DETAILED ACTION
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on Oct 31 2025 has been entered.
Applicant’s response, filed Oct 31 2025, has been fully considered. Rejections and/or objections not reiterated from previous Office Actions are hereby withdrawn. The following rejections and/or objections are either reiterated or newly applied. They constitute the complete set presently being applied to the instant application.
This application has been transferred to a different examiner in AU 1685.
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 .
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
Claim Status
Claims 63-76 and 83-85 are pending.
Claims 1-62 and 77-82 are canceled.
Claims 83-85 are newly added.
Claims 63, 67,70, 72, 83, and 85 are objected to.
Claims 63-76 and 83-85 are rejected.
Priority
The instant Application claims domestic benefit to US provisional application 62/724,622, filed Aug 30 2018.
However, the provisional application does not provide support for
Claim Objections
The claims are objected to because of the following informalities. The instant objection is newly stated and is necessitated by claim amendment.
Claim 63 recites substeps of steps (a), (c), and (d) as, for example, both “i)” and “(i)”. It is recommended to amend the claim to recite either one of the formats consistently.
In claim 63, limitation (a) i), line 5, the term “molecule barcodes” should be amended to recite “molecular barcodes” to maintain consistent claim language.
An “and” should be added to the end of limitation (a) i) in claim 63.
Claim 63, limitation (c) (i), third and second to last line, should be amended to recite “a cell-free nucleic acid molecule of the cell-free nucleic acid molecules”. The other recitations of “a cell-free nucleic acid molecule” in claim 63, step (d), should also be similarly amended. Claims 70 and 85 are similarly objected to.
Claim 67, limitation (c), should be amended to recite “quantifying a number”.
The commas after “processing” and “comprises” in claim 72 should be deleted.
The comma after “computer system” in claim 83 should be deleted.
Claim Rejections- 35 USC § 112
35 USC § 112(a)
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—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 following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
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 63-76 and 83-85 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains 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, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention. The instant rejection is newly stated and is necessitated by claim amendment.
Claim 63 recites “aligning comprising: (i) generating a plurality of Compact Idiosyncratic Gapped Alignment Report (CIGAR) strings from objects stored in a first memory location in a computer system, wherein each CIGAR string of the plurality of CIGAR strings represents at least one or more alternative sequences per position at one or more positions, whereby a genomic start position and a genomic stop position of a cell-free nucleic acid molecule is determined from at least one CIGAR string of the alignment; (iii) assigning at least one barcode from the set of barcodes to an object determinant of the genomic start position and a genomic stop position”. Claim 83 recites “wherein the objects stored in a first memory location in a computer system, each comprise a plurality of nodes and edges, and at least one pointer specifying one or more additional memory locations where one or more adjacent objects are stored”. Claim 84 recites “storing one or more objects determinant of the genomic start position and a genomic stop position at a further additional memory location”. Claim 85 recites “parsing a CIGAR string to identify an object in the first memory location”. The specification as published provides support for: using a CIGAR string to produce a sequence alignment [0169-0170]; a computer system which includes memory and a peripheral device such as other memory [0180]. However, there is not support within the specification, nor has Applicant provided such support, specifically for storing the sequencing reads and the sequences of the molecular barcodes in separate memory as in claim 63; for objects stored in a first memory location in a computer system, each comprise a plurality of nodes and edges, and at least one pointer specifying one or more additional memory locations where one or more adjacent objects are stored as in claim 83; for storing one or more objects determinant of the genomic start position and a genomic stop position at a further additional memory location as in claim 84; or for parsing a CIGAR string to identify an object in the first memory location as in claim 85. Therefore, the limitations introduces new matter. Claims 64-76 are rejected based on their dependence from claim 63.
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.
Claims 63-76 and 83-85 are rejected under 35 U.S.C. 112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor, regards as the invention. The instant rejection is newly stated and is either necessitated by claim amendment or based on further consideration of the claims.
Claim 63, limitation (c), recites “(ii) retrieving the set of molecular barcodes from a second memory location”. However, the claim previously recites in (a) “i) tagging a set of cell-free nucleic acid molecules in each sample with a set of molecular barcodes to generate tagged polynucleotides”, followed by amplifying and sequencing operations. The set of molecular barcodes are therefore interpreted as physical nucleic acid molecules appended to the cell-free nucleic acid molecules in the sample, and cannot be stored or retrieved from a memory location in a computer system. Therefore, the metes and bounds of the limitation are not clear. It is recommended to amend the claim to recite “(ii) retrieving sequences of the set of molecular barcodes from a second memory location”. Claims 64-76 and 83-85 are rejected based on their dependency from claim 63.
Claim 75 recites “wherein the first sample is obtained from a bodily fluid of a subject and the second sample is obtained from the bodily fluid of another subject”. It is unclear whether the wherein clause is intended to require obtaining the first and second samples within the metes and bounds of the claimed invention, or if it is only further limiting the type of samples used in the invention such that obtaining the samples is not required within the metes and bounds of the invention. As set forth in MPEP 2111.04.I, “wherein” clauses raise the question as to the limiting effect of the language in a claim. As the claims do not previously recite an active step of obtaining the samples, the metes and bounds of the claims are unclear. For compact examination, it is assumed that obtaining the samples is not required to be performed. The rejection may be overcome by clarifying what steps are required to be performed. Claim 76 is rejected based on its dependency from claim 75. The rejection is newly stated based upon further consideration of the claims.
Claims 83-85 depend from cancelled claim 1. It is not clear what claim claims 83-85 are intended to depend from. For compact examination, it is assumed that the claims should depend from claim 63. The rejection may be overcome by clarifying the dependency of the claims.
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claims 63-76 and 83-85 are rejected under 35 U.S.C. 101 because the claimed invention is directed to one or more judicial exceptions without significantly more. Any newly recited portions are necessitated by claim amendment.
MPEP 2106 organizes judicial exception analysis into Steps 1, 2A (Prongs One and Two) and 2B as follows below. MPEP 2106 and the following USPTO website provide further explanation and case law citations: uspto.gov/patent/laws-and-regulations/examination-policy/examination-guidance-and-training-materials.
Framework with which to Evaluate Subject Matter Eligibility:
Step 1: Are the claims directed to a process, machine, manufacture, or composition of matter;
Step 2A, Prong One: Do the claims recite a judicially recognized exception, i.e. a law of nature, a natural phenomenon, or an abstract idea;
Step 2A, Prong Two: If the claims recite a judicial exception under Prong One, then is the judicial exception integrated into a practical application (Prong Two); and
Step 2B: If the claims do not integrate the judicial exception, do the claims provide an inventive concept.
Framework Analysis as Pertains to the Instant Claims:
Step 1
With respect to Step 1: yes, the claims are directed to a method, i.e., a process, machine, or manufacture within the above 101 categories [Step 1: YES; See MPEP § 2106.03].
Step 2A, Prong One
With respect to Step 2A, Prong One, the claims recite judicial exceptions in the form of abstract ideas. The MPEP at 2106.04(a)(2) further explains that abstract ideas are defined as:
mathematical concepts (mathematical formulas or equations, mathematical relationships and mathematical calculations);
certain methods of organizing human activity (fundamental economic practices or principles, managing personal behavior or relationships or interactions between people); and/or
mental processes (procedures for observing, evaluating, analyzing/ judging and organizing information).
With respect to the instant claims, under the Step 2A, Prong One evaluation, the claims are found to recite abstract ideas that fall into the grouping of mental processes (in particular procedures for observing, analyzing and organizing information) and mathematical concepts (in particular mathematical relationships and formulas) are as follows:
Independent claim 63: (c) aligning a plurality of sequencing reads from the first sample and the second sample to a reference sequence, aligning comprising: (i) generating a plurality of Compact Idiosyncratic Gapped Alignment Report (CIGAR) strings from objects stored in a first memory location in a computer system, wherein each CIGAR string of the plurality of CIGAR strings represents at least one or more alternative sequences per position at one or more positions, whereby a genomic start position and a genomic stop position of a cell-free nucleic acid molecule is determined from at least one CIGAR string of the alignment; (iii) assigning at least one barcode from the set of barcodes to an object determinant of the genomic start position and a genomic stop position;
(d) for each of the first sample and the second sample, grouping the plurality of sequencing reads into a plurality of families based on: (i) start genomic position and (ii) stop genomic position of the cell-free nucleic acid molecule,
identifying one or more single original cell-free nucleic acid molecules by decoding the encoded sequence reads each based on the assignment of the at least one barcode from the set of barcodes to an object, wherein each family in the sample comprises sequencing reads of progeny polynucleotides amplified from a single original cell-free nucleic acid molecule identified among the set of cell-free nucleic acid molecules in the sample;
(e) generating family identifiers for the plurality of families;
(f) screening for a set of shared family identifiers, wherein a given shared family identifier is a family identifier of the first sample that is identical or substantially identical to a family identifier of the second sample;
(g) determining a quantitative measure of the set of shared family identifiers; and
(h) classifying the first sample as being contaminated with the second sample if the quantitative measure of the set of shared family identifiers is above a predetermined threshold, or as not being contaminated with the second sample if the quantitative measure of the set of shared family identifiers is at or below the predetermined threshold, thereby detecting the presence or absence of contamination.
Dependent claims 64-67, 69, 74, 83, and 85 recite further steps that limit the judicial exceptions in independent claim 63 and, as such, also are directed to those abstract ideas. For example, claims 64-67 and 69 further limit the quantitative measure of the set of shared family identifiers of step (g) in claim 63; claim 74 further limits the predetermined threshold of step (h) in claim 63; claim 83 further limits the stored objects to comprising nodes, edges, and pointers; claim 85 further limits determining a genomic start position and a genomic stop position of a cell-free nucleic acid molecule.
The abstract ideas recited in the claims are evaluated under the Broadest Reasonable Interpretation (BRI) and determined to each cover performance either in the mind and/or by mathematical operation because the method only requires a user to manually classify a sample as being contaminated with another sample based on the presence of shared families of molecular barcode identifiers. Without further detail as to the methodology involved in “aligning”, “generating”, “determining”, “assigning”, “grouping”, “identifying”, “screening”, and “classifying”, under the BRI, one may simply, for example, use pen and paper to align sequence reads to a reference genome by generating CIGAR strings to determine genomic start and stop positions of the nucleic acid molecules, assign barcodes to the genomic start and stop positions, group the sequencing reads into families based on the genomic start and stop positions, identify original nucleic acid molecules based on the assigned barcodes, generate family identifiers, screen for shared family identifiers between samples, determine a quantitative measure of the shared family identifiers, and classify the sample as being contaminated based on the quantitative measure of the shared family identifiers being above or below a threshold. “Aligning” is considered to recite a mental process in part because the claims require amplifying and sequencing only a portion of the tagged polynucleotides, and aligning a plurality of sequencing reads which are not limited. The specification as published discloses an example of generating a CIGAR string for an alignment between two sequences which could be performed mentally at [0170]. At least the step of classifying whether a sample is contaminated requires mathematical techniques as the only supported embodiments because it requires comparing the quantitative measure to a predetermined threshold, where the comparison is a mathematical concept.
Therefore, claim 63 and those claims dependent therefrom recite an abstract idea [Step 2A, Prong 1: YES; See MPEP § 2106.04].
Step 2A, Prong Two
Because the claims do recite judicial exceptions, direction under Step 2A, Prong Two, provides that the claims must be examined further to determine whether they integrate the judicial exceptions into a practical application (MPEP 2106.04(d)). A claim can be said to integrate a judicial exception into a practical application when it applies, relies on, or uses the judicial exception in a manner that imposes a meaningful limit on the judicial exception. This is performed by analyzing the additional elements of the claim to determine if the judicial exceptions are integrated into a practical application (MPEP 2106.04(d).I.; MPEP 2106.05(a-h)). If the claim contains no additional elements beyond the judicial exceptions, the claim is said to fail to integrate the judicial exceptions into a practical application (MPEP 2106.04(d).III).
Additional elements, Step 2A, Prong Two
With respect to the instant recitations, the claims recite the following additional elements:
Independent claim 63: (a) processing the first sample and the second sample, wherein the processing comprises: i) tagging a set of cell-free nucleic acid molecules in each sample with a set of molecular barcodes to generate tagged polynucleotides, wherein the set of molecular barcodes comprise 5-200 different molecular barcode sequences to identify a particular polynucleotide and, wherein the molecule barcodes are configured to generate an at least bi-partite encoded sequence read of a single original cell-free nucleic acid molecules; ii) amplifying a portion of the tagged polynucleotides to generate progeny polynucleotides;
(b) for each of the first sample and the second sample, sequencing a portion of the progeny polynucleotides to generate sequencing reads;
(ii) retrieving the set of molecular barcodes from a second memory location.
Dependent claim 72: enriching a portion of the progeny polynucleotides for specific regions of interest to generate enriched molecules.
Dependent claim 84: storing one or more objects determinant of the genomic start position and a genomic stop position at a further additional memory location.
Dependent claims 68, 70-71, 73, and 75-76 recite steps that further limit the recited additional elements in the claims. For example, claims 68 and 71 further limits the sample to being processed or sequenced in a same flow cell; claims 70 and 73 further limit the attachment of the molecular barcodes to the cell-free nucleic acid molecule; and claims 75-76 further limit the first and second sample to being from body fluid or plasma from separate subjects.
The claims also include non-abstract computing elements. For example, independent claim 63 includes a first and second memory location in a computer system.
Considerations under Step 2A, Prong Two
With respect to Step 2A, Prong Two, the additional elements of the claims do not integrate the judicial exceptions into a practical application for the following reasons. Those steps directed to data gathering, such as “processing” the samples by “tagging” the cell-free nucleic acid molecules in each sample with a set of molecular barcodes and amplifying the tagged polynucleotides, “enriching” and “sequencing” the progeny polynucleotides, and “retrieving” data from a computer memory, and to data outputting, such as “storing” data in a computer memory, perform functions of collecting and outputting the data needed to carry out the judicial exceptions. Data gathering and outputting do not impose any meaningful limitation on the judicial exceptions, or on how the judicial exceptions are performed. Data gathering and outputting steps are not sufficient to integrate judicial exceptions into a practical application (MPEP 2106.05(g)).
Further steps directed to additional non-abstract computer elements do not describe any specific computational steps by which the “computer parts” perform or carry out the judicial exceptions, nor do they provide any details of how specific structures of the computer, such as the computer-readable recording media, are used to implement these functions. The claims state nothing more than a generic computer which performs the functions that constitute the judicial exceptions. Hence, these are mere instructions to apply the judicial exceptions using a computer, and therefore the claim does not integrate that judicial exceptions into a practical application. The courts have weighed in and consistently maintained that when, for example, a memory, display, processor, machine, etc.… are recited so generically (i.e., no details are provided) that they represent no more than mere instructions to apply the judicial exception on a computer, and these limitations may be viewed as nothing more than generally linking the use of the judicial exception to the technological environment of a computer (MPEP 2106.05(f)).
The specification as published discloses that the method is useful for detecting contamination in sample that detect low frequency sequences and are susceptible to sample contamination at [0003-0004], but does not provide a clear explanation for how the additional elements provide these improvements. Therefore, the additional elements do not clearly improve the functioning of a computer, or comprise an improvement to any other technical field. Further, the additional elements do not clearly affect a particular treatment; they do not clearly require or set forth a particular machine; they do not clearly effect a transformation of matter; nor do they clearly provide a nonconventional or unconventional step (MPEP2106.04(d)).
Thus, none of the claims recite additional elements which would integrate a judicial exception into a practical application, and the claims are directed to one or more judicial exceptions [Step 2A, Prong 2: NO; See MPEP § 2106.04(d)].
Step 2B (MPEP 2106.05.A i-vi)
According to analysis so far, the additional elements described above do not provide significantly more than the judicial exception. A determination of whether additional elements provide significantly more also rests on whether the additional elements or a combination of elements represents other than what is well-understood, routine, and conventional. Conventionality is a question of fact and may be evidenced as: a citation to an express statement in the specification or to a statement made by an applicant during prosecution that demonstrates a well-understood, routine or conventional nature of the additional element(s); a citation to one or more of the court decisions as discussed in MPEP 2106(d)(II) as noting the well-understood, routine, conventional nature of the additional element(s); a citation to a publication that demonstrates the well-understood, routine, conventional nature of the additional element(s); and/or a statement that the examiner is taking official notice with respect to the well-understood, routine, conventional nature of the additional element(s).
With respect to the instant claims, the courts have recognized analyzing DNA to provide sequence information and sequencing nucleic acid sequences as well-understood, routine, conventional activity in the life science arts when they are claimed in a merely generic manner [see MPEP§2106.05(d)(II)], i.e., “sequencing" refer to any of a number of technologies used to determine the sequence of a biomolecule, e.g., a nucleic acid such as DNA or RNA. The prior art review to Volik et al. (Molecular Cancer Research, 2016, 14(10):898-908; newly cited) discloses that barcoding, enriching, and sequencing cell-free DNA from plasma is a data gathering element that is routine, well-understood and conventional in the art. Said portions of the prior art are, for example, p. 902, col. 2, par. 2; p. 903, col. 1, par. 2 through p. 906, col. 1, par. 2; entire document is relevant. Further, the courts have found that receiving, storing, and outputting data are well-understood, routine, and conventional functions of a computer when claimed in a merely generic manner or as insignificant extra-solution activity (see Symantec, 838 F.3d at 1321, 120 USPQ2d at 1362 (utilizing an intermediary computer to forward information), buySAFE, Inc. v. Google, Inc., 765 F.3d 1350, 1355, 112 USPQ2d 1093, 1096 (Fed. Cir. 2014) (computer receives and sends information over a network), Versata Dev. Group, Inc. v. SAP Am., Inc., 793 F.3d 1306, 1334, 115 USPQ2d 1681, 1701 (Fed. Cir. 2015), and OIP Techs., 788 F.3d at 1363, 115 USPQ2d at 1092-93, as discussed in MPEP 2106.05(d)(II)(i)). As such, the claims simply append well-understood, routine, conventional activities previously known to the industry, specified at a high level of generality, to the judicial exception (MPEP2106.05(d)). The data gathering steps as recited in the instant claims constitute a general link to a technological environment which is insufficient to constitute an inventive concept which would render the claims significantly more than the judicial exception (MPEP2106.05(g)&(h)).
With respect to claims 63 and those claims dependent therefrom, the computer-related elements or the general purpose computer do not rise to the level of significantly more than the judicial exception. The claims state nothing more than a generic computer which performs the functions that constitute the judicial exceptions. Hence, these are mere instructions to apply the judicial exceptions using a computer, which the courts have found to not provide significantly more when recited in a claim with a judicial exception (Alice Corp., 573 U.S. at 225-26, 110 USPQ2d at 1984; see MPEP 2106.05(A)). The specification also notes that computer processors and systems, as example, are commercially available or widely used at [0179-0189]. The additional elements are set forth at such a high level of generality that they can be met by a general purpose computer. Therefore, the computer components constitute no more than a general link to a technological environment, which is insufficient to constitute an inventive concept that would render the claims significantly more than the judicial exceptions (see MPEP 2106.05(b)I-III).
Taken alone, the additional elements do not amount to significantly more than the above-identified judicial exception(s). Even when viewed as a combination, the additional elements fail to transform the exception into a patent-eligible application of that exception. Thus, the claims as a whole do not amount to significantly more than the exception itself [Step 2B: NO; See MPEP § 2106.05].
Therefore, the instant claims are not drawn to eligible subject matter as they are directed to one or more judicial exceptions without significantly more. For additional guidance, applicant is directed generally to the MPEP § 2106.
Response to Applicant Arguments
At p. 7-10, Applicant submits that the use of CIGAR string alignment provides a computational advantage by invoking positions of differences to identify related sequence reads without requiring object reference and processing of the larger nucleotide sequences. Applicant submits that CIGAR strings are generated from objects in a first memory location that encapsulate sequence read information, and that barcodes are stored in a second memory location, and that upon alignment the CIGAR strings identify positioning using a physical memory addressing scheme, which provides virtually instant identification of relevant sequence reads, thereby eliminating computation inefficiency through a filtering process
It is respectfully submitted that this is not persuasive. Applicant alleges that CIGAR string alignment represents a computation advantage. However, steps directed to CIGAR aligning that provide the supposed improvement in the instant claims are steps that are, themselves, the judicial exceptions and cannot therefore be a practical application of the judicial exception. The courts have made clear that a judicial exception is not eligible subject matter (Bilski, 561 U.S. at 601, 95 USPQ2d at 1005-06 (quoting Chakrabarty, 447 U.S. at 309, 206 USPQ at 197 (1980)) if there are no additional claim elements besides the judicial exception, or if the additional claim elements merely recite another judicial exception that is insufficient to integrate the judicial exception into a practical application. See, e.g., RecogniCorp, LLC v. Nintendo Co., 855 F.3d 1322, 1327, 122 USPQ2d 1377 (Fed. Cir. 2017) ("Adding one abstract idea (math) to another abstract idea (encoding and decoding) does not render the claim non-abstract"); Genetic Techs. v. Merial LLC, 818 F.3d 1369, 1376, 118 USPQ2d 1541, 1546 (Fed. Cir. 2016) (eligibility "cannot be furnished by the unpatentable law of nature (or natural phenomenon or abstract idea) itself."). For a claim reciting a judicial exception to be eligible, it is the additional elements (if any) in the claim that must "transform the nature of the claim" into a patent-eligible application of the judicial exception, Alice Corp., 573 U.S. at 217, 110 USPQ2d at 1981, either at Prong Two or in Step 2B. If there are no additional elements in the claim, then it cannot be eligible. It is submitted here that the instant claims do not include any additional elements that provide for a practical application. Rather, the “additional element” in the instant claims (see exemplary claim 63) includes only the step of “processing the samples” by tagging and amplifying and “sequencing”. As set forth above, said steps operate in the claim as data gathering steps and do not integrate any of the recited judicial exceptions into a practical application, nor do the claims as a whole include any inventive concept beyond well-understood, routine and conventional steps.
Further, the physical memory addressing scheme and associated improvements as argued by Applicant are not convincing because Applicant has not demonstrated how storing the read data and the barcode data in separate memories provides any improvement to the functioning of a computer. Applicant’s arguments are that the CIGAR alignment first locates the position of the read to the reference genome, and then the barcodes are assigned to located reads. Applicant’s arguments regarding the CIGAR alignment eliminating the need to align the entire read are not commensurate with the scope of the claims because the claims require only generating a plurality of CIGAR strings from objects stored in a first memory. The objects are not related to the sequence reads, and the claims do not require only identifying a shared alignment of the CIGAR strings. Further, it is not apparent how the location of the stored data is required for such an action or contributes to an improvement in the functioning of the computer during the performance. Applicant’s argument regarding “filtering” to only aligned reads does not provide an improvement to the functioning of a computer because the filtering as set forth by Applicant actually only demonstrates that the computer is operating on less data, which is not am improvement because the functioning of the computer has remained unchanged.
Applicant’s arguments regarding claims 83-85 are similarly unconvincing, for the reasons discussed above. Claim 83 merely recites that the objects stored in the memory comprise nodes and edges and pointers to the storage of adjacent objects. Claim 84 merely recites storing the genomic start and stop positions at a further memory location, which amounts to data outputting. Claim 85 merely recites that the CIGAR string is parsed to identify a stored object. Claims 83-85 do not recite how those nodes, edges, pointers, or otherwise stored data are used in the claims to provide any sort of advantage as argued by Applicant, and Applicant has not provided any arguments for how storing data in and accessing data from different locations (i.e., a spatial memory addressing) provides an improvement.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
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.
A. Claims 63-76 and 84-85 are rejected under 35 U.S.C. 103 as being unpatentable over Christians et al., (US 20060073506 published April 2006; previously cited) in view of Talasaz et al., (US20160251704 published Sep 2016; previously cited) and Shendure et al. (US 2016/0055293; newly cited). The instant rejection is newly stated and is necessitated by claim amendment.
The claims are drawn to a method for detecting the presence or absence of contamination of a first sample with a second sample among a plurality of samples, comprising: (a) processing the first sample and the second sample, wherein the processing comprises: i) tagging a set of cell-free nucleic acid molecules in each sample with a set of molecular barcodes to generate tagged polynucleotides, wherein the set of molecular barcodes comprise 5-200 different molecular barcode sequences to identify a particular polynucleotide and wherein the molecule barcodes are configured to generate an at least bipartite encoded sequence read of a single original cell-free nucleic acid molecules; ii) amplifying a portion of the tagged polynucleotides to generate progeny polynucleotides;
(b) for each of the first sample and the second sample, sequencing a portion of the progeny polynucleotides to generate sequencing reads;
(c) aligning a plurality of sequencing reads from the first sample and the second sample to a reference sequence, aligning comprising: (i) generating a plurality of Compact Idiosyncratic Gapped Alignment Report (CIGAR) strings from objects stored in a first memory location in a computer system, wherein each CIGAR string of the plurality of CIGAR strings represents at least one or more alternative sequences per position at one or more positions, whereby a genomic start position and a genomic stop position of a cell-free nucleic acid molecule is determined from at least one CIGAR string of the alignment; (ii) retrieving the set of molecular barcodes from a second memory location; (iii) assigning at least one barcode from the set of barcodes to an object determinant of the genomic start position and a genomic stop position;
(d) for each of the first sample and the second sample, grouping the plurality of sequencing reads into a plurality of families based on: (i) start genomic position and (ii) stop genomic position of the cell-free nucleic acid molecule, identifying one or more single original cell free nucleic molecules by decoding the encoded sequence reads each based on the assignment of the at least one barcode from the set of barcodes to an object,, wherein each family in the sample comprises sequencing reads of progeny polynucleotides amplified from a unique cell-free nucleic acid molecule identified among the set of cell-free nucleic acid molecules in the sample;
(e) generating family identifiers for the plurality of families;
(f) screening for a set of shared family identifiers, wherein a given shared family identifier is a family identifier of the first sample that is identical or substantially identical to a family identifier of the second sample;
(g) determining a quantitative measure of the set of shared family identifiers; and
(h) classifying the first sample as being contaminated with the second sample if the quantitative measure of the set of shared family identifiers is above a predetermined threshold, or as not being contaminated with the second sample if the quantitative measure of the set of shared family identifiers is at or below the predetermined threshold, thereby detecting the presence or absence of contamination.
Christians et al., teach a method of detecting contamination of a first sample with a second sample, wherein the first sample is marked with a first barcode and the second sample is marked with a second barcode, wherein a barcode comprises a known combination of tag sequences and said first barcode and said second barcode are different, comprising fragmenting, ligating, amplifying, generating, analyzing and determining that said first sample is contaminated with said second sample if the barcode of the second sample is detected in the first sample [claim 23]. Many methods of genetic analysis require analysis of large numbers of different samples. Each sample may be derived from a different individual or a different source and keeping track of sample identity is frequently essential to analysis of results. Samples may become contaminated by other samples, the identity of a sample may be lost or a sample may become misidentified [para 60]. Teaching claim 75.
Christians et al., teach methods of identifying biological samples. Probes were chosen to flank the central position of the 40 bp sequence at regular intervals; some of the barcodes have only antisense probes, and some have sense and antisense [page 87]. The barcode adaptor preferably has at least one single stranded [para 37]. The two strands of a double stranded DNA molecule or between an oligonucleotide primer and a primer binding site on a single stranded nucleic acid to be sequenced or amplified. Two single stranded RNA or DNA molecules are said to be complementary when the nucleotides of one strand, optimally aligned and compared and with appropriate nucleotide insertions or deletions [para 42].
Christians et al., teach Molecular Barcodes for Internally Marking and Tracking Samples. Each sample may be derived from a different individual or a different source and keeping track of sample identity is frequently essential to analysis of results. Samples may become contaminated by other samples, the identity of a sample may be lost or a sample may become misidentified. Methods of integrally marking a sample in a detectable manner are disclosed [para 60]. It is important that the sample be properly identified as to source of origin and that during subsequent manipulation steps the original sample can be correctly identified. Marking the container in which a sample is placed has been one method of marking but sample mix ups, such as errors in labeling and cross-contamination between samples do occur and can be difficult to detect without a marking method that is integrated within the sample itself. [para 61]. The presently disclosed methods provide mechanisms to mark a sample so that sample identities can be checked to prevent misidentification of a sample and to detect cross contamination between samples [para 61]. Thus, if the barcodes detected on the array differ from the expected barcodes, the researcher could use a small aliquot of the original archived, unamplified, barcoded sample [para 89]. Thus Christians et al., teach permitting subsequent identification of a particular polynucleotide and wherein the molecule barcodes track back to single original nucleic acid molecules as recited by instant claim 63.
Each sample may be marked with a different combination of two or more marker molecules each carrying a different tag nucleic acid sequences. The tag nucleic acid sequences may be random sequences that are not naturally occurring in the nucleic acid sample and do not cross hybridize to sequences naturally occurring in the nucleic acid sample [para 35]. The marker molecules can be added individually to the sample or they may be added in combinations of two or more [para 34]. The barcode construct has restriction sites arranged so that when the sample is cleaved with the selected restriction enzyme the barcode region will be within a fragment that is between 400 and 800 base pairs, the adaptors will ligate to the ends of the barcode fragment and the fragment will be amplified during PCR with the universal primer. The barcode fragments will be labeled during the labeling reaction and the array comprises probes to detect the amplified barcode region [para 75]. Thereby teaching claim 70. As a non-limiting example, a complex population of nucleic acids may be total genomic DNA, total genomic RNA or a combination thereof. Moreover, a complex population of nucleic acids may have been enriched for a given population but include other undesirable populations. For example, a complex population of nucleic acids may be a sample which has been enriched for desired messenger RNA (mRNA) sequences but still includes some undesired ribosomal RNA sequences (rRNA) [para 51]. Teaching claim 72.
Hybridization results for the barcodes have been measured by two methods, both developed based upon calculation of the median intensity of the perfect match probes. First, the GDAS (GeneChip DNA Analysis Software, available from Affymetrix, Inc.) report can be configured to show presence/absence of the barcode based upon intensities above a certain threshold. A safe threshold would be 5000 PM median intensity, which would allow a correct present/absent call in every experiment done to date. The advantage of this GDAS threshold report method is that it is convenient for the user; however, it gives a present/absent answer and does not readily allow for the detection of trace cross-contamination. A second output method is to report the actual PM median intensity. This can be done, for example using a special file in the GDAS folder. The advantage to this second method is that it allows the user more control over the barcode results, including the ability to detect cross-contamination from one sample to another [para 88]. Teaching claim 73. In preferred aspects the methods are capable of detecting cross contamination at very low levels of contamination, for example, 0.4 to 2%, 2-5%, or 5-10% contamination. For example, if a contaminated sample is 95% a first sample and 5% a second sample the first sample is contaminated by the second at 5%. Higher levels of contamination, greater than 10% may also be detected. The two methods may be combined by having the computer system report both the actual barcode intensities as well as a present/absent call, based upon user-tunable thresholds [para 88]. Teaching claims 69 and 74. Christians et al., teach a method for detecting the presence or absence of contamination of a first sample with a second sample among a plurality of samples, comprising: (a) processing the first sample and the second sample, wherein the processing comprises: i) tagging a set of nucleic acid molecules in each sample with a set of molecular barcodes to generate tagged polynucleotides, wherein the set of molecular barcodes comprise 5-200 different molecular barcode sequences to permit subsequent identification of a particular polynucleotide and wherein the molecule barcodes track back to single original nucleic acid molecules; ii) amplifying a portion of the tagged polynucleotides to generate progeny polynucleotides; however Christians et al., do not teach a method for detection of a set of cell-free nucleic acid molecules along with the additional methods steps.
Talasaz et al., teach a system and method for the detection of rare mutations and copy number variations in cell free polynucleotides. Generally, the systems and methods comprise sample preparation, or the extraction and isolation of cell free polynucleotide sequences from a bodily fluid; subsequent sequencing of cell free polynucleotides by techniques known in the art; and application of bioinformatics tools to detect rare mutations and copy number variations as compared to a reference [abstract]. The disclosure provides for a method for detecting copy number variation comprising: a) sequencing extracellular polynucleotides from a bodily sample from a subject, wherein each of the extracellular polynucleotide are optionally attached to unique barcodes; b) filtering out reads that fail to meet a set threshold; c) mapping sequence reads obtained from step (a) to a reference sequence; d) quantifying/counting mapped reads in two or more predefined regions of the reference sequence; e) determining a copy number variation in one or more of the predefined regions by (i) normalizing the number of reads in the predefined regions to each other and/or the number of unique barcodes in the predefined regions to each other; and (ii) comparing the normalized numbers obtained in step (i) to normalized numbers obtained from a control sample [para 4]. 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 comprising: a) sequencing extracellular polynucleotides from a bodily sample from a subject, wherein each of the extracellular polynucleotide generate a plurality of sequencing reads; b) sequencing extracellular polynucleotides from a bodily sample from a subject, wherein each of the extracellular polynucleotide generate a plurality of sequencing reads; sequencing extracellular polynucleotides from a bodily sample from a subject, wherein each of the extracellular polynucleotide generate a plurality of sequencing reads; c) filtering out reads that fail to meet a set threshold; d) mapping sequence reads derived from the sequencing onto a reference sequence; e) identifying a subset of mapped sequence reads that align with a variant of the reference sequence at each mappable base position; f) for each mappable base position, calculating a ratio of (a) a number of mapped sequence reads that include a variant as compared to the reference sequence, to (b) a number of total sequence reads for each mappable base position; g) normalizing the ratios or frequency of variance for each mappable base position and determining potential rare variant(s) or mutation(s); h) and comparing the resulting number for each of the regions with potential rare variant(s) or mutation(s) to similarly derived numbers from a reference sample [para 5]. For example, some sequence reads may originate from contaminant polynucleotides. Sequencing reads with a mapping score at least 90%, 95%, 99%, 99.9%, 99.99% or 99.999% may be filtered out of the data set. In other cases, sequencing reads assigned a mapping scored less than 90%, 95%, 99%, 99.9%, 99.99% or 99.999% may be filtered out of the data set [para 271]. Teaching claims 63 and 65-67. Cell free sequences may be sequenced. Sequencing methods may include, but are not limited to: high-throughput sequencing, pyrosequencing, sequencing-by-synthesis, single-molecule sequencing, nanopore sequencing, semiconductor sequencing, sequencing-by-ligation, sequencing-by-hybridization, RNA-Seq (Illumina), Digital Gene Expression (Helicos), Next generation sequencing, Single Molecule Sequencing by Synthesis (SMSS)(Helicos), massively-parallel sequencing, Clonal Single Molecule Array (Solexa), shotgun sequencing, Maxim-Gilbert sequencing, primer walking, sequencing using PacBio, SOLiD, Ion Torrent, or Nanopore platforms and any other sequencing methods known in the art. In some cases, sequencing reactions various types, as described herein, may comprise a variety of sample processing units. Sample processing units may include but are not limited to multiple lanes, multiple channels, multiple wells, or other mean of processing multiple sample sets substantially simultaneously. Additionally, the sample processing unit may include multiple sample chambers to enable processing of multiple runs simultaneously [para 256-257]. Teaching claims 68 and 71.
The method of inferring frequency of sequence calls in a sample of polynucleotides comprising: a. providing at least one set of first polynucleotides, wherein each set maps to a different reference sequence in one or more genomes, and, for each set of first polynucleotides; i. amplifying the first polynucleotides to produce a set of amplified polynucleotides; ii. sequencing a subset of the set of amplified polynucleotides, to produce a set of sequencing reads; iii. grouping the sequence reads into families, each family comprising sequence reads of amplified polynucleotides amplified from the same first polynucleotide; b. inferring, for each set of first polynucleotides, a call frequency for one or more bases in the set of first polynucleotides, wherein inferring comprises: i. assigning, for each family, confidence score for each of a plurality of calls, the confidence score taking into consideration a frequency of the call among members of the family; and ii. estimating a frequency of the one or more calls taking into consideration the confidence scores of the one or more calls assigned to each family [para 69]. Teaching claim 65.
This disclosure also provides a computer readable medium in non-transitory, tangible form comprising executable code configured to perform the following steps: a. accessing a data file comprising a plurality of sequencing reads, wherein the sequence reads derive from a set of progeny polynucleotides amplified from at least one set of tagged parent polynucleotides; and i. collapsing the sequence reads by: 1. grouping sequences reads sequenced from amplified progeny polynucleotides into families, each family amplified from the same tagged parent polynucleotide and, optionally, 2. determining a quantitative measure of sequence reads in each family. In certain embodiments, the executable code further performs the steps of b. determining a quantitative measure of unique families; c. based on (1) the quantitative measure of unique families and (2) the quantitative measure of sequence reads in each group, inferring a measure of unique tagged parent polynucleotides in the set. In certain embodiments, the executable code further performs the steps of: d. determining a quantitative measure of polymorphic forms among the families; and e. based on the determined quantitative measure of polymorphic forms, inferring a quantitative measure of polymorphic forms in the number of inferred unique tagged parent polynucleotides [para 76]. Teaching claims 65-67.
Cell free polynucleotides may be derived from a variety of sources including human, mammal, non-human mammal sources. Further, samples may be extracted from variety of animal fluids containing cell free sequences, including but not limited to plasma [para 225]. Teaching claims 75-76. After isolation, in some cases, the cell free polynucleotides are pre-mixed with one or more additional materials, such as one or more reagents (e.g., ligase, protease, polymerase) prior to sequencing [para 230]. Similarly, all of the fragments from a single strand of nucleic acid may be tagged, thereby permitting subsequent identification of fragments from the parent strand. In other cases, gene expression products (e.g., mRNA) may be tagged in order to quantify expression, by which the barcode, or the barcode in combination with sequence to which it is attached can be counted [para 233]. Further, using unique sequence data at the beginning (start) and end (stop) portions of individual sequencing reads and sequencing read length may be used, alone or combination, with the use of barcodes [para 235]. The unique identifiers (e.g., oligonucleotide bar-codes, antibodies, probes, etc.) may be introduced to cell free polynucleotide sequences randomly or non-randomly. In some cases, they are introduced at an expected ratio of unique identifiers to microwells. For example, the unique identifiers may be loaded so that more than about 5, 6, 7, 8, 9, 10, 20, 50, 100, unique identifiers are loaded per genome sample [para 241]. Teaching claims 65-66. The unique identifiers may be used to tag a wide range of analytes, including but not limited to RNA or DNA molecules. For example, unique identifiers (e.g., barcode oligonucleotides) may be attached to whole strands of nucleic acids or to fragments of nucleic acids (e.g., fragmented genomic DNA, fragmented RNA). The unique identifiers (e.g., oligonucleotides) may also bind to gene expression products, genomic DNA, mitochondrial DNA, RNA, mRNA, and the like [para 244]. In some embodiments, random barcodes selected from the y different barcode oligonucleotides are ligated to both ends of parent polynucleotides. Random ligation of the y barcodes to one or both ends of the parent polynucleotides can result in production of unique identifiers. For example, a sample comprising about 10,000 haploid human genome equivalents of cfDNA can be tagged with about 36 unique identifiers. The unique identifiers can comprise six unique DNA barcodes. Ligation of 6 unique barcodes to both ends of a polynucleotide can result in 36 possible unique identifiers are produced para 253]. Teaching claims 72-73.
Further, the methods comprise attaching one or more barcodes to the extracellular polynucleotides or fragments thereof prior to any amplification or enrichment step [para 16]. This disclosure also provides for a method comprising: a. providing at least one set of tagged parent polynucleotides, and for each set of tagged parent polynucleotides; b. amplifying the tagged parent polynucleotides in the set to produce a corresponding set of amplified progeny polynucleotides; c. sequencing a subset (including a proper subset) of the set of amplified progeny polynucleotides, to produce a set of sequencing reads; and d. collapsing the set of sequencing reads to generate a set of consensus sequences, each consensus sequence corresponding to a unique polynucleotide among the set of tagged parent polynucleotides. In certain embodiments the method further comprises: e. analyzing the set of consensus sequences for each set of tagged parent molecules [para 38]. Teaching claims 63 & 67.
Talasaz et al., teach performing the following steps: a. providing at least one set of tagged parent polynucleotides, and for each set of tagged parent polynucleotides; b. amplifying the tagged parent polynucleotides in the set to produce a corresponding set of amplified progeny polynucleotides; c. sequencing a subset (including a proper subset) of the set of amplified progeny polynucleotides, to produce a set of sequencing reads; and d. collapsing the set of sequencing reads to generate a set of consensus sequences, each consensus sequence corresponding to a unique polynucleotide among the set of tagged parent polynucleotides and, optionally, e. analyzing the set of consensus sequences for each set of tagged parent molecules [para 65]. The method comprises enriching the set of amplified progeny polynucleotides for polynucleotides mapping to one or more selected reference sequences by: (i) selective amplification of sequences from initial starting genetic material converted to tagged parent polynucleotides; (ii) selective amplification of tagged parent polynucleotides; (iii) selective sequence capture of amplified progeny polynucleotides; or (iv) selective sequence capture of initial starting genetic material [para 55]. In another embodiment collapsing comprises: i. grouping sequences reads sequenced from amplified progeny polynucleotides into families, each family amplified from the same tagged parent polynucleotide; and ii. determining a consensus sequence based on sequence reads in a family [para 64]. The method further comprises correcting for amplification or representational bias between the two sets. In another embodiment the method further comprises using a control or set of control samples to correct for amplification or representational biases between the two sets. In another embodiment the method further comprises determining copy number variation between the sets. In another embodiment the method further comprises (including a, b, c): d. determining a quantitative measure of polymorphic forms among the families; and e. based on the determined quantitative measure of polymorphic forms, inferring a quantitative measure of polymorphic forms in the number of inferred unique tagged parent polynucleotides. The sets derive from a common sample, the method further comprising: a. inferring copy number variation for the plurality of sets based on a comparison of the inferred number of tagged parent polynucleotides in each set mapping to each of a plurality of reference sequences [para 67]. Teaching claims 63 and 67.
Talasaz et al., teach quantifying/counting mapped reads in two or more predefined regions of the reference sequence [para 4]. The amplified progeny polynucleotides are sequenced to produce an average of 5 to 10 sequence reads for each. 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 [para 19]. The method comprises correcting/normalizing/adjusting the quantity of mapped reads using the barcodes or unique properties of individual reads [para 25]. Polynucleotide sequencing can be compared with a problem in communication theory. An initial individual polynucleotide or ensemble of polynucleotides is thought of as an original message. Tagging and/or amplifying can be thought of as encoding the original message into a signal [para 206]. Grouping sequence reads into families derived from original individual molecules can reduce noise and/or distortion from a single individual molecule or from an ensemble of molecules [para 210].
Talasaz et al., an initial individual polynucleotide or ensemble of polynucleotides is thought of as an original message. Tagging and/or amplifying can be thought of as encoding the original message into a signal [para 206]. Noise can be introduced through errors in copying and/or reading a polynucleotide [para 207]. For example, in a sequencing process a single polynucleotide can first be subject to amplification. Amplification can introduce errors, so that a subset of the amplified polynucleotides may contain, at a particular locus, a base that is not the same as the original base at that locus. When a collection of molecules that are all presumed to have the same sequence are sequenced, this noise is sufficiently small that one can identify the original base with high reliability [para 207]. Grouping sequence reads into families derived from original individual molecules can reduce noise and/or distortion from a single individual molecule or from an ensemble of molecules [para 210]. Talasaz et al., teach barcode sequences to permit subsequent identification of a particular polynucleotide and wherein the molecule barcodes track back to single original cell-free nucleic acid molecules
In step 810, the set of sequence reads is collapsed to produce a set of consensus sequences corresponding to unique tagged parent polynucleotides. Sequence reads can be qualified for inclusion in the analysis. Sequence reads can be sorted into families representing reads of progeny molecules derived from a particular unique parent molecule. For example, a family of amplified progeny polynucleotides can constitute those amplified molecules derived from a single parent polynucleotide. By comparing sequences of progeny in a family, a consensus sequence of the original parent polynucleotide can be deduced. This produces a set of consensus sequences representing unique parent polynucleotides in the tagged pool [para 265]. FIG. 9 is a diagram presenting a more generic method of extracting information from a signal represented by a collection of sequence reads. In this method, after sequencing amplified progeny polynucleotides, the sequence reads are grouped into families of molecules amplified from a molecule of unique identity (910). This grouping can be a jumping off point for methods of interpreting the information in the sequence to determine the contents of the tagged parent polynucleotides with higher fidelity, e.g., less noise and/or distortion. Teaching the families of claims 63-67.
In one embodiment collapsing further comprises: 2. determining a quantitative measure of sequence reads in each family. In another embodiment the method further comprises (including a) including a): b. determining a quantitative measure of unique families; and c. based on (1) the quantitative measure of unique families and (2) the quantitative measure of sequence reads in each group, inferring a measure of unique tagged parent polynucleotides in the set [para 67]. Alternatively, one can infer a quantitative measure of families in the population of tagged parent polynucleotides using both a quantitative measure of families and a quantitative measure of family members in each family, e.g., as discussed above. Then, CNV can be determined by comparing the inferred measure of quantity at the plurality of loci. In other embodiments, a hybrid approach can be taken whereby a similar inference of original quantity can be made following normalization for representational bias during the sequencing process, such as GC bias, etc [para. 282]. Teaching claims 64 and 67.
Therefore, it would have been prima facie obvious at the time of applicants’ invention to apply Talasaz et al’s cell free nucleic acid molecules in order to and copy number variations in cell free polynucleotides isolated from a bodily fluid; subsequent sequencing of cell free polynucleotides by techniques known in the art within the method of Christians et al. One of ordinary skill in the art would have a reasonable expectation of success using Christians et al., method for detecting the presence or absence of contamination of a first sample with a second sample among a plurality of samples, comprising: (a) processing the first sample and the second sample, wherein the processing comprises: i. tagging a set of nucleic acid molecules in each sample with a set of molecular barcodes to generate tagged polynucleotides, wherein the set of molecular barcodes comprise 5-200 different molecular barcode sequences; ii. amplifying a portion of the tagged polynucleotides to generate progeny polynucleotides; wherein the application of bioinformatics tools to detect contamination as taught by Talasaz et al., to improve sequencing and techniques to manipulate nucleic acids using cell free DNA to detect and monitor contamination.
Additionally, KSR International Co. v. Teleflex Inc., 127 S. Ct. 1727, 1741 (2007), discloses combining prior art elements according to known methods to yield predictable results, thus the combination is obvious unless its application is beyond that person's skill. KSR International Co. v. Teleflex Inc., 127 S. Ct. 1727, 1741 (2007) also discloses that "The combination of familiar element according to known methods is likely to be obvious when it does no more than yield predictable results". It is well known to take a method of detecting contamination where there is no change in the respective function of the tagging and sequencing steps, thus the combination would have yielded a reasonable expectation of success along with predictable results to one of ordinary skill in the art at the time of the invention. Therefore, it would have been obvious to a person of ordinary skill in the art to combine prior art elements according to known methods that is ready for improvement to yield predictable results. The claimed invention is prima facie obvious in view of the teachings of the prior art, absent any convincing evidence to the contrary.
Neither Christians or Talasz teach that the aligning of step (d) comprises: (i) generating a plurality of Compact Idiosyncratic Gapped Alignment Report (CIGAR) strings from objects stored in a first memory location in a computer system, wherein each CIGAR string of the plurality of CIGAR strings represents at least one or more alternative sequences per position at one or more positions, whereby a genomic start position and a genomic stop position of a cell-free nucleic acid molecule is determined from at least one CIGAR string of the alignment; (ii) retrieving the set of molecular barcodes from a second memory location; and (iii) assigning at least one barcode from the set of barcodes to an object determinant of the genomic start position and a genomic stop position.
However, the prior art to Shendure discloses methods for designing molecular inversion probes (MIP) (abstract). Shendure teaches that MIPs can include a tag or barcode sequence of nucleotides to uniquely identify a MIP [0006]. Shendure teaches that a MIP whose sequence alignment data is stored in an alignment file (i.e., barcodes stored in a second memory location) [0047]. Shendure teaches that data for target sequences is provided to the computing device [0048], and that the computing device can linearly traverse a data file representing MIP alignment with respect to the reference genome, one record at a time [0049]. Shendure teaches that the computing device can parse CIGAR strings of each read to determine insertion, deletions, etc. between the MIP and the reference genome, and fields of the SAM line (namely, start coordinate, CIGAR, sequence, quality and ultimately template length) [0051]. Teaching claim 85. As Shendure teaches start coordinate and template length, it is considered that Shendure also fairly teaches genomic stop positions as instantly claimed. Shendure teaches reads (i.e., objects) are retained in memory (i.e., first memory location) of the computing device until passing the expected coordinate of start of the second targeting arm [0052]. Shendure teaches that tag defined read groups (TDRGs), which may be further stratified by a sample barcode, can be represented either by a read selected at random or by first determining the most frequent CIGAR pattern and drafting a SMC-read determined by a user of the computing device [0053]. Shendure teaches that once all TDRGs have been processed for the MIP site, the computing device can move to the next record in the data file and once total and unique reads have been tallied across samples and smMIP targets, the number of unique capture events (or TDRGs) can be estimated by the computing device using Equation 1 below under an assumption that all MIPs in the oligonucleotide pool are amplified uniformly during PCR [0054-0055]. Teaching step (d) of claim 63. Shendure does not teach storing objects determinant of the genomic start position and a genomic stop position at a further additional memory location, as instantly claimed in claim 84.
Regarding claim 63, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine, in the course of routine experimentation and with a reasonable expectation of success, Christians in view of Talasaz with Shendure because each reference discloses methods for analysis of tagged nucleic acids. The motivation to linearly traverse a data file representing alignment with respect to the reference genome, one record at a time, as taught by Shendure [0049]. Thus, it would have been obvious to one of ordinary skill in the art to replace the alignment method of Christians in view of Talasaz with another known alignment method, such as the CIGAR alignment taught by Shendure, because one of ordinary skill in the art would have been able to carry out such a substitution, and the results of producing an alignment would be reasonably predictable. Regarding claim 84, Shendure is considered to teach objects determinant of the genomic start position and a genomic stop position as discussed above [0051], but does not teach storing this data an additional memory location. However, Shendure teaches storing data in different memory locations [0073; 0119; 0130]. Therefore, it would have been obvious to one of ordinary skill in the art to store the determined data regarding genomic start and stop positions in any memory location, including one where the read and barcode data had not been previously stored as instantly claimed.
B. Claim 83 is rejected under 35 U.S.C. 103 as being unpatentable over Christians in view of Talasaz and Shendure, as applied to claim 63 above, and in further view of Eißler et al. (Bioinformatics, 2011, 27(20):2797-2805; newly cited). The instant rejection is newly stated and is necessitated by claim amendment.
Regarding claim 83, Christians in view of Talasaz and Shendure teach claim 63. Claim 83 further adds that the objects stored in a first memory location in a computer system, each comprise a plurality of nodes and edges, and at least one pointer specifying one or more additional memory locations where one or more adjacent objects are stored, which neither Christians, Talasaz, or Shendure teach.
However, the prior art to Eißler discloses a space-efficient indexing structure for approximate oligonucleotide string matching in nucleic acid sequence data (abstract). Eißler teaches partitioning, truncation and a new suffix tree stream compression to deal with large amounts of aligned and unaligned data by operating in main memory and on secondary storage (abstract). Eißler teaches using their method to perform FindFamily a method for fast and comprehensive sequence similarity searches by oligonucleotide string matching frequencies (p. 2801, col. 1, par. 4). Eißler teaches that suffix trees are comprised of nodes and edges for each partition, with pointers between nodes (p. 2799-2780, section 3.3). Eißler teaches that their method is applicable to mapping short reads produced by next-generation sequencing technologies (p. 2797, col. 1, par. 5).
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine, in the course of routine experimentation and with a reasonable expectation of success, Christians in view of Talasaz and Shendure with Eißler because each reference discloses methods for analysis of tagged nucleic acids. The motivation would have been to use a known space-efficient indexing structure for approximate oligonucleotide string matching in nucleic acid sequence data, as taught by Eißler (abstract). The results of using the method of Eißler would have been predictable in combination with the CIGAR alignment format taught by Shendure because the CIGAR format is merely a string representation of nucleic acid sequence data, which would be expected to be compatible with the oligonucleotide string matching in nucleic acid sequence data method taught by Eißler.
Response to Applicant Arguments
With respect to Applicant’s arguments under 35 USC 103, the arguments have been fully considered but are moot in view of the new grounds of rejection set forth above as necessitated by claim amendment herein.
Conclusion
No claims are allowed.
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/JANNA NICOLE SCHULTZHAUS/Examiner, Art Unit 1685