequencing. Scientific publications from 2012 and later make clear that in order to perform sequencing on ctDNA, large fractions of ctDNA (e.g. >5%) were required (Perakis, Fig. 3; Kirkizlar et al., Detection of Clonal and Subclonal Copy-Number Variants in Cell-Free DNA from Patients with Breast Cancer Using a Massively Multiplexed PCR Methodology, Transl Oncol. 2015 Oct;8(5):407-416. doi: 10.1016/j.tranon.2015.08.004, pg. 408, col. 1). Furthermore, ctDNA extraction and downstream analysis was so inconsistent that it yielded highly variable ctDNA amounts “because the yield and fraction of tumor-derived cfDNA (“tumor fraction”) vary substantially.” (Adalsteinsson et al, Scalable whole-exome sequencing of cell-free DNA reveals high concordance with metastatic tumors, Nature Communications volume 8, Article number: 1324 (2017), pg. 2, col. 1; see also Heidary et al, The dynamic range of circulating tumor DNA in metastatic breast cancer, The dynamic range of circulating tumor DNA in metastatic breast cancer, Breast Cancer Res. 2014 Aug 9;16(4):421. doi: 10.1186/s13058-014-0421-y, pg. 9, col. 1 (discussing highly “variant ctDNA levels” among samples)). No specific, new ctDNA extraction technique to overcome this issue is disclosed. Further, the specification never addresses how to overcome this issue in duplex sequencing as claimed.
Other known sources of unpredictability with cfDNA in the context of sequencing included severe adapter ligation efficiency issues, large sequencing error rates, high rates of false positives, sequence integrity, extraction efficiency, true mutation calling, read depth and bioinformatics analysis.
Basically, methodological approaches for the analysis of ctDNA can be separated in two categories: (i) targeted methods with high resolution, which in most cases interrogate only a single or a few mutations with a high analytical sensitivity and (ii) more comprehensive or untargeted, genome-wide approaches, which require a certain amount of tumor DNA in the circulation, typically 5–10%, to achieve informative results (Fig. 3). The advantages for both approaches are obvious: while comprehensive approaches do not rely on recurrent hotspot mutations or knowledge about the molecular landscape of the respective tumor samples, targeted methods are able to detect mutant alleles even if they are highly underrepresented.
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Owing to high rates of false positives with traditional NGS, most methods interrogate only single or a few targets in cases where the ctDNA fraction is greater than 1–5% of total circulating cfDNA. However, a variety of genomic alterations may be missed if analysis is limited to only hotspots. Therefore, more comprehensive and efficient strategies with high resolution are needed in order to identify all actionable genomic alterations within a sample [58,172]. Conventional NGS approaches and most PCR-based assays have a limited analytical sensitivity and a detection of underrepresented alleles (<5%) cannot be assured. Although error rates and fidelity for sequencing and PCR are well documented, the effects of DNA extraction and various forms of library preparation on downstream sequence integrity have not yet been thoroughly evaluated [173–175]. The fact that, in most cases, only limited amounts of input DNA are available and this DNA is highly fragmented, further aggravates the situation. The key issue of NGS-based methods is the setting and accurate evaluation of detection limits. A mutation can only be considered to be a true mutation if the frequency of a base change at a target locus is higher than a predetermined read error rate. A detection limit of 0.01% can theoretically only be achieved with 100,000 reads covering the target region if the error rate is below 0.01% and the plasma DNA input sample contains approximately 5000 GEs. However, since the error rates of different sequencing platforms are typically in the range of 0.1–0.5% depending on the sequence context, the desired detection cannot be reached without additional molecular barcoding or bioinformatics approaches. The presumptive error rate can be calculated from sequencing data of a sufficient number of normal individuals carrying no mutations. Background errors were shown to be increasingly evident below allele fractions of 0.2% and under an allele fraction of 0.02% in more than 50% of sequenced genomic positions, artifacts are present [51].
In recent years, several bioinformatic approaches for error suppression have been developed. Narayan et al. described a deep sequencing algorithm that demands redundancy within each clonal sequence to produce extremely high quality base calls in short, mutation-prone regions of plasma DNA [176]. Mutation hotspot regions were sequenced by partial overlap of paired-end reads from the forward and reverse strands and read pairs that did not have perfect sequence agreement were discarded, thereby reducing the median error frequency of 0.31% per base to 0.07% [176]. Using the same approach, a Japanese group reported detection limits for EGFR hotspot mutations, i.e., exon 19 deletion, L858R, L861Q, and T790M down to 0.01% at a significance level of p=2x10-5 [177,178]. The detection limits for the exon 19 deletion and the L858R, L861Q, Advances in Circulating Tumor DNA Analysis 101 and T790M substitution mutations at a significance level of p=2x10-5 were less than 0.01%, 0.01%, and 0.05%, respectively.
(Perakis, pgs. 97-103). This is why Perakis concludes that this application would not work:
Another possibility of error reduction involves so-called barcoding strategies, which were first described in 2011 [50,179]. Kinde et al. developed a method called the Safe-Sequencing System, which assigns a unique identifier (UID) to each template molecule by tagging the target-specific primer with an 8 bp degenerated tag. After amplification and redundant sequencing of each uniquely tagged template molecule, UID families are created, thereby enabling the precise tracking of individual molecules and reducing the false positive rate at least 15–24-fold. Since these approaches make use of molecular tagging of single-stranded DNA, they can reduce the frequency of erroneously called variants only by approximately 20-fold. Schmitt et al. further developed this strategy for the preparation of tagged duplex shotgun libraries in order to correct errors that occur in the first round of amplification and are propagated to subsequent copies [180]. After ligating the adapters harboring degenerate molecular tags, the individually labeled strands are PCR-amplified to create sequence families that share the same tag sequences derived from each of the two single parental strands, leading to a >10,000-fold improvement compared with conventional NGS [181,182]. Although the same group used this approach for selective enrichment of small genomic regions, the original protocol has not yet been used with ctDNA [181]. Prospects of success are limited since the method is relatively inefficient when limited amounts of input DNA—as it is most likely the case for cfDNA—are used [182]. Newman et al. combined both barcoding strategies and bioinformatic approaches and further developed their previously published cancer personalized profiling by deep sequencing (CAPP-Seq) methods by tagging each strand of the original DNA duplex molecule with four barcodes: three exogenous barcodes and one endogenous barcode comprising the molecule’s mapped genomic coordinates. Using this integrated digital error suppression (iDES) strategy, they were able to reduce the errors per base down to 9x10-5 [51]. Digital Sequencing TM, another approach using molecular barcoding, is offered by Guardant Health, Inc. (http://www.guardanthealth.com/guardant360/). In a recent publication, the authors report a sensitivity of down to 0.1% mutant allele fraction and an analytic specificity of >99.99%, enabling complete coverage of 54 clinically actionable genes [183]. A similar method combining double-stranded barcoding error correction and rolling circle amplification (RCA)-based target enrichment, termed CypherSeq, was described by Gregory et al. [184]. This method involves the ligation of 102 Samantha Perakis et al. sample DNA into circular vectors which contain double-stranded barcodes for computational error correction and adapters for library preparation and sequencing. The authors demonstrated a reproducible detection of mutations down to a frequency of 2.4x10-7 per base pair in S. cerevisiae genomes. Although the method seems promising for achieving the required sensitivity and specificity for the early detection of disease, it has not yet been transferred to ctDNA [184]
(pg. 103; emphasis added). Schmitt (Detection of ultra-rare mutations by next-generation sequencing, Proc Natl Acad Sci U S A. 2012 Sep 4;109(36):14508-13. doi: 10.1073/pnas.1208715109. Epub 2012 Aug 1) is the NPL on which this ligation-adaptor duplex sequencing application is based. Perakis concludes that it will not work for cfDNA. Further evidence that this is true: none of these cfDNA sequencing issues identified in the art are addressed in this specification. For example, ligation efficiency in ctDNA or cfDNA is never addressed; nor adaptor configuration; nor tag configuration; nor ligation reactions; nor cfDNA end preparation; nor cfDNA isolation. In fact, the examples in the specification never use ctDNA or cfDNA, rather genomic DNA, which is present in much higher amounts than cfDNA. In other words, the specification at best mentions miRNA as an afterthought, with no specific details to overcome these known critical limitations.
Finally, Perkais makes clear that
Most of the early studies on ctDNA have employed PCR techniques and its derivatives thereof. PCR assays have been widely used for quantification of cfDNA and the detection of single mutations, aberrant methylation patterns, MSI, or LOH. However, the introduction of next-generation sequencing (NGS) in 2005 has had a tremendous impact on the liquid biopsy field. Today it is possible to reconstruct individual cancer genomes noninvasively from plasma. While the establishment of SCNAs can easily be done via low-coverage WGS in a fast and cost-effective manner given a tumor fraction of more than 5–10%, the identification of mutations at the nucleotide level for large gene panels is much more expensive and time-consuming since it requires a much higher sequencing depth, in particular for the detection of underrepresented mutations.
(pgs. 96-97; emphasis added). Therefore, cfDNA alone requires extensive, painstaking optimization and experimentation, none of which is evident in the specification.
Yet, more evidence of the overwhelming difficulty of cfDNA adaptor ligation is found in the art. As explained above, the invention would find particular utility in analysing DNA from tumors, so that somatic mutations can be detected by sequencing tumor-derived cfDNA rather than needing to sample DNA directly from cells in the tumor. In a cfDNA sample, however, the vast majority of cfDNA will be derived from non-tumor cells because these are much more abundant than tumor cells, and their cfDNA is released as a result of the everyday cellular turnover. Moreover, even within the tumor-derived cfDNA, most of the fragments will not contain mutations (because
most of the genome is not mutated). Thus the technical challenge is to identify those few cfDNA molecules which are derived from tumor cells and which contain a mutation, in a population wherein they are massively outnumbered - the proverbial needle in the haystack, which creates a high risk of false negatives.
To add to this challenge, the error rate in typical sequencing techniques is higher than the mutation rate underlying tumorigenesis, so the level of noise (polymerase errors, etc.) will typically be much higher than the level of signal (true mutations). As noted in Schmitt, (p.14510, top-left), ">99.9% of the apparent mutations identified by standard sequencing are erroneous" (also outlined in [0072] to [0074] of US9598731). Thus there is also a very high risk of false positives.
Accordingly, when analyzing cfDNA for true mutations, the assay must be (i) sensitive enough to ensure that those few true mutations are not lost in the processing steps, and (ii) specific enough to be able to distinguish these true mutations from the errors introduced in the processing steps. To achieve the sensitivity required, the method therefore must reduce data loss at every available step of the workflow, including in both the wet-lab processing steps and in the bioinformatics analysis. The combination of steps must permit low-abundance cfDNA molecules to be reliably sequenced, avoiding the noise which arises from amplification and sequencing techniques.
In contrast, the prior art had not applied these techniques to cfDNA. This is because cfDNA molecules are generated by enzymatic digestion of nucleosomal structures during cell-death, and this process means that cfDNA fragments do not have a random structure. Rather, their lengths vary periodically (rather than continuously), and the structural differences between cfDNA and randomly-sheared genomic DNA are shown in Figure 4 of Leary - the upper panel shows that cfDNA molecules show peaks & troughs in length, whereas the lower panel shows that randomly-sheared DNA varies continuously, with a central peak. Therefore cfDNA molecules and randomly-sheared DNA fragments are not analogous. Thus cfDNA shows a fragmentation pattern which is not seen when random shearing is used, and the end points of cfDNA molecules are not evenly distributed. Compared to randomly-sheared fragments, therefore, cfDNA molecules show lower sequence diversity at their termini. Furthermore, very few cfDNA molecules are longer than 200 bp (see Figure 3 of Leary). This patterned nature and narrow size-range also means that each cfDNA derived from a single haploid genome is essentially unique, because chromosomal sequences typically do not include long repeated sequences, and where such repeats occur their periodicity does not match the periodicity of cfDNA fragmentation. Conversely, two haploid genomes will give rise to very similar cfDNA fragments as each other so, as the number of haploid genome equivalents increases, the chance of seeing duplicate cfDNA molecules also increases (US9598731 [0113]).
Here, the specification discloses that “[s]heared double-stranded DNA that has been end-repaired and T-tailed is combined with A-tailed SMI adaptors and ligated according to one embodiment” (para. 0013). This is the only embodiment disclosed. As explained above, this shearing of cfDNA would not work. The specification simply never contemplates how to ligate tags/UIDs onto cfDNA.
As explained above in relation to Perakis, the method claimed here would not be expected to work with cfDNA (explaining that Schmitt would not work with cfDNA). The reasons for this view are clear. The method of the claims prepares DNA analytes by shearing with the "Covaris AFA system", which uses ultrasonication (see 'Sequencing Library Preparation' on page 1 of Forshew et al, Noninvasive identification and monitoring of cancer mutations by targeted deep sequencing of plasma DNA, Sci Transl Med. 2012 May 30;4(136):136ra68. doi: 10.1126/scitranslmed.3003726). Subjecting cfDNA to such acoustic shearing techniques would destroy most of the DNA and further exacerbate the problems caused by having low concentrations of analyte. Sonication is an essential and deliberate step of the claimed method.
In addition, after sonication the specification discloses that Applicants select a fraction of fragments which would exclude almost all cfDNA. The specifictaion focuses on DNA fragments within "the optimal range of ~200-500 bp" (Forshew, sentence bridging the two columns on page 1). Fragments longer than 500 bp are discarded, and then remaining fragments longer than 200 bp are retained. The peak size of cfDNA, however, is ~160 nucleotides (see [0113] of US9598731), so the claimed method would discard the large majority of cfDNA. Furthermore, a sonication step would have reduced the size of cfDNA even further, leading to even great loss of original cfDNA molecules.
Aside from these practical issues, the claimed method would also be seen as unsuitable for use in a 'needle in a haystack' scenario because of its high rate of data loss. As noted above, the inherent error rate in next-generation sequencing means that the long tags used here lead to around ¼ of sequence reads being discarded. The shearing and selection steps would essentially eliminate any remaining useful data, so the claimed method is effectively useless in analyzing cfDNA. This level of data loss can be acceptable for detecting rare mutations in cellular genomic DNA, which is relatively abundant, but the skilled person would understand that this is not tolerable in the context of cfDNA (where a rare mutation is not merely the result of a rare event, but is also rare within the total analyte DNA because of the dominant background of cfDNA from non-mutated cells, see US9598731: [0240]).
All of this is recognized by Schwarzenbach et al, Cell-free nucleic acids as biomarkers in cancer patients, Nature Reviews Cancer volume 11, pages426–437 (2011)
The major problem with this approach has been assay specificity and sensitivity. Assays targeting cfDNA mutations require that the mutation in the tumour occurs frequently at a specific genomic site. A major drawback of cfDNA assays is the low frequency of some of the mutations that occur in tumours. In general, wild-type sequences often interfere with cfDNA mutation assays. This is due to the low level of cfDNA mutations and the dilution effect of DNA fragments and wild-type DNA in circulation. . . .
[ . . . ]
. . . As new approaches in the assessment of cfDNA, such as next-generation sequencing, are being developed, the issue of extraction of DNA will continue to complicate cfDNA biomarker assay development and regulatory group approval.
(pgs. 428 & 432). Similarly, US9598731 states
The concentrations of tumor-shed nucleic acids are typically so low that current next-generation sequencing technologies can only detect such signals sporadically or in patients with terminally high tumor burden. The main reason being that such technologies are plagued by error rates and bias that can be orders of magnitude higher than what is required to reliably detect de novo genetic alterations associated with cancer in circulating DNA
(col. 60, ll. 20-25). And Lennon et al, Technological considerations for genome-guided diagnosis and management of cancer, Genome Med. 2016 Oct 26;8(1):112. doi: 10.1186/s13073-016-0370-4 summarizes in 2016, that
the sensitivity to detect variants from low purity samples will be limited by the total yield or genome equivalents of cfDNA that are available for sequencing. Thus, the accurate profiling of tumor DNA or RNA in a sample that contains non-tumor DNA or RNA is challenging and requires specialized methods, such as error-correcting with molecular barcodes (tags of parsable (separable by software) sequence that are used to label individual starting molecules), also known as unique molecular indexes (UMI) [35], high efficiency library preparation kits for low input material [36, 37], or mutation enrichment [38])
(pg. 3). None of these “specialized methods” for sequencing cfDNA are disclosed here.
Thus, this factor weighs against enablement because the state of the art at the time of effective filing until at least 2016 demonstrates that there were innumerable unpredictable high hurdles to achieve the claimed cfDNA ligation tagging and sequencing, and the specification fails to disclose any details essential to make and use it.
D. The Level Of One Of Ordinary Skill: PhD (High)
Generally, skilled artisans in biotechnology are highly-skilled with a PhD. Enzo Biochem, Inc. v. Calgene, Inc., 188 F.3d 1362, 1373 (Fed. Cir. 1999) (citing Enzo Biochem, Inc. v. Calgene, Inc., 14 F. Supp. 2d 536, 567 (D. Del 1998)) (district court did not abuse discretion in finding that “a person of ordinary skill in the art would be ‘a junior faculty member with one or two years of relevant experience or a postdoctoral student with several years of experience’”). Thus, this factor generally weighs in favor of enablement.
However, in light of the extensive experimentation required in this specific field of cfDNA ligation-adaptor sequencing , and the complete lack of guidance in the Specification as to cfDNA, this factor as applied here weighs against enablement.
E. The Level Of Predictability In The Art: Low
Generally, the level of predictability in the biotechnology arts is low. C.f. In re Kubin, 561 F.3d 1351 (Fed. Cir. 2009); Pfizer, Inc. v. Apotex, Inc., 480 F.3d 1348 (Fed. Cir. 2007). This finding is further evidenced by the state of the prior art as explained above.
In fact, the instant claim seems to encompass a nascent technology of ligation-tagged cfDNA sequencing of notoriously difficult cfDNA. See Chiron Corp. v. Genentech Inc., 363 F.3d 1247, 1254 (Fed. Cir. 2004) (“Nascent technology, however, must be enabled with a ‘specific and useful teaching.’ The law requires an enabling disclosure for nascent technology because a person of ordinary skill in the art has little or no knowledge independent from the patentee’s instruction. Thus, the public’s end of the bargain struck by the patent system is a full enabling disclosure of the claimed technology.” (citations omitted)); MPEP § 2164.03 (“The amount of guidance or direction needed to enable the invention is inversely related to the amount of knowledge in the state of the art as well as the predictability in the art. In re Fisher, 427 F.2d 833, 839, 166 USPQ 18, 24 (CCPA 1970).”). As explained above, the prior art demonstrates that limited amounts of input DNA (e.g. ctDNA, which is well-known to be present in very low amounts in samples), yield even more difficult ligation-tagging sequencing design compared to standard DNA samples. Therefore, this factor weighs against enablement.
F. The Amount Of Direction Provided By The Inventor: Zero
Applicants’ specification provides no guidance. The specification is completely silent as to cfDNA, much less its isolation and ligation-tagging. None of the issues raised above for cfDNA sequencing are addressed in the specification. At most the specification mentions the genomic DNA duplex sequencing technique can be applied to miRNA. Thus, this factor weighs heavily against enablement.
G. The Existence Of Working Examples: Zero
The specification provides zero working examples for cfDNA. Therefore, in light of the evidence that highly cfDNA sequencing and ligation-tagging of cfDNA required extensive experimentation for each application, and the lack of such guidance in the specification, this factor weighs against enablement.
H. The Quantity Of Experimentation Needed To Make Or Use The Invention Based On The Content Of The Disclosure: Extensive
Thus, the above factors make clear that a skilled artisan would be required to engage in extensive optimization that amounts to undue experimentation. Although “an extended period of experimentation may not be undue if the skilled artisan is given sufficient direction or guidance,” yet here, the specification and prior art provide little guidance. See In re Colianni, 561 F.2d 220, 224 (CCPA 1977). In fact, the specification and state of the art indicate that a skilled artisan would expect that accomplishing highly-multiplexed directed sequencing of ctDNA would require extensive, painstaking experimentation based on a generic, vague primer-dimer roadmap. “Section 112 of the Patent Act reflects Congress’s judgment that if an inventor claims a lot, but enables only a little, the public does not receive its benefit of the bargain.” Amgen Inc., v. Sanofi, No. 21-757, slip op. at 19 (Supreme Court, May 18, 2023).
Our decisions in Morse, Incandescent Lamp, and Holland Furniture reinforce the simple statutory command. If a patent claims an entire class of processes, machines, manufactures, or compositions of matter, the patent’s specification must enable a person skilled in the art to make and use the entire class. In other words, the specification must enable the full scope of the invention as defined by its claims. The more one claims, the more one must enable. See §112(a); see also Continental Paper Bag Co. v. Eastern Paper Bag Co., 210 U. S. 405, 419 (1908) (“[T]he claims measure the invention.”).
[ . . . ]
Decisions such as Wood and Minerals Separation establish that a specification may call for a reasonable amount of experimentation to make and use a patented invention. What is reasonable in any case will depend on the nature of the invention and the underlying art. See Minerals Separation, 242 U. S., at 270–271; see also Mowry v. Whitney, 14 Wall. 620, 644 (1872) (“[T]he definiteness of a specification must vary with the nature of its subject. Addressed as it is to those skilled in the art, it may leave something to their skill in applying the invention.”). But in allowing that much tolerance, courts cannot detract from the basic statutory requirement that a patent’s specification describe the invention “in such full, clear, concise, and exact terms as to enable any person skilled in the art” to “make and use” the invention. §112(a). Judges may no more subtract from the requirements for obtaining a patent that Congress has prescribed than they may add to them. See Bilski v. Kappos, 561 U. S. 593, 602–603, 612 (2010).
Id. at 13, 15. Here, the specification is devoid of any guidance as to how to accomplish the claimed invention for cfDNA. Stated differently, Applicants claim much, but disclose very little. See id. (“The more one claims, the more one must enable”). Therefore, this factor weighs heavily against enablement.
Conclusion: Factors Weigh Strongly Against Enablement
Taken together, the factors weigh heavily against patentability of the claims.
The Constitution vests Congress with the power to “promote the Progress of Science and useful Arts, by securing for limited Times to Authors and Inventors the exclusive Right to their respective Writings and Discoveries.” Art. I, §8, cl. 8. Right there in the text, one finds the outline of what this Court has called the patent “bargain.” Bonito Boats, Inc. v. Thunder Craft Boats, Inc., 489 U. S. 141, 150 (1989). In exchange for bringing “new designs and technologies into the public domain through disclosure,” so they may benefit all, an inventor receives a limited term of “protection from competitive exploitation.” Id., at 151; see also The Federalist No. 43, p. 272 (C. Rossiter ed. 1961) (J. Madison) (explaining that in such cases “[t]he public good fully coincides . . . with the claims of individuals”).
Congress has exercised this authority from the start. The Patent Act of 1790 promised up to a 14-year monopoly to any applicant who “invented or discovered any useful art, manufacture, . . . or device, or any improvement therein not before known or used.” Act of Apr. 10, 1790, §1, 1 Stat. 110. Reflecting the quid-pro-quo premise of patent law, the statute required the applicant to deposit with the Secretary of State a “specification . . . so particular . . . as not only to distinguish the invention or discovery from other things before known and used, but also to enable a workman or other person skilled in the art or manufacture . . . to make, construct, or use the same.” §2, ibid. The statute made clear that this disclosure would ensure “the public may have the full benefit [of the invention or discovery], after the expiration of the patent term.” Ibid.
Even as Congress has revised the patent laws over time, it has left this “enablement” obligation largely intact. See 35 U. S. C. §§111, 112. Section 111 of the current Patent Act provides that a patent application “shall include . . . a specification as prescribed by section 112.” §111(a)(2)(A). Section 112, in turn, requires a specification to include “a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art . . . to make and use the same.” §112(a). So today, just as in 1790, the law secures for the public its benefit of the patent bargain by ensuring that, “upon the expiration of [the patent], the knowledge of the invention [i]nures to the people, who are thus enabled without restriction to practice it.” United States v. Dubilier Condenser Corp., 289 U. S. 178, 187 (1933); see also Grant v. Raymond, 6 Pet. 218, 247 (1832) (Marshall, C. J.) (“This is necessary in order to give the public, after the privilege shall expire, the advantage for which the privilege is allowed, and is the foundation of the power to issue a patent.”); Whittemore v. Cutter, 29 F. Cas. 1120, 1122 (No. 17,600) (CC Mass. 1813) (Story, J.) (“If therefore [the disclosure] be so obscure, loose, and imperfect, that this cannot be done, it is defrauding the public of all the consideration, upon which the monopoly is granted.”)
Amgen Inc., v. Sanofi, No. 21-757, slip op. at 7-8 (Supreme Court, May 18, 2023). Applicants have not met this essential obligation. As demonstrated by the art cited above, a skilled artisan would have been required to engage in extensive experimentation in order to make and use the claimed invention. In contrast, the specification is “so obscure, loose, and imperfect, that [enablement of public use of the claimed invention] cannot be done, it is defrauding the public of all the consideration, upon which the monopoly is granted.” The state-of-the-art reveals that ligation-tagged sequencing of cfDNA would have required extensive experimentation for each application. However, the specification fails to “contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same.” See 35 USC § 112, first paragraph (pre-AIA ). The specification only mentions miRNA. No cfDNA adaptors are disclosed. No cfDNA isolation technique. No cfDNA tags. No cfDNA ligation reaction conditions. ctDNA is never discussed, much less how to overcome its innumerable high hurdles to use it in sequencing. And no working examples of cfDNA. Thus, the claims are not enabled.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. § 102 that form the basis for the rejections under this section made in this Office action (AIA and pre-AIA ):
(A) A person shall be entitled to a patent unless –
(1)the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention; or
(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
A person shall be entitled to a patent unless —
(a) the invention was known or used by others in this country, or patented or described in a printed publication in this or a foreign country, before the invention thereof by the applicant for patent, or
(b) the invention was patented or described in a printed publication in this or a foreign country or in public use or on sale in this country, more than one year prior to the date of the application for patent in the United States
Claims 21-53 are rejected under 35 U.S.C. § 102(a)(1) or 102(b) as being anticipated by TALASAZ (US 20140066317).
Claims 21 and 36 differ only in that claim 21 recites “partially double-stranded adapters,” whereas claim 36 recites “adaptors.” Thus, the rejection of claim 21 applies to claim 36 as a species of claim 36.
As to claim 21, TALASAZ teaches a method of sequencing double-stranded circulating DNA molecules, the method comprising (para. 0005- “The disclosure also provides for a method for detecting a rare mutation in a cell-free or substantially cell free sample obtained from a subject”):
(a) ligating partially double-stranded adapters (blunt-end ligation, sticky end ligation; para. 0045) to both ends of the double-stranded circulating DNA molecules to form adapter-target complexes (para. 0214- “This disclosure provides methods of converting initial polynucleotides into tagged polynucleotides with a conversion efficiency of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 80% or at least 90%. The methods involve, for example, using any of blunt-end ligation, sticky end ligation, molecular inversion probes, PCR, ligation-based PCR, multiplex PCR, single strand ligation and single strand circularization. The methods can also involve limiting the amount of initial genetic material. For example, the amount of initial genetic material can be less than 1 ug, less than 100 ng or less than 10 ng. These methods are described in more detail herein”), wherein
(i) the double-stranded adapters each comprise an identifier sequence (para. 0014- “In some embodiments, the method may comprise comprising attaching one or more barcodes to the extracellular polynucleotides or fragments thereof prior to sequencing, in which the barcodes comprise are unique. In other embodiments barcodes attached to extracellular polynucleotides or fragments thereof prior to sequencing are not unique”),
(ii) at least two adapters comprise the same identifier sequence and are ligated to different circulating DNA molecules (id.), and
(iii) circulating DNA end sequences together with associated identifier sequences form tags at both ends of the circulating DNA molecules (para. 0017- “In some embodiments, the barcode is a polynucleotide, which may further comprise random sequence or a fixed or semi-random set of oligonucleotides that in combination with the diversity of molecules sequenced from a select region enables identification of unique molecules and be at least a 3, 5, 10, 15, 20 25, 30, 35, 40,45, or 50 mer base pairs in length”; para. 0019- “In some embodiments, sequence reads of unique identity may be detected based on sequence information at the beginning (start) and end (stop) regions of the sequence read and the length of the sequence read. In other embodiments sequence molecules of unique identity are detected based on sequence information at the beginning (start) and end (stop) regions of the sequence read, the length of the sequence read and attachment of a barcode”); and
(iv) individual adapter-target complexes comprise different pairs of tags (paras. 0014-17- “identification of unique molecules”);
(b) amplifying the adapter-target complexes to produce a plurality of adapter-target amplification products from each of a first strand and a complementary second strand of the adapter-target complexes (parent and progeny strands tagged; paras. 0232ff);
(c) sequencing the adapter-target amplification products to produce a plurality of first-strand sequencing reads and a plurality of second-strand sequencing reads (id.);
(d) comparing the first-strand sequencing reads with the second-strand sequencing reads for each of a plurality of the adapter-target complexes (para. 0063- “In some embodiments collapsing comprising detecting and/or correcting errors, nicks or lesions present in the sense or anti-sense strand of the tagged parent polynucleotides or amplified progeny polynucleotides”); and
(e) generating error-corrected sequences for each of a plurality of the double-stranded circulating DNA molecules by distinguishing erroneous nucleotides in one strand that lack a matched base change in the complementary strand (id.)
As to claim 22, TALASAZ teaches the identifier sequence is a random identifier sequence (para. 0017).
As to claim 23, TALASAZ teaches the identifier sequence is not completely random (id.)
As to claim 24, TALASAZ teaches the identifier sequence is at an end of the partially double-stranded adapter (id.)
As to claim 25, TALASAZ teaches the identifier sequence is about 5 to about 20 nucleotides in length (id.)
As to claim 26, TALASAZ teaches the identifier sequence is 5 to 10 nucleotides in length (id.)
As to claim 27, TALASAZ teaches the circulating DNA end sequences comprise 10 terminal nucleotides of the double-stranded circulating DNA molecules (paras. 0017, 0019, 0050, 0100, 0243).
As to claim 28, TALASAZ teaches the erroneous nucleotides comprise a polymerase error that arose during amplification or sequencing (para. 0120, 0123).
As to claim 29, TALASAZ teaches identifying a true mutation that is present in both strands of one of the double-stranded circulating DNA molecules (para. 0198).
As to claim 30, TALASAZ teaches the true mutation is identified when it is present in substantially all first-strand sequencing reads and second-strand sequencing reads for the double-stranded circulating DNA molecule (id.)
As to claim 31, TALASAZ teaches the true mutation is identified when it is present in all first-strand sequencing reads and second-strand sequencing reads for the double-stranded circulating DNA molecule (id.)
As to claim 32, TALASAZ teaches generating error-corrected sequences comprises comparing the first-strand sequencing reads with the second-strand sequencing reads within each of a plurality of groups of reads, wherein the groups of reads are distinguishable by the different pairs of tags (grouping into families; paras. 0064-70).
As to claim 33, TALASAZ teaches the circulating DNA molecules are isolated from a blood sample of a subject (para. 0009).
As to claim 34, TALASAZ teaches the circulating DNA molecules are isolated from a subject having cancer (paras. 0012-13).
As to claim 35, TALASAZ teaches comprising detecting a circulating DNA molecule from a cancer (id.)
As to claim 36, TALASAZ teaches method of sequencing double-stranded circulating DNA molecules, the method comprising:
(a) ligating adapters to both ends of the double-stranded circulating DNA molecules to form adapter-target complexes, wherein
(i) the adapters each comprise a double-stranded identifier sequence,
(ii) at least two adapters comprise the same identifier sequence and are ligated to different circulating DNA molecules, and
(iii) circulating DNA end sequences together with associated identifier sequences form tags at both ends of the circulating DNA molecules; and
(iv) individual adapter-target complexes comprise different pairs of tags;
(b) amplifying the adapter-target complexes to produce a plurality of adapter-target amplification products from each of a first strand and a complementary second strand of the adapter-target complexes;
(c) sequencing the adapter-target amplification products to produce a plurality of first-strand sequencing reads and a plurality of second-strand sequencing reads;
(d) comparing the first-strand sequencing reads with the second-strand sequencing reads for each of a plurality of the adapter-target complexes; and
(e) generating error-corrected sequences for each of a plurality of the double-stranded circulating DNA molecules by distinguishing erroneous nucleotides in the first strand that lack a matched base change in the complementary second strand (see claim 21, above).
As to claim 37, TALASAZ teaches the identifier sequence is a random identifier sequence (claim 22, above).
As to claim 38, TALASAZ teaches the identifier sequence is not completely random (claim 23, above).
As to claim 39, TALASAZ teaches the identifier sequence is at an end of the adapter (claim 24, above).
As to claim 40, TALASAZ teaches the identifier sequence is about 3 to about 20 nucleotides in length (claim 25, above).
As to claim 41, TALASAZ teaches the identifier sequence is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length (claim 26, above).
As to claim 42, TALASAZ teaches the identifier sequence is 5, 6, 7, 8, 9, or 10 nucleotides in length.
As to claim 43, TALASAZ teaches the identifier sequence is about 5 to about 20 nucleotides in length (claim 27, above).
As to claim 44, TALASAZ teaches the identifier sequence is 1, 2, 3, 4, 5, 6, 7 or 8 nucleotides in length (claim 28, above).
As to claim 45, TALASAZ teaches the circulating DNA end sequences comprise 10 terminal nucleotides of the double-stranded circulating DNA molecules (claim 29, above).
As to claim 46, TALASAZ teaches the erroneous nucleotides comprise a polymerase error that arose during amplification or sequencing (claim 30, above).
As to claim 47, TALASAZ teaches comprising identifying a true mutation as a mutation that is present in both the first strand and the complementary second strand of one of the double-stranded circulating DNA molecules (claim 31, above).
As to claim 48, TALASAZ teaches the true mutation is identified when it is present in substantially all first-strand sequencing reads and second-strand sequencing reads for the double-stranded circulating DNA molecule (claim 32, above).
As to claim 49, TALASAZ teaches the true mutation is identified when it is present in all first-strand sequencing reads and second-strand sequencing reads for the double-stranded circulating DNA molecule (claim 32, above).
As to claim 50, TALASAZ teaches generating error-corrected sequences comprises comparing the first-strand sequencing reads with the second-strand sequencing reads within each of a plurality of groups of reads, wherein the groups of reads are distinguishable by the different pairs of tags (claim 33, above).
As to claim 51, TALASAZ teaches at least a portion of the circulating DNA molecules are nucleic acid-based blood biomarkers (claim 34, above).
As to claim 52, TALASAZ teaches the circulating DNA molecules are isolated from a subject having cancer (claim 35, above).
As to claim 53, TALASAZ teaches comprising detecting a circulating DNA molecule from a cancer (claim 36, above).
Double Patenting- Obvious Type
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory obviousness-type double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); and In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on a nonstatutory double patenting ground provided the conflicting application or patent either is shown to be commonly owned with this application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement.
Effective January 1, 1994, a registered attorney or agent of record may sign a terminal disclaimer. A terminal disclaimer signed by the assignee must fully comply with 37 CFR 3.73(b).
Instant claims 21-53 are rejected on the ground of nonstatutory obviousness-type double patenting as being unpatentable over conflicting claims 1-30 of US 10604804.
The instant claims are obvious over the conflicting claims because the conflicting claims anticipate the instant claims. More specifically, the conflicting claims teach:
1. A method for quantifying single nucleotide variant cancer biomarkers in circulating nucleic acid from a subject, comprising:
(a) providing a plurality of circulating nucleic acid molecules obtained from a bodily sample of the subject;
(b) attaching tags comprising barcodes selected from a plurality of distinct barcode sequences to said circulating nucleic acid molecules obtained from said bodily sample of the subject, to generate non-uniquely tagged parent polynucleotides, wherein each non-uniquely tagged parent polynucleotide is substantially unique with respect to other non-uniquely tagged parent polynucleotides in the bodily sample;
(c) amplifying the non-uniquely tagged parent polynucleotides to produce amplified non-uniquely tagged progeny polynucleotides;
(d) sequencing the amplified non-uniquely tagged progeny polynucleotides to produce a plurality of sequence reads from each non-uniquely tagged parent polynucleotide, wherein each sequence read comprises a barcode sequence and a sequence derived from a circulating nucleic acid molecule;
(e) grouping the plurality of sequence reads produced from each non-uniquely tagged parent polynucleotide into families based on i) the barcode sequence and ii) sequence information derived from the circulating nucleic acid molecule, whereby each family comprises sequence reads of non-uniquely tagged progeny polynucleotides amplified from a unique polynucleotide among the non-uniquely tagged parent polynucleotides;
(f) comparing the sequence reads grouped within each family to each other to determine consensus sequences for each family, wherein each of the consensus sequences corresponds to a unique polynucleotide among the non-uniquely tagged parent polynucleotides;
(g) providing a reference sequence, said reference sequence comprising one or more loci;
(h) identifying consensus sequences that map to a given locus of said one or more loci; and
(i) calculating a number of consensus sequences that map to the given locus that include a cancer-associated single nucleotide variant thereby quantifying single nucleotide variant cancer biomarkers in said circulating nucleic acid from said subject.
Thus, the conflicting claims anticipate the instant claims because the conflicting claims teach the same double-stranded adaptors ligated to circulating DNA, the same identifier sequence-end sequence tags, and the same error-correction based on bi-directional/paired-end sequencing.
Instant claims 21-53 are rejected on the ground of nonstatutory obviousness-type double patenting as being unpatentable over conflicting claims 1-27 of US 10689699.
The instant claims are obvious over the conflicting claims because the conflicting claims anticipate the instant claims. More specifically, the conflicting claims teach:
1. A method, comprising:
a) providing a population of circulating DNA molecules obtained from a bodily sample from a subject;
b) converting the population of circulating DNA molecules into a population of non-uniquely tagged parent polynucleotides, wherein each of the non-uniquely tagged parent polynucleotides comprises (i) a sequence from a circulating DNA molecule of the population of circulating DNA molecules, and (ii) an identifier sequence comprising one or more polynucleotide barcodes, such that each non-uniquely tagged parent polynucleotide is substantially unique with respect to other non-uniquely tagged parent polynucleotides in the population;
c) amplifying the population of non-uniquely tagged parent polynucleotides to produce a corresponding population of amplified progeny polynucleotides;
d) sequencing at least a portion of the population of amplified progeny polynucleotides to produce a set of sequence reads;
e) grouping the sequence reads into families, each of the families comprising sequence reads comprising the same identifier sequence and having the same start and stop positions, whereby each of the families comprises sequence reads amplified from the same non-uniquely tagged parent polynucleotide; and
f) collapsing sequence reads in each family to yield a base call for each family corresponding to one or more genetic loci.
20. A method, comprising:
a) attaching a set of molecular tags to a population of circulating DNA molecules obtained from a bodily sample of a subject to produce a population of tagged original DNA molecules, wherein a plurality of the tagged original DNA molecules has identical molecular tags, and wherein each tagged original DNA molecule is substantially unique with respect to other tagged original DNA molecules in the population;
b) amplifying the population of tagged original DNA molecules to produce a corresponding population of DNA molecule amplicons;
c) sequencing at least a portion of the population of DNA molecule amplicons to produce a set of sequence reads;
d) grouping the sequence reads into families based on i) the molecular tag and ii) sequence information derived from the circulating DNA molecule, whereby each of the families comprises sequence reads amplified from the same tagged original DNA molecule; and
e) collapsing sequence reads in each family to provide an error-corrected consensus sequence read for each family corresponding to one or more at the genetic loci.
Thus, the conflicting claims anticipate the instant claims because the conflicting claims teach the same double-stranded adaptors ligated to circulating DNA, the same identifier sequence-end sequence tags, and the same error-correction based on bi-directional/paired-end sequencing.
Instant claims 21-53 are rejected on the ground of nonstatutory obviousness-type double patenting as being unpatentable over conflicting claims 1-30 of US 11047006.
The instant claims are obvious over the conflicting claims because the conflicting claims anticipate the instant claims. More specifically, the conflicting claims teach:
1. A method of sequencing DNA, comprising:
(a) preparing a sequence library from a sample comprising a plurality of double-stranded DNA fragments from a biological source, wherein preparing the sequence library comprises ligating adapter molecules to the plurality of double-stranded DNA fragments to generate adapter-DNA molecules having a first strand and a second strand;
(b) sequencing first and second strands of at least a portion of the adapter-DNA molecules to provide a first strand sequence read and a distinct yet related second strand sequence read for each of a plurality of adapter-DNA molecules;
(c) for individual adapter-DNA molecules in the plurality, comparing the first strand sequence read and the second strand sequence read to identify one or more correspondences between the first and second strand sequence reads; and
(d) analyzing the one or more correspondences between the first and second strand sequence reads for the individual adapter-DNA molecules and comparing said one or more correspondences to a reference sequence to determine a presence or absence of a true mutation present in the biological source, wherein the true mutation is identified when one or more correspondences does not correspond with the reference sequence.
14. A method of sequencing nucleic acid molecules extracted from a biological source, comprising:
providing a sample from the biological source, wherein the sample comprises a plurality of double-stranded nucleic acid molecules;
attaching adapter molecules to individual double-stranded nucleic acid molecules to generate a plurality of adapter-nucleic acid molecules; and
for each adapter-nucleic acid molecule among at least a portion of the adapter-nucleic acid molecules:
generating a set of copies of an original first strand of the adapter-nucleic acid molecule and a set of distinct yet related copies of an original second strand of the adapter-nucleic acid molecule;
sequencing one or more copies of the original first and second strands to provide a first strand sequence and a second strand sequence;
comparing a series of base calls from the first strand sequence to a corresponding series of base calls from the second strand sequence to determine if the base calls are in agreement; and
maintaining a sequence base call at a given position only if the base call from the first strand sequence agrees with the base call from the second strand sequence.
24. A method of generating a sequence read of a double-stranded target nucleic acid molecule comprising:
amplifying each original strand of the double-stranded target nucleic acid molecule resulting in each original strand generating a distinct yet related set of amplified target nucleic acid products;
sequencing the amplified target nucleic acid products generated from each original strand;
confirming the presence of at least one sequence read of an amplified target nucleic acid product generated from each of the original strands;
comparing the at least one sequence read obtained from the amplified target nucleic acid products generated from one original strand with the at least one sequence read obtained from the amplified target nucleic acid products generated from the other original strand; and
identifying correspondences in base calls between the compared sequence reads obtained from the amplified target nucleic acid products generated from each of the original strands, wherein each base call that is in agreement is identified as true.
Thus, the conflicting claims anticipate the instant claims because the conflicting claims teach the same double-stranded adaptors ligated to circulating DNA, the same identifier sequence-end sequence tags, and the same error-correction based on bi-directional/paired-end sequencing.
Instant claims 21-53 are rejected on the ground of nonstatutory obviousness-type double patenting as being unpatentable over conflicting claims 1-35 of US 11098359.
The instant claims are obvious over the conflicting claims because the conflicting claims anticipate the instant claims. More specifically, the conflicting claims teach:
1. A method of generating high accuracy sequencing reads of nucleic acid molecules from a sample, comprising:
(a) attaching adapters to both ends of double-stranded nucleic acid fragments in the sample to provide adapter-nucleic acid products,
wherein the adapters each comprise a double-stranded hybridized region, a single-stranded 5′ arm, a single-stranded 3′ arm, and a physical unique molecular identifier (UMI) on at least one of the single-stranded 5′ arm and the single-stranded 3′ arm, and
wherein a physical UMI is an oligonucleotide sequence that can be used to identify an individual molecule of a double-stranded nucleic acid fragment in the sample;
(b) amplifying both strands of adapter-nucleic acid products from (a), thereby obtaining a plurality of amplified polynucleotides;
(c) sequencing the plurality of amplified polynucleotides, thereby obtaining a plurality of sequence reads each associated with a physical UMI;
(d) identifying a plurality of physical UMIs associated with the plurality of reads; and
(e) determining high accuracy sequence reads of the double-stranded nucleic acid fragments in the sample using the plurality of sequences obtained in (c) and the plurality of physical UMIs identified in (d).
Thus, the conflicting claims anticipate the instant claims because the conflicting claims teach the same double-stranded adaptors ligated to circulating DNA, the same identifier sequence-end sequence tags, and the same error-correction based on bi-directional/paired-end sequencing.
Instant claims 21-53 are rejected on the ground of nonstatutory obviousness-type double patenting as being unpatentable over conflicting claims 1-22 of US 11198907.
The instant claims are obvious over the conflicting claims because the conflicting claims anticipate the instant claims. More specifically, the conflicting claims teach:
1. A method of generating a high accuracy sequence read of a double-stranded target nucleic acid molecule comprising:
amplifying each original strand of the double-stranded target nucleic acid molecule resulting in each original strand generating a distinct yet related set of amplified target nucleic acid products;
sequencing the amplified target nucleic acid products generated from each original strand;
confirming the presence of at least one sequence read of an amplified target nucleic acid product generated from each of the original strands;
comparing the at least one sequence read obtained from the amplified target nucleic acid products generated from one original strand with the at least one sequence read obtained from the amplified target nucleic acid products generated from the other original strand; and
generating a consensus sequence of the double-stranded target nucleic acid molecule wherein each position in the consensus sequence is identified as true if the particular position in the at least one sequence read of both strands of the double-stranded target nucleic acid molecule is complementary.
10. A method of generating an error-corrected consensus sequence comprising:
ligating adapter molecules to individual duplex DNA molecules to form tagged DNA molecules;
for each of one or more tagged DNA molecules
generating a first set of duplicates of an original forward strand of the tagged DNA molecule and a second set of duplicates of an original complement strand of the tagged DNA molecule;
creating a single strand consensus sequence (SSCS) from the first set of duplicates of the original forward strand and a single strand consensus sequence (SSCS) from the second set of duplicates of the original complement strand;
comparing the SSCS of the original forward strand to the SSCS of the original complement strand; and
generating an error-corrected consensus sequence having only nucleotide bases at which the sequence of both the SSCS of the original forward strand and the SSCS of the original complement strand are complementary.
20. The method of claim 10, wherein at least some of the duplex DNA molecules are derived from a tumor or circulating neoplastic cells in a subject, and wherein the method further comprises:
comparing the error-corrected consensus sequence to a respective reference sequence; and
detecting the tumor or circulating neoplastic cells by identifying one or more nucleotide sequence variations present in the error-corrected consensus sequence and not present in the reference sequence.
Thus, the conflicting claims anticipate the instant claims because the conflicting claims teach the same double-stranded adaptors ligated to circulating DNA, the same identifier sequence-end sequence tags, and the same error-correction based on bi-directional/paired-end sequencing.
Instant claims 21-53 are rejected on the ground of nonstatutory obviousness-type double patenting as being unpatentable over conflicting claims 1-42 of US 11242562.
The instant claims are obvious over the conflicting claims because the conflicting claims anticipate the instant claims. More specifically, the conflicting claims teach:
1. A method for reducing an error rate in sequencing reads, comprising:
(a) preparing a sequencing library, wherein preparing a sequencing library comprises—
providing a set of hairpin adapters having a double-stranded region and a linker region, wherein at least a portion of the hairpin adapters comprise a single molecule identifier (SMI) sequence;
ligating the hairpin adapters to a plurality of double-stranded target DNA molecules to generate a sequencing library comprising a plurality of adapter-target DNA complexes; and
transitioning the adapter-target DNA complexes from a double-stranded form to a linear single-stranded form, wherein each linear single-stranded adapter-target DNA complex comprises at least a first strand of the target DNA molecule and a second strand of the same target DNA molecule separated by an adapter sequence;
(b) sequencing at least a portion of the linear single-stranded adapter-target DNA complexes to obtain a plurality of sequence reads;
(c) grouping the plurality of sequence reads into a plurality of families based on the SMIs; and
(d) comparing the sequence reads within each family to generate a consensus sequence for each of the families.
9. The method of claim 1, wherein at least some of the double-stranded target DNA molecules are derived from a tumor or circulating neoplastic cells.
20. A method for preparing a sequencing library, comprising:
(a) obtaining a sample comprising a plurality of double-stranded DNA (dsDNA) fragments comprising a forward strand and a reverse complement strand;
(b) ligating one or more hairpin adapter molecules comprising single molecule identifier (SMI) sequences selected from a plurality of distinct SMI sequences to at least a portion of the plurality of dsDNA fragments to form non-uniquely tagged adapter-dsDNA constructs, wherein each non-uniquely tagged adapter-dsDNA construct is substantially unique with respect to other non-uniquely tagged adapter-dsDNA constructs in the sample; and
(c) transitioning the non-uniquely tagged adapter-dsDNA constructs to produce a plurality of linear adapter-single-stranded DNA (ssDNA) constructs comprising the forward strand and the reverse complement strand linked by an adapter sequence to generate a sequencing library.
25. The method of claim 20, wherein at least some of the dsDNA fragments are derived from a tumor or circulating neoplastic cells.
27. A method for detecting one or more rare variants in a biological sample, the method comprising:
(a) preparing a sequencing library comprising a plurality of double-stranded DNA (dsDNA) molecules, wherein preparing the sequencing library comprises—
providing a set of hairpin adapters having a double-stranded region and a linker region, wherein at least a portion of an adapter sequence comprises a single molecule identifier (SMI) sequence;
ligating the hairpin adapters to the plurality of dsDNA molecules to generate a plurality of adapter-DNA complexes; and
transitioning the adapter-DNA complexes from a double-stranded form to a linear single-stranded form, wherein each linear single-stranded adapter-target DNA complex comprises at least a first strand of a parent dsDNA molecule and a second strand of the parent dsDNA molecule separated by the adapter sequence;
(b) sequencing at least a portion of the sequencing library to obtain a plurality of sequencing reads;
(c) grouping the plurality of sequencing reads into a plurality of families based on the SMIs, wherein each of the families comprise a first set of first strand sequence reads, each having a first SMI, and a second set of second strand sequencing reads, each having a second SMI, wherein the first SMI is relatable to the second SMI;
(d) comparing the sequence reads within each family to generate one or more consensus sequences for each of the families;
(e) aligning the one or more consensus sequences to a reference sequence; and
(f) identifying a consensus sequence as a rare variant if the consensus sequence differs from the reference sequence at one or more nucleotide positions.
32. The method of claim 27, wherein at least some of the dsDNA molecules are derived from a tumor or circulating neoplastic cells.
33. A method for reducing an error rate in sequencing reads, comprising:
(a) preparing a sequencing library, wherein preparing a sequencing library comprises—
providing a set of hairpin adapters having a double-stranded region and a linker region, wherein at least a portion of the hairpin adapters comprise a single molecule identifier (SMI) sequence;
ligating the hairpin adapters to a plurality of double-stranded target DNA molecules to generate a sequencing library comprising a plurality of adapter-target DNA complexes; and
transitioning the adapter-target DNA complexes from a double-stranded form to a single-stranded form, wherein each single-stranded adapter-target DNA complex comprises at least a first strand of the target DNA molecule and a second strand of the same target DNA molecule separated by an adapter sequence;
(b) sequencing at least a portion of the single-stranded adapter-target DNA complexes in the sequencing library to obtain a plurality of sequence reads;
(c) grouping the plurality of sequence reads into a plurality of families based on the SMIs; and
(d) comparing the sequence reads within each family to generate a consensus sequence for each of the families, wherein—
each nucleotide base is identified at a given position in the consensus sequence when a specific nucleotide is complementary between at least one sequence read of the first strand and at least one sequence read of the second strand of the same target DNA molecule, and
identifying nucleotide positions where the compared sequence read of the first strand and the sequence read of the second strand are non-complementary and scoring the identified non-complimentary nucleotide positions as potential artifacts.
36. A method for detecting one or more rare variants in a heterogenous biological sample, the method comprising:
(a) preparing a sequencing library comprising a plurality of double-stranded DNA (dsDNA) molecules, wherein preparing the sequencing library comprises—
providing a set of hairpin adapters having a double-stranded region and a linker region, wherein at least a portion of an adapter sequence comprises a single molecule identifier (SMI) sequence;
ligating the hairpin adapters to the plurality of dsDNA molecules to generate a plurality of adapter-DNA complexes; and
transitioning the adapter-DNA complexes from a double-stranded form to a single-stranded form, wherein each single-stranded adapter-target DNA complex comprises at least a first strand of a parent dsDNA molecule and a second strand of the parent dsDNA molecule separated by the adapter sequence;
(b) sequencing at least a portion of the sequencing library to obtain a plurality of sequencing reads;
(d) grouping the plurality of sequencing reads into a plurality of families based on the SM Is, wherein each of the families comprise a first set of first strand sequence reads, each having a first SMI, and a second set of second strand sequencing reads, each having a second SMI, wherein the first SMI is relatable to the second SMI;
(e) comparing at least one first strand sequence read from the first set with at least one second strand sequence read from the second set within one or more families to generate consensus sequences, each consensus sequence representing a parent dsDNA molecule, wherein nucleotide positions where the compared first strand sequence read and the second strand sequence read are non-complementary are scored as potential artifacts;
(f) aligning one or more consensus sequences to a reference sequence; and
(g) identifying a rare variant in a consensus sequence if the consensus sequence differs from the reference sequence at one or more nucleotide positions.
Thus, the conflicting claims anticipate the instant claims because the conflicting claims teach the same double-stranded adaptors ligated to circulating DNA, the same identifier sequence-end sequence tags, and the same error-correction based on bi-directional/paired-end sequencing.
Instant claims 21-53 are rejected on the ground of nonstatutory obviousness-type double patenting as being unpatentable over conflicting claims 1-34 of US 12258629.
The instant claims are obvious over the conflicting claims because the conflicting claims anticipate the instant claims. More specifically, the conflicting claims teach:
1. A method of sequencing DNA comprising:
preparing a sequencing library from a sample comprising a plurality of double-stranded DNA fragments, wherein preparing the sequence library comprises ligating adapter molecules to the plurality of double-stranded DNA fragments to generate a plurality of double-stranded adapter-DNA molecules;
sequencing first and second strands of the adapter-DNA molecules to provide a first strand sequence read derived from an original first strand and a distinct, yet related second strand sequence read derived from an original second strand for individual adapter-DNA molecules in the plurality;
for at least some of the adapter-DNA molecules:
comparing the first strand sequence read with the distinct, yet related second strand sequence read; and
generating a consensus sequence of the double-stranded DNA fragment wherein each position in the consensus sequence is identified as correct if the particular position in the first strand sequence read and the particular position in the distinct, yet related second strand sequence read is in agreement.
14. The method of claim 1, wherein the plurality of double-stranded DNA fragments are circulating nucleic acid molecules.
15. The method of claim 14, wherein the circulating nucleic acid molecules comprise nucleic acid-based biomarkers from serum or plasma.
18. A method of sequencing DNA comprising:
ligating adapter molecules comprising a region of non-complementarity to a plurality of target double-stranded DNA fragments to generate a plurality of double-stranded adapter-DNA molecules;
amplifying an original first strand and an original second strand of at least some of the double-stranded adapter-DNA molecules, resulting in a set of copies of the original first strand and a set of copies of the original second strand;
for the at least some of the adapter-DNA molecules:
sequencing the set of copies of the original first strand and the set of copies of the original second strand to generate a set of first strand sequence reads and a set of second strand sequence reads that are distinguishable from the set of first strand sequence reads by the region of non-complementarity; and
confirming the presence of at least one sequence read derived from each of the original first strand and the original second strand of the adapter-DNA molecule;
comparing the first strand sequence read with the second strand sequence read; and
generating a consensus sequence of the target double-stranded DNA fragment wherein each position in the consensus sequence is identified as correct if the particular position in the first strand sequence read and the second strand sequence read is in agreement.
26. The method of claim 24, wherein the plurality of target double-stranded DNA fragments are circulating nucleic acid molecules.
Thus, the conflicting claims anticipate the instant claims because the conflicting claims teach the same double-stranded adaptors ligated to circulating DNA, the same identifier sequence-end sequence tags, and the same error-correction based on bi-directional/paired-end sequencing.
Conclusion
No claims are allowed.
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/AARON A PRIEST/Primary Examiner, Art Unit 1681
1 ctDNA is used as an example of cfDNA in order to demonstrate the point that not Applicants fail not only to enable any species of cfDNA, and their application to the claimed duplex sequencing, even more Applicants fail to enable the full scope of cfDNA. The specification simply never contemplates any cfDNA species, or their enablement.