DETAILED ACTION
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
This application is the national phase of PCT/US2022/073956, filed 20 July 2022, claiming priority to US provisional application 63/223,664, filed 20 July 2021. The International Search Report and Written Opinion issued in the PCT application have been received and reviewed.
Election/Restrictions
Applicant’s election without traverse of Group I, and of the species of synthetic adaptors, in the reply filed on 07 July 2026 is acknowledged.
Claim 39 is withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 07 July 2026.
Claims 1, 5-6, 8, 11, 14, 17, 21-22, and 29-38 correspond to the elected invention and are under consideration herein.
Information Disclosure Statement
With regard to the Information Disclosure Statement filed 19 January 2024, it is noted that the examiner has corrected a typographical error in the date of cite no. 2.
Nucleotide and/or Amino Acid Sequence Disclosures
Summary of Requirements for Patent Applications Filed On Or After July 1, 2022, That Have Sequence Disclosures
37 CFR 1.831(a) requires that patent applications which contain disclosures of nucleotide and/or amino acid sequences that fall within the definitions of 37 CFR 1.831(b) must contain a “Sequence Listing XML”, as a separate part of the disclosure, which presents the nucleotide and/or amino acid sequences and associated information using the symbols and format in accordance with the requirements of 37 CFR 1.831-1.835. This “Sequence Listing XML” part of the disclosure may be submitted:
1. In accordance with 37 CFR 1.831(a) using the symbols and format requirements of 37 CFR 1.832 through 1.834 via the USPTO patent electronic filing system (see Section I.1 of the Legal Framework for Patent Electronic System (https://www.uspto.gov/PatentLegalFramework), hereinafter “Legal Framework”) in XML format, together with an incorporation by reference statement of the material in the XML file in a separate paragraph of the specification (an incorporation by reference paragraph) as required by 37 CFR 1.835(a)(2) or 1.835(b)(2) identifying:
a. the name of the XML file
b. the date of creation; and
c. the size of the XML file in bytes; or
2. In accordance with 37 CFR 1.831(a) using the symbols and format requirements of 37 CFR 1.832 through 1.834 on read-only optical disc(s) as permitted by 37 CFR 1.52(e)(1)(ii), labeled according to 37 CFR 1.52(e)(5), with an incorporation by reference statement of the material in the XML format according to 37 CFR 1.52(e)(8) and 37 CFR 1.835(a)(2) or 1.835(b)(2) in a separate paragraph of the specification identifying:
a. the name of the XML file;
b. the date of creation; and
c. the size of the XML file in bytes.
SPECIFIC DEFICIENCIES AND THE REQUIRED RESPONSE TO THIS NOTICE ARE AS FOLLOWS:
Specific deficiency - The incorporation by reference paragraph required by 37 CFR 1.834(c)(1), 1.835(a)(2), or 1.835(b)(2) is missing, defective or incomplete. In particular, it is noted that the required incorporation by reference statement must include all of the name of the XML file, the date of creation, and the size in bytes.
Required response - Applicant must:
• Provide a substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3), and 1.125 inserting the required incorporation by reference paragraph, consisting of:
• A copy of the previously-submitted specification, with deletions shown with strikethrough or brackets and insertions shown with underlining (marked-up version);
• A copy of the amended specification without markings (clean version); and
• A statement that the substitute specification contains no new matter.
Specific deficiency - Sequences appearing in the drawings are not identified by sequence identifiers in accordance with 37 CFR 1.831(c); see in particular Figure 2B. Sequence identifiers for sequences (i.e., “SEQ ID NO:X” or the like) must appear either in the drawings or in the Brief Description of the Drawings. It is noted that as the Drawings filed 19 January 2024 are otherwise acceptable they have been approved; however, correction of either Figure 2B or of the description thereof is required.
Required response – Applicant must provide:
Amended drawings in accordance with 37 CFR 1.121(d) inserting the required sequence identifiers;
AND/OR
A substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3), and 1.125 inserting the required sequence identifiers (i.e., “SEQ ID NO:X” or the like) into the Brief Description of the Drawings, consisting of:
• A copy of the previously-submitted specification, with deletions shown with strikethrough or brackets and insertions shown with underlining (marked-up version);
• A copy of the amended specification without markings (clean version); and
• A statement that the substitute specification contains no new matter.
Claim Interpretation
Regarding dependent claim 14, it is noted that the specification at paragraph 60 of the corresponding published application states with regard to the term “Unique Molecular Index” tag” that “UMIs, also known as ‘Molecular Barcodes’, are short sequences or molecular tags that are added to DNA or RNA fragments in some long-read sequencing library preparation protocols to identify the individual input DNA or RNA molecule in a population of DNA or RNA molecules”. Thus, the term as used in the claim is consistent with the well-known meaning of UMI as it applies to methods such as those of the claims.
Regarding dependent claims 30-32, it is noted that the recitation in claim 30 of ‘the processing step” has been interpreted as referring to the “processing” of b set forth in claim 29 (as this is the only previously referenced “processing”); i.e., the use of the term “step” in this manner does not render the claims indefinite.
Regarding claim 30-32, it is noted that the reference to “the long reads” in claim 31 have been interpreted as referring to the “long reads representing full-length transcripts” recited in b of claim 30. As this type of “long read” is defined in the claim itself, the use of the term in these claims is not indefinite (in contrast to the other uses of this term in the claims discussed below).
Claim Rejections - 35 USC § 112(b)/second paragraph
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1, 5-6, 11, 14, 17, 21-22, and 29-38 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Regarding all of claims 1, 5-6, 11, 14, 17, 21-22, and 29-38, independent claim 1 at line 1 recites “an all-in-one RNA-sequencing assay for, comprising”, with nothing stated following the recitation of “for”. The claim appears to have been intended to include some type of further limitation that is missing, and it is therefore unclear what additional limitations (if any) are required. Clarification is therefore needed.
Regarding all of claims 1, 5-6, 11, 14, 17, 21-22, and 29-38, independent claim 1 in e) recites “thereby obtaining a sequence for each of the RNA molecules that is target captured from the original RNA sample”. This limitations lacks sufficient antecedent basis, as the claim previously recites and requires “target capturing one or more cDNAS of interest”, but not an RNA or RNAs (including “RNA…target captured from the original RNA sample”). Further clarification is therefore needed with regard to what the claims require with respect to “target captured” RNAs. It is noted that for purposes of comparing the claims to the prior art, given that the claim previously refers to “target capturing one or more cDNAs of interest…”, the claim at e) has been interpreted as simply requiring “sequencing the one or more cDNAs” that are referenced in the prior step/activity of d).
Regarding all of claims 1, 5-6, 11, 14, 17, 21-22, and 29-38, independent claim 1 at the last two lines recites the limitation “wherein the assay simultaneously provides long read sequencing and sequencing depth of the one or more RNAs of interest”. First, the language “assay….provides” is vague, and does not make sufficiently clear whether this language recites a further/additional concrete requirement imposed on the method claimed, or whether the “wherein” language is simply descriptive of what the previously recited method as performed “provides”. Second, the term “long read” is a relative term, rendering the claim indefinite. The term is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. It is noted that the specification provides an example of “long read” sequencing as referring to sequencing of cDNAs produced from non-fragmented RNAs such that reads correspond to full length transcripts (see, e.g., paragraph 7 of the published application); however, as this embodiment is referenced in dependent claim 6, it appears that the scope of “long read sequencing” encompasses other types of sequencing that are not clearly defined (and this term may have different meanings in different prior art contexts). Third, the statement “assay…provides…sequencing depth” is also confusing, because “sequencing depth” is a characteristic of any type of sequencing; the term as used lacks meaning unless some kind of actual requirements/ criteria are provided (as is done in dependent claim 6).
Claim 5 is indefinite because the use of “and/or’ and “or” within what appears to be a list of alternatives creates confusion with regard to what the possible alternatives are, and what limitations apply to the claim (as it appears that there may be different subgrouping within the larger group, but it is unclear what those subgroupings are). Clarification is therefore required with regard to what the different alternatives embraced by the claim are.
Claim 5 is also indefinite over the recitation of the limitation “pest sequence within a host organism”, as it is not clear what is (and is not) embraced by this language. It is noted that the specification provides an example of “malaria RNAs….out of an infected person”, such that “pest sequence” appears to constitute a broad reference to infectious agents. However, it is unclear whether the claim simply encompasses such “pest sequences” (e.g., a “pre-selected locus” that is simply “malaria RNAs” that may be found in a host cell), or whether the claim requires some type of further limitation pertaining to a “host organism” (as is suggested by the language ‘within a host organism”). Clarification is therefore required, as there are multiple reasonable interpretations of the claim language that impart different boundaries on what is claimed.
Claims 6 and 23 (dependent from claim 6) are indefinite over the recitation in claim 6 of the limitation “long read sequencing comprises full-length transcripts” because “long read sequencing” is a process/method, while transcripts are products that may potentially be sequenced. It is not clear how this language of claim 6 further limits the characteristics of “long read sequencing” as recited in claim 1. If the claim is intended to require that “long read sequencing” refers to or requires the sequencing of full-length transcripts by the claimed method, this should be stated in the claim.
Claim 11 is indefinite over the recitation of the limitation “wherein the synthetic adapter is the Universal miRNA Cloning Linker”. While it is noted that only one embodiment of claim 1 requires a “synthetic adapter”, to the extent that the claims require such an adapter, what is required by claim 11 is not sufficiently clear. First, there is insufficient antecedent basis for this limitation in the claims, as there is no prior reference to this linker. Second, it is noted that while the terminology in the claim is not trademarked, the specification discloses a preferred linker defined by a nucleotide sequence (see, e.g., Figure 2B) that may or may not correspond to/limit what is being claimed. Accordingly, further clarification is needed with regard to how the reference to “the Universal miRNA Cloning Linker” further limits what is claimed.
Claim 14 is also indefinite over the recitation “wherein generating a cDNA library comprises using a DNA oligonucleotide complementary to the synthetic adapter….”, because claim 1 as written does not require use of a synthetic adapter (rather, this is one alternative recited in the claims). The language of claim 14 does not clearly state that the method of claim 1 must employ a “synthetic adapter”, as would appear to be necessary for the more preferred method of claim 14. Accordingly, clarification is required, particularly with regard to whether a “synthetic adapter” is or is not required, and to the extent that it is not, how the claim is further limiting of the other alternative embodiments of claim 1 (if it is).
Regarding dependent claim 17, while it is noted that the language “wherein the multiplex index sequence permits pooling and subsequent demultiplexing of the indexed cDNA libraries” is only further limiting of a property/capability of “the multiplex index sequence” of claim 1 (as the claim as written does not actual require performance of any further actions/steps), the claim is indefinite over the recitation of the limitation “the indexed cDNA libraries” as there is insufficient antecedent basis for this language in claim 1. While claim 1 does recite generation of sequencing libraries including index sequences as well as target capturing and sequencing cDNAs within such libraries, given the references in claim 1 to multiple different populations of cDNAs that may be considered “libraries”, further clarification is needed to ensure that it is clear what “libraries” are being referenced in claim 17.
Claim 21 is indefinite over the recitation of the limitation “further comprising amplifying the libraries using primers that do not contain the tags or the multiplex index sequences, permitting amplification of all pool libraries at the same time”. First, as there are multiple different groups of cDNAs recited in claim 1 that constitute “libraries”, it is not clear what library/libraries are being referenced in claim 21. Second, as the term “pool libraries” is not previously employed, it is unclear what is being referenced/required by that term (i.e., clear antecedent basis is lacking).
Regarding claim 22, the recitation of “the multiplex indexes for multiplexing” is unclear, as it is not clear what is being further limited, and how. Claim 1 includes a reference to generation of a cDNA sequencing library in which the library includes “a multiplex index sequence identifying the library”; however, there is no recitation of “multiplexing” in the claims, nor is it clear whether/how the “multiplex index sequence” of claim 1 relates to the “multiplex indexes for multiplexing” of claim 22. Further clarification is therefore required.
Claim 23 is indefinite because it recites a further limitation on “obtaining long reads”; however, the claims from which it depends do not recite or require such an action/step. Clarification is therefore required with regard to what aspect of claims 1/6 is/are being further limited, and how.
Claim 23 contains the trademark/trade name Oxford Nanopore. Where a trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph. See Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982). The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product. A trademark or trade name is used to identify a source of goods, and not the goods themselves. Thus, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name. In the present case, the trademark/trade name is used to identify/describe particular sequencing assays/materials and, accordingly, the identification/description is indefinite.
Claim 23 is also indefinite over the recitation the limitation “sequencing of the cDNAs”, because there are multiple cDNAs/cDNA libraries previously recited (such that further clarification is needed regarding what cDNAs are being referenced).
Claims 29-38 are indefinite because it is unclear whether the claims do or do not require performing the method of claim 1, from which the claims depend. Claim 29 recites “obtaining or having obtained long read sequences of RNA transcripts expressed from at least one pre-selected locus using the all-in-one RNA-sequencing assay of claim 1”. First, the use of the relative terminology “long read” is indefinite for the reasons indicated above, and it is reiterated that the language of claim 1 does not make clear what is required by that claim with regard to “long read sequencing” (such that that nature of what is required by claim 29 is also unclear). Second, claim 29 refers to “obtaining or having obtained” long read sequences “using the all-in-one RNA sequencing assay of claim 1”; however, this language is vague, and fails to make clear how the assay is “used” to result in “obtaining or having obtained long read sequences” (which further suggests that the performance of the entirety of the method of claim 1 may not be a required part of the method of claim 29, raising the issue of improper dependency, addressed below). Further clarification is therefore needed.
Claim 32 is indefinite over the recitation “further comprising determining the features of RNA products, wherein the features comprise…” because there is no indication in the claim as to what “RNA products” are being referenced, and how those “RNA products” relate to the method being claimed. Further clarification is therefore needed.
Claim 33 is indefinite over the recitation of the “wherein the gene is a transgene and determination of the transgene expression stability leads to…” because there is insufficient antecedent basis for the limitation “the gene”, and it is also unclear how the prior claims relate to “the gene” and to “determination of the transgene expression stability”. Further clarification is needed.
Claims 34-35 are indefinite over the recitation in claim 34 of the limitation “comprising selecting a transgenic event that has gene-like transgene expression patterns in a transgenic organism”, because it is entirely unclear how this relates to the claims from which claims 34-35 depend (as those claims do not reference a “transgenic event”, “gene-like transgene expression patterns”, etc. Further, it is not clear what type of “selecting” is being referenced (and again, how such a “selecting” relates to the claims). It is noted that claim 35 recites further more particular types of “gene-like transgene expression”, but again it is not clear how this relates to the claimed methods.
Claim 36 is indefinite over the recitation of the limitation “further comprising identifying off-type RNAs that trigger RNA decay, RNA degradation, transcriptional or post-transcriptional silencing”. First, it is not clear how such an “identifying” of “off-type RNAs” relates to the claimed methods (e.g., where does this occur, and how does it relate to the recited “analyzing gene expression”, etc.). Second, it is not clear whether the claim requires all of the recited elements, or whether these were intended to be alternatives (as the present wording of the claim is ambiguous).
Claims 37-38 are indefinite over the recitation of the limitation “the long reads” in claim 37, as again this terminology is indefinite as used in this manner (as discussed above), such that it is not clear what must be “compared” to meet the requirements of the claims.
Claim Rejections - 35 USC § 112(d)/fourth paragraph
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claims 29-38 are rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends. As noted above, claims 29-38 are indefinite, and it is unclear from the present language of claim 29 whether the claim does nor does not actually require performing the method of claim 1. To the extent that claims 29-38 do not require performing the method of claim 1, they are not proper dependent claims. Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
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.
Claim(s) 1, 5-6, 8, 11, 14, 17, 21-22, and 29-38 are rejected under 35 U.S.C. 103 as being unpatentable over Abvitro (US 2020/0354784 A1 [12 Nov 2020]; cited in IDS) in view of Boone et al (Nucleic Acids Research 46(6):2701 [2018]; cited herein).
Abvitro teach methods “for target gene sequencing and single cell barcoding in conjunction with analysis of gene expression”, which methods “can be used to carry out transcriptome sequencing, e.g., RNA sequencing, to capture transcriptome of single cells paired with full receptor immune receptor sequences such that information about the immune repertoire and transcriptome of a cell can be determined” (see entire reference, particularly the Abstract). Abvitro teach that their method provides advantages over prior art methods in capturing full length immune receptor sequences, teaching that “one or more full-length barcoded target polynucleotide sequences or selected fragments thereof are produced, while co-generating a multitude of barcoded polynucleotide sequences, the collection of which substantially represents the transcriptome or genome of a cell or plurality of cells”, with the barcoding permitting “analysis and quantification of expression or presence of polynucleotides from the same cell”, and which methods also allow for transcriptome analysis from a single cell or multitude of cells (see, e.g., paragraphs 4-5).
With regard to the recitation in independent claim 1 of an “all-in-one RNA sequencing assay” and the analysis of RNAs of interest “transcribed from at least one pre-selected locus of an organisms genome”, Abvitro teach targeting a cell “that expresses a particular gene or genes or interest, such as a target gene” (providing the example of an immune receptors” (see, e.g., paragraph 82), and teach that such target sequences ( as well as the genome or transcriptome sequences of a plurality of cells) may be “produced in one simultaneous reaction” providing “a mechanism for linking sequence information of sequences derived from same cell” in “one single reaction assay”, further stating that “one can sequence any number of cells and any number of targeted regions per cell” (paragraph 83). Abvitro also teach a “one pot reaction” for preparation of sequencing libraries prior to sequencing “using any of a variety of sequence platforms” (paragraph 88). Thus, Abvitro disclose embodiments corresponding to an “all-in-one RNA-sequencing assay” that targets full length RNAs transcribed for a target locus.
With regard to the process of RNA library preparation, Abvitro teach “adding a second adaptor to each of a plurality of barcoded single-stranded polynucleotides at or near a terminal end that is opposite a first adapter attached to each of the barcoded single stranded polynucleotides”, and further that such barcoded single-stranded polynucleotides may contain “a molecular barcode that is unique to each single-stranded polynucleotide” (see, e.g., paragraphs 6-7; paragraph 111). Thus, Abvitro teach incorporation of multiple adaptors into collections/libraries of cDNAs, including a barcode identifying a features such as “cell of origin”/source of the group/population of RNAs, as well as a barcode that “is unique to each” polynucleotide, where those barcodes may be incorporated into adaptors attached to opposite ends of a strand. These features correspond to an “unique tag” for a strand (as recited in b) of claim 1) and a type of “multiplex index sequence” that identifies a collection/library as recited in c) of claim 1. With further regard to “ligating” an adaptor as recited in a) of claim 1, Abvitro teach that their methods may comprise conversion of RNA to DNA via a “prior ligation step, to ligate a linker fragment to the RNA, thereby permitting use of universal primers to initiate reverse transcription” (paragraph 219). Abvitro also depict/illustrate amplification and sequencing of dual barcoded transcripts, including teaching target transcripts and transcriptome sequences into which 3’ adaptors and 5’ sequencing adapters having been incorporating, stating that sequencing uses “a primer specific to the universal priming sequence of the first adapter and a target specific primer” (Figure 2 and the description thereof). Regard “target capturing” as recited in d) of claim 1, it is reiterated that Abvitro teach targeting a cell “that expresses a particular gene or genes or interest, such as a target gene” (providing the example of an immune receptor) (see, e.g., paragraph 82), and Abvitro teach the use of “target specific primer” in this embodiment of their methods (see, e.g., paragraph 76, as well as paragraph 100, 349 and 358), which meets the requirement for enriching for cDNAs of interest using “oligonucleotide probes that hybridize to cDNAs comprising sequences specific” to RNA(s) of interest. Regarding e) of claim 1, Abvitro teach sequencing their collections/libraries of cDNAs via, e.g., high throughput sequencing of libraries that may include “full-length target sequences” (paragraph 84; see also paragraph 96), teaching that sequencing “can be performed by any sequencing method known in the art”, including nanopore sequencing (see, e.g., paragraphs 362-364), and with Abvitro also teaching using a “long-read sequencer” to sequence their polynucleotides (paragraph 317). Abvitro thus teach sequencing as recited in e) of claim 1, and further teach a variety of sequencing methods including “long-read” sequencing, which inherently includes some type of “sequencing depth”.
Abvitro thus teaches methods meeting all of the requirements of the claims, with the exception of explicitly teaching the ligating of an adaptor “to the 3’ end” of their target RNAs prior obtaining full-length cDNA transcripts.
Boone et al provide an overview of “technical developments in the field” RNA and DNA library construction, noting that a primary application of library construction is in construction of sequencing libraries (see entire reference). Boone et al teach that construction of RNA libraries may employ ligation of adaptors to the 3’ end of the RNA molecule, further teaching that such an approach “preserves the directionality of RNA molecules” (see page 2705, right column, under the heading “RNA ligation”, through page 2707, including Figure 2). Boone et al also address the use of barcoding/tagging, teaching that barcoding is “a common and recommended approach” in such sequencing methods that increases cost efficiency, and further that “the earlier samples are barcoded……the better”; Boone et al further teach that it was known in the prior art to incorporate a barcode (i.e., a tag) during RNA ligation (as well as during cDNA synthesis) (see page 2712, right column under “Barcodes, Molecular Tags and Frameshifts”, through page 2713). Regarding the elected species of “synthetic adapters”, adaptors/barcodes of the type disclosed by Boone et al inherently constitute “synthetic adapters”.
In view of the teachings of Abvitro and Boone et al, 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 have employed ligation of synthetic adapters to the 3’ end of each RNA molecule, as taught by Boone et al, when preparing libraries of full-length RNAs as taught by Abvitro. First, an ordinary artisan would have recognized that any of a variety of well-known techniques (including those disclosed by Boone et al) could have been employed successfully in the methods of Abvitro to achieve the predictable result of cDNA preparation, as well as tagging/identification of cDNAs. Additionally, given Boone et al’s teachings of the benefits provided by the use of 3’ adapter ligation, as well as barcoding/tagging, an ordinary artisan would have been further motivated to have made such a modification for those benefits (e.g., preserving directionality, cost-efficiency). Given the detailed guidance provided by Abvitro and Boone et al, an ordinary artisan also would have had a reasonable expectation of success in performing such methods.
With further regard to claim 5, it is reiterated that Abvitro teaches targets including a gene or genes of interest (see, e.g., paragraph 82).
Regarding claims 6 and 23, claim 6 (from which claim 23 depends) is further limiting only of what the “wherein” clause at the end of claim 1 states regarding what performance of the method “provides” (as opposed to adding any further required steps to the methods claimed); thus, as Abvitro in view of Boone et al suggest the assay/method actually claimed, they also suggest what this claim requires (see also MPEP 2111.04 regarding such “wherein” clauses). With further regard to claim 23, it is also noted that to the extent that the claim is intended to require “nanopore” sequencing, this is an alternative disclosed by Abvitro, as discussed above.
Regarding dependent claim 8, it is reiterated that Abvitro teach that their libraries may be generated from cellular RNA including, e.g., polyadenylated or “not polyadenylated” RNA (see paragraph 182).
Regarding claims 11 and 14, it is noted that both Abvitro and Boone et al teach use of synthetic adapters including tags/barcodes, as well as unique molecular index tags (see in particular Boone et al at page 2713); given the indefiniteness of claim 11, the adapters taught by Boone et al are sufficient to meet the requirements of the claims.
Regarding claim 17, the claim recites a feature that is provided by the use of a “multiplex index sequence” (but does not actually require further steps such as pooling and demultiplexing); thus, the teachings of Abvitro and Boone et al are sufficient to suggest what is claimed (as they provide teachings of such sequences; it is also noted that Boone et al describe such benefits/applications of unique molecular indexes at page 2712, right column-page 2713).
Regarding claim 21, while it noted that it is unclear what “libraries” are to be amplified “using primers that do not contain” tags, it is reiterated that Abvitro teaches a variety of applications of amplification, including the target-specific amplification noted above (which is sufficient to meet the requirements of the claim).
Regarding claim 22, which includes an additional requirement for end-repair of cDNA (followed by incorporation of adapters including indexes as already addressed above), Boone et al teach that such end repair is often performed following second strand synthesis (page 2707, right column, second full paragraph).
Regarding claim 29 and claims dependent therefrom, it is again noted that the manner in which these claims further limit claim 1 is entirely unclear; the limitations of obtaining transcripts of a pre-selected locus, as well as long-read sequencing, are addressed above, such that the teachings of Abvitro and Boone et al are sufficient to suggest what is claimed. With further regard to claims 30-32 and 36-38, which recite instructional limitations related to data analysis rather than any further actions/ manipulations, such non-functional descriptive material is not given patentable weight when comparing a claimed invention to the prior art (although it is noted that Boone et al do teach pooling/demultiplexing, and orienting sequencing reads, as discussed above, while Abvitro et al teach mapping sequence reads and “determining features” as a component of “transcriptome sequence data analysis [paragraphs 726-733])(see MPEP 2111.05).
With further regard to claims 33-35, which appear to require a target that is a transgene and determination of transgenic expression/events, Abvitro teach that a source of their target polynucleotides includes transgenic animals, including animals whose “endogenous immunoglobulin loci” have been inactivated and replaced by human immunoglobulin loci (see the discussion of target polynucleotides in paragraphs 133-179, and particularly paragraph 154 regarding transgenic animals/transgenes). Accordingly, Abvitro et al in view of Boone et al also suggest methods as set forth in claims 33-35.
Conclusion
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure. Oikonomopoulos et al (Frontiers in Genetics 11:606 [July 2020]; cited herein) provide an overview of methodologies “for transcript profiling using long-read technologies” (see entire reference). Yan et al (Nature Communications 9:3676 [2018]; cited herein) teach a method “called SMRT-Cappable-seq that combines the isolation of unfragmented primary transcripts with single-molecule long read sequencing” (see entire reference). Singh et al (Nature Communications 10:3120 [2019]; cited herein) teaches a method that combines target capture and long-read sequencing of target transcripts with short-read transcriptome profiling of barcoded single-cell libraries (see entire reference). Eckert et al (Microbial Genomics doi:10.1099/mgen.0.000087, 10 pages [2016]; cited herein) describes enriching target long DNA fragments by hybridization prior to nanopore sequencing (see entire reference).
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/DIANA B JOHANNSEN/Primary Examiner, Art Unit 1682
