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 .
Applicants Amendment
Applicants’ preliminary amendment filed 11/13/2022 has been received and entered. Claims 1-176 have been cancelled and claims 177-206 have been added.
Claims 177-206 are currently pending.
Priority
This application filed 10/13/2022 is a continuation of application 17/37400 filed 7/31/2021 which is a divisional of 15/329683, filed 1/27/2017 which is a national stage filing of PCT/US2015/042556 filed 7/29/2015 which claims benefit to US provisional applications 62/142653 filed 4/3/2015, 62/050636 filed 9/15/2014 and 62/031062 filed 7/30/2014;
and is related to 18/184967 filed (now US Patent 11959075) filed 3/16/2023, to US applications 18/932298 filed 3/16/2023, now US Patent 12,473546 and to 18/932378 filed 4/7/2023 now US Patent 12,522819, 18/932442 now US Patent 12,522820 and 18/185048 now US Patent 12,104151 through claim of priority to 18/046409 filed 10/13/2023;
and is related to 18/297378 filed 4/7/2023 and 19/552704 filed 2/27/2026 through claim of priority to 17/37400 filed 7/31/2021 (now US Patent 12209237);
and is related to US Applications 15/329651 (now US Patent 10240146) and 16/253919 filed 1/22/2019 now Abandoned and continuation 18/915641 filed 10/15/2024 through claim of benefit to the same PCT applications and US provisional applications listed above.
It is noted that the information above is taken from the ADS form, and that in the specification the application claims benefit of only US Provisional applications listed above.
Information Disclosure Statement
The five information disclosure statements (IDS) submitted between 10/13/2022 and 9/20/2023 are in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statements are being considered by the examiner.
It is noted that multiple references that are cited are not in English and/or only have partial translations of abstracts. For clarity of the record, it is noted that only the portions in English have been reviewed, and the other non-English portions have not been considered.
Further, US applications have been cited without any specific indication of what in the file is to be considered. For clarity of the record, the applications are noted and published applications have been searched and reviewed using the office’s search engine, but the complete file has not been reviewed for this examination.
Additionally, it appears that some of the citations have been cited in both documents, for example the Chinese office action mailed Jan 16, 2019 for application no: 201280052678.7.
Finally, it is noted that the listing of references in the specification is not a proper information disclosure statement. See for example top of page 31 of the specification. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
Double Patenting
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 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. 2065); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 2062); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); 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 nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claims 177-206 are rejected on the ground of nonstatutory double patenting as being unpatentable over the claims of U.S. Patent No. 11,098303 (Application 15/329683 filed 1/27/2017). Although the claims at issue are not identical, they are not patentably distinct from each other because each provide for contacting a sample with a probe that can be detected serial and translated into a code word. The independent claim of ‘303 is broader, but dependent claims provide for using fluorescence and the sample being a fixed cell, where the detection provides the basis of codewords that can be error correcting.
Claim 1 of ‘303:
A method for detecting nucleic acid targets, comprising:
(a) exposing a sample suspected of containing at least some of a plurality of nucleic acid targets to a plurality of encoding nucleic acid probes, wherein at least some of the plurality of encoding nucleic acid probes comprise a target sequence that specifically hybridizes to a nucleic acid target in the plurality of nucleic acid targets and one or more read sequences, wherein unique combinations of one or more read sequences correspond to valid codewords, each of the valid codewords associated to each of the plurality of nucleic acid targets, wherein the valid codeword consists of a series of positions and values for each position, and each read sequence is associated with each of the values, or a subset thereof, at each of the positions of the valid codewords;
(b) exposing the sample to a first round of one or more readout nucleic acid probes, wherein at least some of the readout nucleic acid probes comprise a first portion and a second portion, the first portion comprising a sequence that specifically hybridizes to one of the read sequences, and the second portion comprising a fluorescent signaling entity;
(c) identifying nucleic acid targets in the sample, comprising: detecting fluorescent signals emitted from the fluorescent signaling entities of bound readout nucleic acid probes in at least some locations within the sample to determine a codeword in at least some locations within the sample based on the read sequences present on the bound encoding nucleic acid probes;
identifying the codeword at each location by comparing the codeword to the valid codewords associated with the plurality of nucleic acid targets, and if no match is found, applying error correction to the codeword to match one of the valid codewords or discarding the codeword; and
assigning the identity of some of the plurality of nucleic acid targets in the sample based on the identity of the nucleic acid targets associated with the valid codewords to which the identified codewords were matched.
Claims 177-206 are rejected on the ground of nonstatutory double patenting as being unpatentable over the claims of copending Application No. 18/185048 now US Patent 12,104151. Although the claims at issue are not identical, they are not patentably distinct from each other because each provide for contacting a sample with a probe that can be detected serial and translated into a code word. The independent claim of ‘303 is broader, but dependent claims provide for using fluorescence and the sample being a fixed cell, where the detection provides the basis of codewords that can be error correcting.
Claims 177-206 are rejected on the ground of nonstatutory double patenting as being unpatentable over the claims of copending Application No. 17/37400 filed 7/13/2021 now US Patent 12,209237 (parent application). Although the claims at issue are not identical, they are not patentably distinct from each other because the methods 122 and 126 of ‘000 and the instant application each provide for contacting a sample with a probe that can be detected serial and translated into a code word. The independent claim of ‘000 is broader, but dependent claims provide for using fluorescence and the sample being a fixed cell, where the detection provides the basis of codewords that can be error correcting. The products of ‘000 are found obvious and necessary for the practice of the methods as claimed.
Claim 122 of ‘000:
A method, comprising:
associating a plurality of targets with a plurality of target sequences and a plurality of codewords, wherein the codewords comprise a number of positions and values for each position, and the codewords form an error-checking and/or error-correcting code space;
associating a plurality of distinguishable read sequences with the plurality of codewords such that each distinguishable read sequence represents a value of a position within the codewords; and
forming a plurality of nucleic acid probes, each comprising a target sequence and one or more read sequences.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Claims 177-206 are rejected on the ground of nonstatutory double patenting as being unpatentable over the claims of US Patent 11,959075 (Application No. 18/184967 filed 3/16/2023). Although the claims at issue are not identical, they are not patentably distinct from each other because the method in claim 195 and the instant application each provide for contacting a sample with a probe that can be detected serial and translated into a code word. The claims vary slightly in terminology, but dependent claims provide for using fluorescence and the sample being a fixed cell, where the detection provides the basis of codewords that can be error correcting for obvious claim requirements.
Independent claim 195 of ‘0967:
A method for determining a plurality of nucleic acid targets within a sample by in situ hybridization, comprising:
(a) contacting a sample comprising a plurality of nucleic acid targets with a plurality of primary nucleic acid probes, wherein each primary nucleic acid probe comprises (i) a target sequence and (ii) one or more read sequences, wherein the target sequence hybridizes to the nucleic acid target in the sample to form a primary probe-hybridized complex;
(b) contacting the sample to a second round of hybridization using a plurality of secondary nucleic acid probes wherein the secondary nucleic acid probes comprise a first portion that hybridizes to a subset of the read sequences of the primary nucleic acid probe and a second portion comprising a fluorescent label to form a secondary probe-hybridized complex;
(c) detecting a fluorescent signal from each of the secondary probe-hybridized complexes;
(d) removing the secondary nucleic acid probes or inactivating the fluorescent signal from each of the secondary probe-hybridized complexes; and
(e) repeating steps (b) through (d) with multiple sets of differing secondary nucleic acid probes to generate a pattern of binding of the secondary nucleic acid probes; wherein the pattern of binding of the secondary nucleic acid probes is converted to a codeword, wherein the codeword determines the identity of the nucleic acid target, and wherein the codeword facilitates error correction.
Claims 177-206 are rejected on the ground of nonstatutory double patenting as being unpatentable over the claims of copending Application No. 18/297378 filed 4/7/2023 now US Patent 12,522819. Although the claims at issue are not identical, they are not patentably distinct from each other because the method of the instant claims requires the products set forth in ‘378.
Claim 1 of ‘378:
A composition, comprising:
a plurality of nucleic acid probes, each comprising a first portion comprising a target sequence and a plurality of read sequences,
wherein the plurality of read sequences are distributed on the plurality of nucleic acid probes so as to define an error-correcting code.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Claims 177-206 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over the claims of copending Application No. 18/297378 filed 4/7/2023. Although the claims at issue are not identical, they are not patentably distinct from each other because the method of the instant claims requires the products set forth in ‘378.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Claims 177-206 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over the claims of copending Application No. 19/552704 filed 2/27/704. Although the claims at issue are not identical, they are not patentably distinct from each other because the method of the instant claims requires the products set forth in ‘409.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Claims 177-206 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over the claims of copending Application No. 18/915641 filed 10/15/2024. Although the claims at issue are not identical, they are not patentably distinct from each other because the method of the instant claims requires the products set forth in ‘409.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
For completeness and clarity of the record it is noted that related U.S. Patent No. 10,240146, (Application 15/329651 filed 1/2/2017) and 18/915641 filed 10/15/2024, were reviewed and that the claims were found to be directed to a method of creating DNA oligonucleotides from RNA which appears unobvious over the present 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 177-206 are rejected under 35 U.S.C. 101 because the claimed invention is directed to a judicial exception (i.e., a law of nature, a natural phenomenon, or an abstract idea) without significantly more.
Claim analysis
Independent claim 177 is generally directed to spatially imaging RNA in a sample by using a target specific probe with a tail that can be identified by multiple alternative detectable probes. More specifically, nucleic acid probes comprising a target specific sequence and a tag read sequences are hybridized to a sample and a second probe that detects the tag read sequence is used to visualize the location of the probe. The steps to detect the tag can be repeated for a given sample. Dependent claims provide for associating the detection of the tag to correlating it to a code, and given the guidance of the specification this is accomplished through knowledge of the tag read sequence and probe used, where through sequential hybridization with tag read sequences and probes that detect the tag read sequence an expected association of the signal imaged can be correlated to the target sequence of the probe and its location in the sample.
For step 1 of the 101 analysis, the claims are found to be directed to a statutory category of a method.
For step 2A of the 101 analysis, the judicial exception of the claims are the steps of accessing sequence data based on target probe hybridization and imaging the target probe with a second probe specific for the tag read sequence associated with the target portion. The step of assessing the image labels and associating the images with a ‘binary code’ are instructional steps. The judicial exception is a set of instructions for analysis of sequence image data derived from probing RNA in a sample, and appears to fall into the category of a Mental Processes, that is concepts performed in the human mind (including an observation, evaluation, judgment, opinion). Here, after observing a fluorescent signal in a location and if repeated and if it exists, associating the resulting images into a binary code representing the target sequence’s location in the sample.
Recent guidance from the office requires that the judicial exception be evaluated under a second prong to determine whether the judicial exception is practically applied. In the instant case, the claims do not have an additional element to which the judicial exception is applied. In review of the claims and evidence provided in the specification, it appears that data obtained is simply assessed as provided in Figure 5 for example. This assessment appears to be possible only through pre-knowledge of the tag read sequence and associated target sequence of a given probe. This judicial exception requires steps recited at high level of generality and appears to provide only information, and is not found to be a practical application of the judicial exception as broadly set forth.
For step 2B of the 101 analysis, each of the independent claims recites additional elements and are found to be the steps of obtaining hybridization data in the form of an image. Imaging RNA is a sample, for example a cell was known and performed in the art as evidenced by Cai who analyze single cells using multiple probes to visualize the location of mRNA that might be present, or Lubeck et al which uses serial hybridization to provide a barcode in the analysis of RNA in a cell. As such, the claims do not provide for any additional element to consider under step 2B where the judicial exception is applied. While it is acknowledged that the images can be digitally evaluated and analyzed to represent a binary code using a computer, it is noted that in explaining the Alice framework, the Court wrote that "[i]n cases involving software innovations, [the step one] inquiry often turns on whether the claims focus on the specific asserted improvement in computer capabilities or, instead, on a process that qualifies as an abstract idea for which computers are invoked merely as a tool." The Court further noted that "[s]ince Alice, we have found software inventions to be patent-eligible where they have made non-abstract improvements to existing technological processes and computer technology." Moreover, these improvements must be specific -- "[a]n improved result, without more stated in the claim, is not enough to confer eligibility to an otherwise abstract idea . . . [t]o be patent-eligible, the claims must recite a specific means or method that solves a problem in an existing technological process." Here, associating colors on images and correlating a binary code based on their presence or absence doe not appear to be an improve result in a computer environment. Dependent claims set forth additional steps which are more specifically define the considerations and steps of calculating, and comparing, and do not add additional elements which result in significantly more to the claimed method for the analysis.
One way to overcome a rejection for non-patent-eligible subject matter is to persuasively argue that the claimed subject matter is not directed to a judicial exception. Another way for the applicants to overcome the rejection is to persuasively argue that the claims contain elements in addition to the judicial exception that either individually or as an ordered combination are not well understood, routine, or conventional. Another way for the applicants to overcome the rejection is to persuasively argue that the claims as a whole result in an improvement to a technology. Persuasive evidence for an improvement to a technology could be a comparison of results of the claimed subject matter with results of the prior art, or arguments based on scientific reasoning that the claimed subject matter inherently results an improvement over the prior art. The applicants should show why the claims require the improvement in all embodiments.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 177-206 are rejected under 35 U.S.C. 103 as being unpatentable over Fan and Zhang (US Patent 10,829,814), Gunderson et al. (Genome Res. 14(5):870-877 (2004)), Chee (Patent Publication No. US2007/0231824A1), Curry (8765642) and Hamady (Patent Publication No. US2010/0323348) (all of record).
The claims encompass a method for detecting a plurality of different nucleic acid targets within a sample by in situ hybridization as set forth in the preamble. Fan et al provide for methods and compositions for single cell genomics teaching “Presented herein are methods and compositions for obtaining sequence information from one or more individual cells. The methods are useful for obtaining sequence information for a single nucleotide sequence, and for multiplex generation of sequence information from an individual cell. In some embodiments presented herein, the methods comprise first amplifying one or more nucleic acid molecules in situ within a cell, and subsequently hybridizing a barcode probe to the amplified nucleic acid molecules. The identity of the amplified nucleic acid molecules is then determined by decoding of the barcode probes. In other embodiments presented herein, the methods comprise first hybridizing one or more different barcode probes to one or more nucleic acid molecule in situ within a cell, and subsequently amplifying the barcode probe, thereby generating an amplified barcode probe. The identity of the nucleic acid molecules is then determined by decoding of the amplified barcode probes.” (Fan at 1:58-2:7).
More specifically, claim 177 requires producing a plurality of first hybridized complexes within a sample containing a plurality of different nucleic acid targets by exposing the sample to a plurality of different encoding nucleic acid probes. Fan teaches producing a plurality of first hybridized complexes within a sample containing a plurality of different nucleic acid targets by exposing the sample to a plurality of different encoding nucleic acid probes. For example: “In some embodiments presented herein, the methods comprise first amplifying one or more nucleic acid molecules in situ within a cell, and subsequently hybridizing a barcode probe to the amplified nucleic acid molecules. The identity of the amplified nucleic acid molecules is then determined by decoding of the barcode probes. In other embodiments presented herein, the methods comprise first hybridizing one or more different barcode probes to one or more nucleic acid molecule in situ within a cell, and subsequently amplifying the barcode probe, thereby generating an amplified barcode probe. The identity of the nucleic acid molecules is then determined by decoding of the amplified barcode probes.” Fan at 1:63-2:7 (emphasis added). “FIG. 1 describes one embodiment of padlock capture and hybridization decoding of colonies. (A) Molecular barcodes are attached to colonies in a gene specific manner using padlock probes. (B) After padlock capture, each colony will have a unique barcode in thousands of copies. These barcodes can be accurately decoded by hybridization similar to the decoding of Illumina bead array, using a combinatorial chemistry approach (Gunderson et al. Genome Res. 14(5): 870-877 (2004)).” Fan, Fig. 1 and 3:32-40. “As an example, one or more target nucleic acid species can be amplified, either specifically or non-specifically, to generate an amplified molecule containing many repeats of the amplified nucleic acid in tandem. In the embodiment shown in FIG. 1, cDNA is generated in situ, using either random hexamers or oligo dT primers or both. Then the cDNA molecules are amplified using rolling circle amplification (RCA) to generate a ‘DNA ball’ or ‘colony’. The amplified nucleic acids can then be hybridized with one or more unique padlock probes with unique molecular barcodes. When a padlock probe specifically hybridizes to a sequence of interest, the probe is ligated to form, for example, a circularized barcoded padlock probe. The amplified nucleic acid molecules and/or the hybridized padlock probes are immobilized within the cell using the crosslinking methods described herein, and decoding of the barcode probe takes place by multiple rounds of sequential hybridization. In the example shown in FIG. 1, decoding takes place via a set of barcode probes having decoder sequences that can be decoded by eight rounds of sequential hybridization with fluorescently labeled oligos and imaging of fluorescent images.” (Fan at 9:1-22). With respect to the probe design set forth in the claims, Fan teaches that each of the plurality of encoding nucleic acid probes comprises (i) a target sequence that specifically hybridizes to a different nucleic acid target or a different location of an identical nucleic acid target of the plurality of different nucleic acid targets. For example: “The methods provided herein further comprise annealing in situ a barcode probe to the amplified nucleic acid molecule. As used herein, the term barcode probe refers to a nucleic acid molecule which can hybridize specifically to a nucleic acid of interest, and which comprises a barcode sequence. As used herein, the terms barcode, barcode sequence and like terms refer to a unique identifier sequence on a probe molecule. The unique identifier sequence can be, for example, a nucleic acid sequence which associates the barcode probe with the nucleic acid of interest to which it hybridizes. The barcode probes presented herein can be any probe which specifically hybridizes to a nucleic acid species of interest and which comprises at least a first and a second decoder sequence. In some embodiments presented herein, the barcode probes are used as a template for further amplification, thus generating a clonal object that comprises many repeating copies of the barcode, as described in greater detail in the disclosure of WO 2011/123246, the entire content of which is incorporated herein by reference in its entirety.” Fan at 5:15-35 (emphasis added). “Barcode probes used in the methods presented herein can include padlock probes. Padlock probes are specialized ligation probes and are known in the art, as exemplified generally by Nilsson et al, 1994, Science 265:2085, hereby incorporated by reference. These probes have a first ligation domain that is identical to a first ligation probe, in that it hybridizes to a first target sequence domain, and a second ligation domain, identical to the second ligation probe, that hybridizes to an adjacent target sequence domain. Again, as for OLA, the detection position can be either at the 3′ end of the first ligation domain or at the 5′ end of the second ligation domain. However, the two ligation domains are connected by a linker, frequently nucleic acid. The configuration of the system is such that upon ligation of the first and second ligation domains of the padlock probe, the probe forms a circular probe, and forms a complex with the target sequence wherein the target sequence is "inserted" into the loop of the circle.” Fan at 7:36-53. “The at least first and second decoding sequences can be any suitable length to ensure specific hybridization. Thus, for example, in some embodiments, each decoding sequence can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or greater than 20 nucleotides in length.” Fan at 9:44-48. Fan teaches one or more read sequences (decoder sequences) such that each of the plurality of first hybridized complexes comprises multiple encoding nucleic acid probes of the different encoding nucleic acid probes located in its different locations. For example, “The methods and compositions presented herein make use of unique barcode probes having decoder sequences to identify multiple nucleic acid targets in a multiplex fashion. The instant innovations overcome limitations of existing colony identification methodologies by surprisingly and successfully applying decoding strategies that entail sequential hybridizations of labeled decoder probes to decoder sequences found in immobilized nucleic acids within an individual cell. Such decoding strategies using decoder sequences have until now been used for decoding of randomly assembled arrays of beads. Thus, the decoding strategies presented herein are flexible and scalable and can be used to detect and decode thousands of unique amplified nucleic acid species. In some embodiments, a barcode probe can comprise at least a first decoder sequence and a second decoder sequence. In some embodiments, a barcode probe can comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 decoder sequences. As used herein, the terms decoder sequence, stage, slot and like terms refer to a unique sequence which is part of a plurality of unique sequences are designed to each specifically hybridize to a labeled decoder probe. During decoding, labeled decoder probe oligonucleotides are sequentially hybridized to the decoder sequences to specifically determine the identity of each barcode sequence. Sequential hybridization of decoder probes to decoder sequences is described by Gunderson et al. (Genome Res. (2004) 14:870-877), the content of which is incorporated by reference in its entirety. Additional description of decoding algorithms and the use of sequential hybridization useful in the methods presented herein can be found in U.S. Pat. Nos. 7,060,431; 7,033,754; 7,226,734; and 7,455,971, the disclosures of each of which are hereby incorporated by reference in their entireties. The principles of sequential hybridization to decode bead arrays are surprisingly robust and effective when applied to amplified nucleic acids in situ according to the methods provided herein.” Fan at 8:31-67 (emphasis added). “As an example, one or more target nucleic acid species can be amplified, either specifically or non-specifically, to generate an amplified molecule containing many repeats of the amplified nucleic acid in tandem. In the embodiment shown in FIG. 1, cDNA is generated in situ, using either random hexamers or oligo dT primers or both. Then the cDNA molecules are amplified using rolling circle amplification (RCA) to generate a ‘DNA ball’ or ‘colony’. The amplified nucleic acids can then be hybridized with one or more unique padlock probes with unique molecular barcodes. When a padlock probe specifically hybridizes to a sequence of interest, the probe is ligated to form, for example, a circularized barcoded padlock probe. The amplified nucleic acid molecules and/or the hybridized padlock probes are immobilized within the cell using the crosslinking methods described herein, and decoding of the barcode probe takes place by multiple rounds of sequential hybridization. In the example shown in FIG. 1, decoding takes place via a set of barcode probes having decoder sequences that can be decoded by eight rounds of sequential hybridization with fluorescently labeled oligos and imaging of fluorescent images.” Fan at 9:1-22. “An amplicon typically contains multiple, tandem copies of the circularized nucleic acid molecule of the corresponding sample nucleic acid. That is, each amplicon contains multiple, tandem copies of a single sample nucleic acid molecule, which was circularized. The number of copies can be varied by appropriate modification of the amplification reaction including, for example, varying the number of amplification cycles run, using polymerases of varying processivity in the influence amplification yield. Generally, the number of copies of a nucleic acid in an amplicon is at least 100, 200, 500, 1000,2000,3000,4000, 5000,6000, 7000, 8000, 9000 and 10,000 copies, and can be varied depending on the particular application. As disclosed herein, one particular form of an amplicon is as a nucleic acid "ball" having amplification reaction and/or varying the length of time that the amplification reaction is run, as well as modification of other conditions known in the art to desired dimensions. The number of copies of the nucleic acid molecule can therefore provide a desired size of a nucleic acid "ball" or a sufficient number of copies for efficient subsequent analysis of the amplicon, for example, sequencing.” Fan at 6:8-29. “Useful methods for clonal amplification from single molecules include, for example, rolling circle amplification (RCA) (Lizardi et al., Nat. Genet. 19:225- 232 (1998), which is incorporated herein by reference) and oligonucleotide ligation assay (OLA) (See generally U.S. Pat. Nos. 7,582,420, 5,185,243, 5,679,524 and 5,573,907; EP 0 320 308 B1; EP 0 336 731 B1; EP 0 439 182 B1; WO 90/01069; WO 89/12696; and WO 89/09835, all of which are incorporated by reference) technologies. The enhanced signal-to-noise ratio provided by clonal amplification provides a great advantage over existing detection methodologies. In a particularly useful embodiment, amplicons are generated by rolling circle amplification (RCA), which can be used to generate amplicons having multiple copies of a nucleic acid sequence and which can be used to create nucleic acid ‘balls,’ as disclosed herein. It will be understood that these ‘balls’ need not be perfectly spherical and can include other globular or packed conformations. In a particular embodiment, RCA is primed using the at least one universal primer attached to the sample nucleic acid molecule.” Fan at 17:15-35. To the extent that Fan is argued by Vizgen or found by the Court not to explicitly teach that each of the plurality of first hybridized complexes comprises multiple encoding nucleic acid probes of the different encoding nucleic acid probes located in its different locations, such limitations would have been inherent and/or obvious in view Cai and/or Raj as described in 10x’s Invalidity Contentions. See Attachments 1 and 4, limitation 1[a][ii].
With respect to method step and the limitation requiring producing a plurality of readout probe-hybridized complexes by exposing each of the plurality of first hybridized complexes within the sample to a first readout nucleic acid probe, Fan teaches producing a plurality of readout probe-hybridized complexes (the decoder probe-detection reagent-analyte conjugate) by exposing each of the plurality of first hybridized complexes within the sample to a first readout nucleic acid probe (decoder probe). The first readout nucleic acid probe (decoder probe) comprises a first portion and a second portion, the first portion comprising a sequence that specifically hybridizes to one of the read sequences (decoder sequence), and the second portion comprising a fluorescent signaling entity capable of producing a fluorescent signal. For example: “The methods and compositions presented herein make use of unique barcode probes having decoder sequences to identify multiple nucleic acid targets in a multiplex fashion. The instant innovations overcome limitations of existing colony identification methodologies by surprisingly and successfully applying decoding strategies that entail sequential hybridizations of labeled decoder probes to decoder sequences found in immobilized nucleic acids within an individual cell. Such decoding strategies using decoder sequences have until now been used for decoding of randomly assembled arrays of beads. Thus, the decoding strategies presented herein are flexible and scalable and can be used to detect and decode thousands of unique amplified nucleic acid species. In some embodiments, a barcode probe can comprise at least a first decoder sequence and a second decoder sequence. In some embodiments, a barcode probe can comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 decoder sequences. As used herein, the terms decoder sequence, stage, slot and like terms refer to a unique sequence which is part of a plurality of unique sequences are designed to each specifically hybridize to a labeled decoder probe. During decoding, labeled decoder probe oligonucleotides are sequentially hybridized to the decoder sequences to specifically determine the identity of each barcode sequence. Sequential hybridization of decoder probes to decoder sequences is described by Gunderson et al. (Genome Res. (2004) 14:870-877), the content of which is incorporated by reference in its entirety. Additional description of decoding algorithms and the use of sequential hybridization useful in the methods presented herein can be found in U.S. Pat. Nos. 7,060,431; 7,033,754; 7,226,734; and 7,455,971, the disclosures of each of which are hereby incorporated by reference in their entireties. The principles of sequential hybridization to decode bead arrays are surprisingly robust and effective when applied to amplified nucleic acids in situ according to the methods provided herein.” Fan at 8:31-67.
For the step that requires detecting a fluorescent signal from each of the plurality of readout probe hybridized complexes, Fan teaches detecting a fluorescent signal from each of the plurality of readout probe hybridized complexes. For example: “As an example, one or more target nucleic acid species can be amplified, either specifically or non-specifically, to generate an amplified molecule containing many repeats of the amplified nucleic acid in tandem. In the embodiment shown in FIG. 1, cDNA is generated in situ, using either random hexamers or oligo dT primers or both. Then the cDNA molecules are amplified using rolling circle amplification (RCA) to generate a "DNA ball" or "rolony". The amplified nucleic acids can then be hybridized with one or more unique padlock probes with unique molecular barcodes. When a padlock probe specifically hybridizes to a sequence of interest, the probe is ligated to form, for example, a circularized barcoded padlock probe. The amplified nucleic acid molecules and/or the hybridized padlock probes are immobilized within the cell using the crosslinking methods described herein, and decoding of the barcode probe takes place by multiple rounds of sequential hybridization. In the example shown in FIG. 1, decoding takes place via a set of barcode probes having decoder sequences that can be decoded by eight rounds of sequential hybridization with fluorescently labeled oligos and imaging of fluorescent images.” Fan at 9:1-22 (emphasis added). “The decoding strategies presented herein provide several key advantages over existing sequence identification methods. For example, one advantage is the simplicity and robustness of the decoding process, which involves hybridization and washing of oligo pools at room temperature. As such, heating and cooling steps are not necessary, thus minimizing damage to the cellular structure and reducing the potential drift of the specimen throughout the imaging process. As an additional advantage, the signals that are generated are more even and have higher intensity inside a structured tissue than methods that rely on other detection techniques. Further, in many embodiments, only two fluors are needed for decoding, which provides convenient spectral distinction using simple detection devices and leaves enough spectrum for labeling cellular structure using other fluors. Of course more than two colors can be used to expand the number of sequences that are decoded, using modifications of the method set forth in further detail elsewhere herein and in the references that are incorporated by reference above as describing sequential hybridization of decoder probes to decoder sequences.” Fan at 9:23-43. “The signals generated by fluorescent imaging of one or more probes during decoding, sequencing and other detection methods carried out in situ and can be detected using any suitable imaging and detection technologies which allow for detection of individual amplicons in situ.” Fan at 11:11-15.
With respect to the step of removing the fluorescent signal from each of the plurality of readout probe-hybridized complexes by inactivating the fluorescent signaling entity or removing the fluorescent signaling entity from each of the plurality of readout probe hybridized complexes, Fan teaches removing the fluorescent signal from each of the plurality of readout probe-hybridized complexes by inactivating the fluorescent signaling entity or removing the fluorescent signaling entity from each of the plurality of readout probe hybridized complexes. For example: “In some aspects of the above embodiments, the first labeled decoder probe is removed prior to hybridizing the second labeled decoder probe. In some aspects, the second labeled decoder probe is hybridized prior to the first labeled decoder probe.” Fan at 2:56-60. “The decoding strategies presented herein provide several key advantages over existing sequence identification methods. For example, one advantage is the simplicity and robustness of the decoding process, which involves hybridization and washing of oligo pools at room temperature. As such, heating and cooling steps are not necessary, thus minimizing damage to the cellular structure and reducing the potential drift of the specimen throughout the imaging process.” Fan at 9:23-31.
For the step of repeating the process of producing a different plurality of readout probe-hybridized complexes by exposing the sample to a different readout nucleic acid probe, wherein the different readout nucleic acid probe comprise a first portion and a second portion, the first portion comprising a sequence that specifically hybridizes to one of the read sequences, and the second portion comprising a fluorescent signaling entity capable of producing a fluorescent signal; Fan teaches after removing or inactivating the fluorescent signal, producing a different plurality of readout probe-hybridized complexes by exposing the sample to a different readout nucleic acid probe. For example: “As an example, one or more target nucleic acid species can be amplified, either specifically or non-specifically, to generate an amplified molecule containing many repeats of the amplified nucleic acid in tandem. In the embodiment shown in FIG. 1, cDNA is generated in situ, using either random hexamers or oligo dT primers or both. Then the cDNA molecules are amplified using rolling circle amplification (RCA) to generate a ‘DNA ball’ or ‘rolony’. The amplified nucleic acids can then be hybridized with one or more unique padlock probes with unique molecular barcodes. When a padlock probe specifically hybridizes to a sequence of interest, the probe is ligated to form, for example, a circularized barcoded padlock probe. The amplified nucleic acid molecules and/or the hybridized padlock probes are immobilized within the cell using the crosslinking methods described herein, and decoding of the barcode probe takes place by multiple rounds of sequential hybridization. In the example shown in FIG. 1, decoding takes place via a set of barcode probes having decoder sequences that can be decoded by eight rounds of sequential hybridization with fluorescently labeled oligos and imaging of fluorescent images.” Fan at 9:1-22; see also Fig. 1. Fan teaches that the readout nucleic acid probe (decoder probe) comprises a first portion and a second portion, the first portion comprising a sequence that specifically hybridizes to one of the read sequences, and the second portion comprising a fluorescent signaling entity capable of producing a fluorescent signal. Fan teaches detecting a fluorescent signal from each of the different plurality of readout probe hybridized complexes. For example: “As an example, one or more target nucleic acid species can be amplified, either specifically or non-specifically, to generate an amplified molecule containing many repeats of the amplified nucleic acid in tandem. In the embodiment shown in FIG. 1, cDNA is generated in situ, using either random hexamers or oligo dT primers or both. Then the cDNA molecules are amplified using rolling circle amplification (RCA) to generate a ‘DNA ball’ or ‘rolony’. The amplified nucleic acids can then be hybridized with one or more unique padlock probes with unique molecular barcodes. When a padlock probe specifically hybridizes to a sequence of interest, the probe is ligated to form, for example, a circularized barcoded padlock probe. The amplified nucleic acid molecules and/or the hybridized padlock probes are immobilized within the cell using the crosslinking methods described herein, and decoding of the barcode probe takes place by multiple rounds of sequential hybridization. In the example shown in FIG. 1, decoding takes place via a set of barcode probes having decoder sequences that can be decoded by eight rounds of sequential hybridization with fluorescently labeled oligos and imaging of fluorescent images.” Fan at 9:1-22 (emphasis added). “The decoding strategies presented herein provide several key advantages over existing sequence identification methods. For example, one advantage is the simplicity and robustness of the decoding process, which involves hybridization and washing of oligo pools at room temperature. As such, heating and cooling steps are not necessary, thus minimizing damage to the cellular structure and reducing the potential drift of the specimen throughout the imaging process. As an additional advantage, the signals that are generated are more even and have higher intensity inside a structured tissue than methods that rely on other detection techniques. Further, in many embodiments, only two fluors are needed for decoding, which provides convenient spectral distinction using simple detection devices and leaves enough spectrum for labeling cellular structure using other fluors. Of course more than two colors can be used to expand the number of sequences that are decoded, using modifications of the method set forth in further detail elsewhere herein and in the references that are incorporated by reference above as describing sequential hybridization of decoder probes to decoder sequences.” (Fan at 9:23-43). Fan teaches removing the fluorescent signal from each of the different plurality of readout probe hybridized complexes by inactivating the fluorescent signaling entity or removing the fluorescent signaling entity from each of the different plurality of readout probe hybridized complexes. See disclosure for element 1[d]. The inactivation or removal of the fluorescent signaling entity is performed after each detecting step in a readout round. For example: “In some aspects of the above embodiments, the first labeled decoder probe is removed prior to hybridizing the second labeled decoder probe. In some aspects, the second labeled decoder probe is hybridized prior to the first labeled decoder probe.” Fan at 2:56-60. “The decoding strategies presented herein provide several key advantages over existing sequence identification methods. For example, one advantage is the simplicity and robustness of the decoding process, which involves hybridization and washing of oligo pools at room temperature. As such, heating and cooling steps are not necessary, thus minimizing damage to the cellular structure and reducing the potential drift of the specimen throughout the imaging process.” Fan at 9:23-31. Moreover, Fan teaches after removing or inactivating the fluorescent signal, producing a different plurality of readout probe-hybridized complexes by exposing the sample to a different readout nucleic acid probe. For example: “As an example, one or more target nucleic acid species can be amplified, either specifically or non-specifically, to generate an amplified molecule containing many repeats of the amplified nucleic acid in tandem. In the embodiment shown in FIG. 1, cDNA is generated in situ, using either random hexamers or oligo dT primers or both. Then the cDNA molecules are amplified using rolling circle amplification (RCA) to generate a ‘DNA ball’ or ‘rolony’. The amplified nucleic acids can then be hybridized with one or more unique padlock probes with unique molecular barcodes. When a padlock probe specifically hybridizes to a sequence of interest, the probe is ligated to form, for example, a circularized barcoded padlock probe. The amplified nucleic acid molecules and/or the hybridized padlock probes are immobilized within the cell using the crosslinking methods described herein, and decoding of the barcode probe takes place by multiple rounds of sequential hybridization. In the example shown in FIG. 1, decoding takes place via a set of barcode probes having decoder sequences that can be decoded by eight rounds of sequential hybridization with fluorescently labeled oligos and imaging of fluorescent images.” Fan at 9:1-22 (emphasis added). Fan teaches that the readout nucleic acid probe (decoder probe) comprises a first portion and a second portion, the first portion comprising a sequence that specifically hybridizes to one of the read sequences, and the second portion comprising a fluorescent signaling entity capable of producing a fluorescent signal.
With respect to the probe design and method steps resulting in a ‘codeword’ Fan teaches producing codewords (temporal sequence of signal signature) representing the plurality of different nucleic acid targets at locations within the sample, wherein each of the codeword represents one of the plurality of different nucleic acid targets and is obtained when the fluorescent signal is detected from one of the plurality of readout probe hybridized complexes or one of the different plurality of readout probe hybridized complexes at a respective location within the sample. For example: “In some aspects, detecting can further comprise evaluating a signal to determine the presence, quantity, frequency, and/or spatial distribution of a disease-causing mutation, allele or common variant within a tissue.” Fan at 3:21-24. “Spatial information is also desirable when interpreting transcriptional heterogeneity of individual cells in a structured tissue. However, RNA sequencing is typically performed on isolated cells, such that spatial information is lost. One existing approach to link transcriptome information from isolated cells to their context is to first acquire the spatial information as well as other ‘phenotypes’ of individual cells within the intact tissue using fluorescent microscopy, and then extract specific cells (or cellular content) for subsequent transcriptional profiling. Thus, there is a need for methods which provide high resolution mapping of a large number of transcripts in situ.” Fan at 3:61-4:5. “The methods and compositions set forth herein will be exemplified for detection of nucleic acids in a single cell. However, the methods can be extended to the simultaneous detection of nucleic acids in several individual cells. Parallel detection can be carried out such that several individual cells are resolved with regard to nucleic acid sequences that are present. In some embodiments the spatial location of nucleic acids can be simultaneously determined for several individual cells in a tissue or other cell-containing sample.” Fan at 4:26-34.
With respect to targets of interest, design and assessing the results of the hybridization steps, Fan et al provide examples for Design and Optimization of a Set of Padlock Probes and Hybridization Oligos for Targeted Detection and Quantification of a Subset of Genes This example presents an innovative method based on padlock circularization and hybridization-based decoding (FIG. 1). This method is built upon ultra-high degree multiplexed capture (>300,000 per reaction) using padlock probes for useful for genomic, transcriptomic and methylomic analyses, as well as technology for accurate decoding of randomly assembled bead arrays. In some embodiments, colonies for an entire transcriptome can be generated using either random hexamers or oligo-dT primers (FIG. 1A). In some embodiments, colonies for a specific subset of genes of interest can then be tagged with gene-specific barcodes using a library of padlock probes (FIG. 1B). Padlock probes are designed in a similar manner as the full transcriptome probe sets previously developed for digital RNA allele-typing according to the incorporated materials disclosed in Zhang, K. et al. Nat Methods 6, 613-618 (2009) and Noggle, S. et al. Nature 478, 70-75 (2011). A neural network, trained for predicting probe efficiency using experimental data accumulated on over 400,000 probes, is integrated into a probe design algorithm called ppDesigner. The entire probe design, synthesis and production process has been highly optimized. Long oligonucleotides (up to 300 nt in length) acquired from Agilent Technologies contain amplification adaptors to allow production of essentially an unlimited amount of functional padlock probes from a set of seed oligos. The design of the barcoding region incorporates the error-checking scheme described in the incorporated materials of Gunderson et al. (Genome Res. (2004) 14:870-877). For up to 1700 target genes, eight rounds of hybridization decoding are performed. In each round, a colony can have three states: two fluorescent (FAM and Cy3) ‘ON’ states and one nonfluorescent OFF state. In this three-state, eight-stage decoding scheme, the valid barcode for each gene is required to have exactly two OFF stages and six ON stages for error checking Each padlock probe only needs six slots for hybridization (corresponding to the six ON stages). For the hybridization sequences used in these six slots, a set of 16 (8 stages x 2 ON states) 20-mers is selected that are orthogonal to the human genome and have uniform annealing parameters. This eight-stage barcoding scheme is sufficient to decode up to 1792 transcripts with a low error rate Fan at Example 1, 14:1-48. “Example 2 Design and Optimization of a Set of Padlock Probes for Targeted Detection and Quantification of BRAF V600E, EGFR T790M and EGFR L858R Mutations In this example, allele-specific (padlock) probes are generated for disease causing mutations. Specifically, point mutations are detected in genomic DNA and the decoding scheme is utilized to measure the frequency and spatial distribution of these mutations within tissue sections at single cell resolution. For each of the specifically targeted mutations described below, a padlock probe is designed for specific hybridization to either the wild-type or mutant version of the gene. Then, the padlock probe serves as a template for RCA as described in greater detail herein. The resulting amplicon is detected and decoded using a barcode sequence integrated into the padlock probe.” Fan at Example 2, 14:49-67.
Fan explicitly incorporates Gunderson, et al., Decoding Randomly Ordered DNA Arrays, 14 Genome Res, 870-877 (2004) for its decoding and error-checking methods, which discloses codewords of multiple binary values 1 and 0, wherein a value of 1 is obtained when the fluorescent signal is detected during a stage of sequential hybridization and a value of 0 is obtained when a fluorescent signal is not detected (or a different signal is detected) in another stage of sequential hybridization. For example, “Our algorithm uses sequential hybridizations of dye-labeled oligonucleotides, or decoders, complementary to bead sequences to create a combinatorial decoding scheme for arrays. It is distinct from sequencing by hybridization (SBH), which has been used successfully to characterize sequences de novo by hybridization to all n-mers or a well-chosen subset, typically in the range of 4- to 10-mers (Drmanac et al. 1996, 1998; Gunderson et al. 1998; Brenner et al. 2000). Our approach uses longer sequences, each designed to hybridize to a defined target with high specificity. It is capable of decoding, with high accuracy, many 1000s of bead types. Each bead type is defined by a unique DNA sequence that is recognized by a complementary decoder.” Gunderson, at 870. “Decoding process. (A) The sequential hybridization process is illustrated for a single bead, of bead type 2. In stage 1, a complementary decoder hybridizes to the oligonucleotide probe that is attached to the bead (for details of the procedure, see Methods). The decoder is labeled with a fluorophore (green in stage 1, red in stage 2, and green in stage 3). The fluorescent signal is read by imaging the entire array. The array is then dehybridized, and the process is repeated for two more stages. (B) A scanning electron micrograph of an array of beads, artificially colored to represent three sequential hybridization stages. The images, taken collectively, reveal a combinatorial code for each bead. Note that the bead circled in yellow has the color signature GRG or code 010. (C) Colors, or states, are assigned to individual decoder sequences in each stage to produce a unique combination across stages. This signature, or code, identifies each bead type. As indicated in the parity code column, an extra decoding stage (data not shown) can be performed to provide an error checking parity bit. After three stages of decoding, all the beads are uniquely identified by their color.” Gunderson, at 872, Figure 2. To the extent that Gunderson is not considered to be an express disclosure of Fan, the motivation to combine Fan with the error correction/detection methods of Gunderson is explicit. Both Fan and Gunderson rely on a three-state, eight-stage decoding scheme. Gunderson, at 871-2, Section "Error Checking": “The error-checking concepts that we have developed, and that underlie the robustness of our hybridization-based approach, are based on algorithms from digital information theory (Hamming 2066). ... In our implementation of three-state decoding, the most common errors are transitions from an ON state, one or two, to the OFF state, zero. Less common are transitions from OFF to ON. Transitions from one ON state to the other ON state are extremely rare. This can be explained by the fact that such transitions require two simultaneous classification errors: a mistaken ON in one color channel and a mistaken OFF call in the other color channel. We used these biases in error rates by designing the decode stages so that every valid code has a fixed number of OFF states. ... The space of all possible color codes is divided into three categories: used valid, unused valid, and invalid (table 1).” See Gunderson at 872, Table 1 (summarizing a codebook with respect to the distribution and use of 6561 codes in the three-state, eight-stage system.)
To the extent that Fan and incorporated Gunderson do not explicitly teach codewords comprising multiple binary values 1 and 0, wherein a value of 1 is obtained when the fluorescent signal is detected from one of the plurality of readout probe hybridized complexes or one of the different plurality of readout probe-hybridized complexes at a respective location within the sample while a value of 0 is obtained when the fluorescent signal is not detected from one of the plurality of readout probe-hybridized complexes or one of the different plurality of readout probe-hybridized complexes at the respective location within the sample, such limitations would have been inherent and/or obvious in view of Curry. Moreover, the use of binary codewords representing the plurality of different nucleic acid targets at locations within the sample were well known in the art at the time of the invention and it would have been obvious to combine the teachings of Fan/Gunderson with Curry, Hamady, and/or Chee. Hamady (Patent Publication No. US2010/0323348) teaches the use of Hamming code to create an error detection system for nucleic acid barcodes detection to identify nucleic acid targets. See e.g., Hamady at [0097]-[0100], [0108]-[0110], [0135]-[0145], Figs. 1B and 1C (showing error correction of binary barcodes). Chee (Patent Publication No. US2007/0231824A1) teaches the use of binary code to encode bioactive agents such as nucleic acids. For example: “In a preferred embodiment, each subpopulation of beads comprises a plurality of different IBLs. By using a plurality of different IBLs to encode each bioactive agent, the number of possible unique codes is substantially increased. That is, by using one unique IBL per bioactive agent, the size of the array will be the number of unique IBLs (assuming no ‘reuse’ occurs, as outlined below). However, by using a plurality of different IBLs per bead, n, the size of the array can be increased to 2n , when the presence or absence of each IBL is used as the indicator. For example, the assignment of 10 IBLs per bead generates a 10 bit binary code, where each bit can be designated as ‘1’ (IBL is present) or ‘0’ (IBL is absent). A 10 bit binary code has 210 possible variants.” Chee at [0102]. This further supports the teaching of Fan teaches matching the codewords (the temporal order of signal signatures) with valid codewords in a codebook (list of identifiers identifying the probe reagent corresponding to a specific analyte) by comparing the codewords to the valid codewords in the book, wherein the codebook comprises the valid codewords of the plurality of nucleic acid targets. For example: “These barcodes can be accurately decoded by hybridization similar to the decoding of Illumina bead array, using a combinatorial chemistry approach (Gunderson et al. Genome Res. 14(5):870-877 (2004.)” Fan at 3:36-40. “During decoding, labeled decoder probe oligonucleotides are sequentially hybridized to the decoder sequences to specifically determine the identity of each barcode sequence. Sequential hybridization of decoder probes to decoder sequences is described by Gunderson et al. (Genome Res. (2004) 14:870-877), the content of which is incorporated by reference in its entirety. Additional description of decoding algorithms and the use of sequential hybridization useful in the methods presented herein can be found in U.S. Pat. Nos. 7,060,431; 7,033,754; 7,226,734; and 7,455,971, the disclosures of each of which are hereby incorporated by reference in their entireties.” Fan at 8:53-64. “The design of the barcoding region incorporates the error-checking scheme described in the incorporated materials of Gunderson et al. (Genome Res. (2004) 14:870-877). For up to 1700 target genes, eight rounds of hybridization decoding are performed. In each round, a rolony can have three states: two fluorescent (FAM and Cy3) "ON" states and one nonfluorescent OFF state. In this three-state, eight-stage decoding scheme, the valid barcode for each gene is required to have exactly two OFF stages and six ON stages for error checking Each padlock probe only needs six slots for hybridization (corresponding to the six ON stages). For the hybridization sequences used in these six slots, a set of 16 (8 stages x 2 ON states) 20-mers is selected that are orthogonal to the human genome and have uniform annealing parameters. This eight-stage barcoding scheme is sufficient to decode up to 1792 transcripts with a low error rate. Fan at Example 1, 14:32-48. To the extent that Gunderson is not considered to be an express disclosure of Fan, the motivation to combine Fan with the error correction/detection methods of Gunderson is explicit. Both Fan and Gunderson rely on a three-state, eight-stage decoding scheme. Gunderson, at 871-2, Section "Error Checking": “The error-checking concepts that we have developed, and that underlie the robustness of our hybridization-based approach, are based on algorithms from digital information theory (Shanon 1948a,b; Hamming 2066). ... In our implementation of three-state decoding, the most common errors are transitions from an ON state, one or two, to the OFF state, zero. Less common are transitions from OFF to ON. Transitions from one ON state to the other ON state are extremely rare. This can be explained by the fact that such transitions require two simultaneous classification errors: a mistaken ON in one color channel and a mistaken OFF call in the other color channel. We used these biases in error rates by designing the decode stages so that every valid code has a fixed number of OFF states. ... The space of all possible color codes is divided into three categories: used valid, unused valid, and invalid (table 1).” See Gunderson at 872, Table 1 (summarizing a codebook with respect to the distribution and use of 6561 codes in the three-state, eight-stage system.) To the extent Fan/Gunderson is argued by Vizgen or found by the Court not to explicitly teach applying an error detection or correction system, matching the one of the codewords with another of the valid codewords in the codebook, or discarding the one of the codewords, such limitations would have been inherent and/or obvious in view of Curry, Hamady, and/or Chee as described in 10x’s Invalidity Contentions. See Attachment 5, limitation 1[j]. Hamady teaches the use of Hamming code to create an error detection system for nucleic acid barcodes detection to identify nucleic acid targets. See e.g., Hamady at [0097]-[0100], [0108]-[0110], [0135]-[0145], Figs. 1B and 1C (showing error correction of binary barcodes). Chee teaches the use of parity analysis during decoding to detect errors in binary codes. See e.g., Chee at [0241]-[0250]. Fan teaches determining the plurality of nucleic acid targets in the sample based on results from the matching step. For example: “Presented herein are methods and compositions for obtaining sequence information from one or more individual cells. The methods are useful for obtaining sequence information for a single nucleotide sequence, and for multiplex generation of sequence information from an individual cell. In some embodiments presented herein, the methods comprise first amplifying one or more nucleic acid molecules in situ within a cell, and subsequently hybridizing a barcode probe to the amplified nucleic acid molecules. The identity of the amplified nucleic acid molecules is then determined by decoding of the barcode probes. In other embodiments presented herein, the methods comprise first hybridizing one or more different barcode probes to one or more nucleic acid molecule in situ within a cell, and subsequently amplifying the barcode probe, thereby generating an amplified barcode probe. The identity of the nucleic acid molecules is then determined by decoding of the amplified barcode probes.” Fan at 1:58-2:7 (emphasis added). “A nucleic acid sample that is amplified, sequenced or otherwise manipulated in a method disclosed herein can be, for example, DNA or RNA. Exemplary DNA species include, but are not limited to, genomic DNA (gDNA), mitochondrial DNA, chloroplast DNA, episomal DNA, viral DNA and copy DNA (cDNA). One non-limiting example of a subset of genomic DNA is one particular chromosome or one region of a particular chromosome. Exemplary RNA species include, without limitation, coding RNA such as messenger RNA (mRNA), and non-coding RNA (ncRNA) such as transfer RNA (tRNA), microRNA (miRNA), small nuclear RNA (snRNA) and ribosomal RNA (rRNA). Further species of DNA or RNA include fragments or portions of the species listed above or amplified products derived from these species, fragments thereof or portions thereof. The methods described herein are applicable to the above species encompassing all or part of the complement present in a cell. For example, using methods described herein the sequence of a substantially complete genome can be determined or the sequence of a substantially complete targeted nucleic acid sequences such as mRNA or cDNA complement of a cell can be determined.” Fan at 6:60-7:14.
Thus, the claimed invention as a whole was clearly prima facie obvious.
Conclusion
No claim is allowed.
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/Joseph Woitach/ Primary Examiner, Art Unit 1687
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