DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claim Status and Action Summary
This action is in response to the papers filed on August 12, 2024.
Currently, claims 1-3, 5-6, 12-16, 18, 20-22, 25-27, and 36-38 are pending and under examination.
Priority
The present application, filed on September 13, 2023, is a 371 of PCT/US2022/071272, filed on March 22, 2022 and claims priority to U.S. Provisional Patent Application No: 63/164,958, filed on March 23, 2021.
Drawings
The drawings filed on September 13, 2023 are acceptable.
Specification
The use of the terms “Helicos Biosciences Corporation”, “Lightning Terminator”, “Bst 2.0”, and “Bst 3.0”, which are each a trade name or a mark used in commerce, has been noted in this application. Each term should be accompanied by the generic terminology; furthermore the term should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the term.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
It is noted that Sequence ID Nos: 2 and 3, present in the sequence listing part of the disclosure, are not specifically named or described in the sequence listing and do not appear in the specification.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
Claims 21 and 25 are rejected under 35 U.S.C. 112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor, regards as the invention.
Regarding claim 21, the claim recites “the nucleotides used in each sequencing flow step of the first… or the second plurality of sequencing flow steps comprise a single type of nucleotide base.” As presently recited, the open-ended claim language (i.e. “comprise”) renders the claim indefinite because it encompasses embodiments wherein the flow steps can comprise additional types of nucleotide bases. Therefore, the claim as written is not limited to contacting the hybrid with a single type of nucleotide base to the exclusion of other types of nucleotide bases. Furthermore, the “or” term emphasized above renders the claim indefinite because it encompasses embodiments wherein only one of the first or the second plurality of sequencing flow steps comprise[s] a single type of nucleotide base (i.e. at least one type) and encompasses embodiments wherein only one of the first or second plurality of flow steps comprises a single type of nucleotide base (i.e. only one of the first and second plurality of flow steps occurs).
Regarding claim 25, the claim recites “the nucleotides used in each dark sequencing flow step comprises a single type of nucleotide base.” As presently recited, the open-ended claim language (i.e. “comprise”) renders the claim indefinite because it encompasses embodiments wherein the flow steps can comprise additional types of nucleotide bases. Therefore, the claim as written is not limited to contacting the hybrid with a single type of nucleotide base to the exclusion of other types of nucleotide bases.
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.
Claim 21 and 25 are rejected under 35 U.S.C. 112(d) as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 21 depends upon claim 1. Claim 1 recites “each sequencing step in the first [and second] plurality of sequencing flow steps compris[es] combining the hybrid with nucleotides. Claim 21 recites “the nucleotides used in each sequencing flow step of the first [or second] plurality of sequencing flow steps comprise a single type of nucleotide base.”
As presently recited, the open-ended claim language (i.e. “comprise”) encompasses embodiments wherein the flow steps can comprise additional types of nucleotide bases (i.e. at least one single type of nucleotide base). Accordingly, the claim as written is not limited to contacting the hybrid with a single type of nucleotide base to the exclusion of other types of nucleotide bases. Therefore, claim 21 does not further limit claim 1 which recites that each sequencing flow step comprises combining the hybrid with nucleotides.
Furthermore, the “or” term encompasses embodiments wherein only one of the first or the second plurality of sequencing flow steps comprise[s] a single type of nucleotide base (i.e. at least one type) and encompasses embodiments wherein only one of the first or second plurality of flow steps comprises a single type of nucleotide base (i.e. only one of the first and second plurality of flow steps occurs). Therefore, as presently written, claim 21 also does not include all of the limitations of claim 1 (i.e. each sequencing flow step of the first and second plurality of sequencing flow steps comprises contacting the hybrid with nucleotides).
Claim 25 depends upon claim 1. Claim 1 recites “…using a set of one or more dark sequencing flow steps comprising combining the hybrid with nucleotides…” Claim 25 recites “…the nucleotides used in each dark sequencing flow step comprises a single type of nucleotide base.”
As presently recited, the open-ended claim language (i.e. “comprise”) encompasses embodiments wherein the dark sequencing flow step(s) can comprise additional types of nucleotide bases (i.e. at least one single type of nucleotide base). Accordingly, the claim as written is not limited to contacting the hybrid with a single type of nucleotide base to the exclusion of other types of nucleotide bases. Therefore, claim 25 does not further limit claim 1 which recites that each dark sequencing flow step comprises combining the hybrid with nucleotides.
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
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 1-3, 5-6, 12-16, 18, 20-22, 25-27, and 36-38 are rejected under 35 U.S.C. 103 as being unpatentable over Belgrader et al., US 2020/0002764 A1 (published January 2, 2020) in view of Rigatti et al., US 2013/0281306 A1 (published October 24, 2013).
Regarding claim 1, Belgrader et al. teach methods of sequencing polynucleotides comprising two or more barcode regions on the same end of a polynucleotide relative to a region of interest (Figures 9A, 51A, and paragraph 0673).
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Belgrader et al. further teach hybridizing a sequencing primer to the polynucleotide, wherein the polynucleotide comprises an intervening (i.e. spacer) sequence between the first and second barcode sequences (Belgrader et al., paragraph 0604-0605), and sequencing the barcode sequences (Belgrader et al., paragraph 0052-0054 and paragraphs 0604-0605) by known sequencing techniques such as “Illumina sequencing” (Belgrader et al., paragraph 0004). (i.e. combining the hybrid with labeled nucleotides and detecting the presence of incorporated nucleotides in a plurality of sequencing flow steps).
Belgrader et al. does not teach extending the sequencing primer through the intervening sequence using a set of one or more dark sequencing flow steps wherein nucleotides are incorporated into the polynucleotide without detecting the presence or absence of the incorporated nucleotide(s).
However, Rigatti et al. teach methods of sequencing polynucleotides comprising “fast forward” “limited dark steps” (Rigatti et al., figure 5 and paragraphs 0325-0328) (i.e. extending a sequencing primer through an intervening region without detecting the presence or absence of incorporated nucleotide(s)).
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Rigatti et al. further teach that “a barcode sequence can include an ordered series of determined regions and at least one dark region” (Rigatti et al., paragraphs 0306-0307) and “Sequence information obtained using iterations of at least one limited dark extension step and at least one limited read extension step can produce a molecular signature for a target nucleic acid that is predictable and informative (Rigatti et al., paragraph 0165).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have modified the method taught by Belgrader et al. comprising identifying uniquely barcoded nucleic acids from single cells wherein two or more barcode sequences are separated by a spacer (i.e. intervening) sequence with the teachings of Rigatti et al. that limited dark sequencing flow steps can be implemented to “fast forward” through non-informative sequence between barcode sequences that “produce a molecular signature for a target nucleic acid that is predictable and informative” (Rigatti et al., paragraph 0165). The ordinary artisan would have been motivated to modify the sequencing methods taught by Belgrader et al. with the limited dark sequencing steps taught by Rigatti et al. by the suggestion of Rigatti et al. that target nucleic acids can be identified by their molecular signatures comprising iterations of read extension steps and dark extension steps. Additionally, the ordinary artisan would have recognized the predictable advantages of reducing the amount of sequencing data required to identify particular target sequences in sequencing methods wherein the total number of read cycles is limited per flow cell/sequencing run and by extension, determines the total cost and time required for obtaining said sequencing data.
Regarding claim 2, Belgrader et al. teach that the barcode and intervening sequences are known (Belgrader et al., paragraphs 0551-0557, 0608, and Figure 51A). Rigatti et al. also teaches the “fast forward” sequence synthesized by the limited dark extension steps is known (Figure 5 and paragraphs 0362-0364).
Regarding claim 3, Rigatti et al. teach the dark sequencing flow steps extend the primer during each dark sequencing flow step (Rigatti et al., Figure 2A and paragraph 0353).
Regarding claim 5, Rigatti et al. teach sequencing the region of interest (Rigatti et al., paragraphs 0073 and 0288). Belgrader et al. also teach sequencing the region of interest (Belgrader et al., paragraph 0274)
Regarding claim 6, Belgrader et al. teach sequencing the two barcodes (i.e. “BC” and “UMI” from the “Read 1 primer” prior to sequencing the region of interest (i.e. V D J C) (Belgrader et al., figure 84B).
Regarding claim 12, Belgrader et al. teach associating each sequencing read (i.e. region of interest) with its barcode sequences (Belgrader et al., paragraph 0064).
Regarding claims 13-14, Rigatti et al. teach the nucleotides used in the sequencing flow steps are non-terminating nucleotides (Rigatti et al., paragraph 0166-0171).
Regarding claim 15, Rigatti et al. teach that the nucleotides used in the sequencing by synthesis steps can be labeled or unlabeled (Rigatti et al., paragraph 0242).
Regarding claim 16, Rigatti et al. teach that dark extensions (i.e. dark sequencing flow steps) include incorporating nucleotide monomer(s) without detecting the incorporation (Rigatti et al., paragraph 0162). Furthermore, Rigatti et al. teaches an example wherein a dark extension step is performed with nucleotide monomers only and two read extension steps are performed with nucleotide monomers comprising labels (Rigatti et al., paragraph 0325) (i.e. the nucleotides in the read extension, but not the dark extension step comprise labels).
Regarding claim 18, Belgrader et al. teach that the polynucleotide may further comprise a unique molecular identifier (Belgrader et al., paragraph 0011 and 0029).
Regarding claim 20, Belgrader et al. teach the polynucleotide can be a cDNA molecule (Belgrader et al., paragraph 0031).
Regarding claim 21, Rigatti et al. teach that each of first or second plurality of sequencing flow steps comprise a single type of nucleotide base (either mixtures of combinations of labeled nucleotides, or sequential addition of a single type of each of the four labeled nucleotides) (Rigatti et al., paragraph 0168).
Regarding claim 22, Rigatti et al. teach the nucleotides used in the first and second plurality of sequencing flow steps can comprise limited read extension steps including “doublet” and “triplet” deliveries of nucleotide monomers (i.e. comprising two or three different types of nucleotide bases) (Rigatti et al., paragraph 0170).
Regarding claims 25-26, Rigatti et al. teach examples of “limited dark extension” wherein “no more than one type of nucleotide monomer is incorporated without being detected” or “two or more types of nucleotide monomers are incorporated without being detected” or “three or more types of nucleotide monomers are incorporated without being detected” (Rigatti et al., paragraph 0162).
Regarding claim 27, Belgrader et al. teach the barcode regions, in combination, uniquely identify a cell of origin for the polynucleotide (Belgrader et al., paragraph 0317).
Regarding claim 36, Belgrader et al. teach the sequence of each of a plurality of polynucleotides each having different sequences are determined in parallel (Belgrader et al., paragraph 0317 and 0406).
Regarding claim 37, Belgrader et al. teach that cDNAs (i.e. polynucleotides) derived from the same cell will include common barcode sequences as well as a unique molecular identifier enabling quantitation of the different mRNA molecules present in that particular single cell (Belgrader et al., paragraph 0317).
Regarding claim 38, Belgrader et al. teach associating polynucleotides having the same barcode sequences to the same cell of origin (Belgrader et al., paragraph 0317).
Claims 1-3, 5-6, 12-16, 18, 20-22, 26-27, and 36-38 are rejected under 35 U.S.C. 103 as being obvious over Belgrader et al., US 2020/0002764 A1 (published January 2, 2020) in view of Pratt et al., US 2020/0377937 A1 (published December 3, 2020)
The applied reference (Pratt et al.) has a common inventor and applicant with the instant application AND names other inventors. Based upon the earlier effectively filed date of the reference, it constitutes prior art under 35 U.S.C. 102(a)(2).
Regarding claim 1, Belgrader et al. teach methods of sequencing polynucleotides comprising two or more barcode regions on the same end of a polynucleotide relative to a region of interest (Figures 9A, 51A, and paragraph 0673).
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Belgrader et al. further teach hybridizing a sequencing primer to the polynucleotide, wherein the polynucleotide comprises an intervening (i.e. spacer) sequence between the first and second barcode sequences (Belgrader et al., paragraph 0604-0605), and sequencing the barcode sequences (Belgrader et al., paragraph 0052-0054 and paragraphs 0604-0605) by known sequencing techniques such as “Illumina sequencing” (Belgrader et al., paragraph 0004). (i.e. combining the hybrid with labeled nucleotides and detecting the presence of incorporated nucleotides in a plurality of sequencing flow steps).
Belgrader et al. does not teach extending the sequencing primer through the intervening sequence using a set of one or more dark sequencing flow steps wherein nucleotides are incorporated into the polynucleotide without detecting the presence or absence of the incorporated nucleotide(s).
However, Pratt et al. teach “fast forward” or “dark” sequencing steps for an intervening sequence between two sequences of interest that are sequenced in “light” sequencing steps wherein the identity of the nucleotides are determined (Pratt et al., figure 1 and paragraphs 0078 and 0146). Pratt et al. further teach the “dark” or “fast forward” steps are faster than the “light” sequencing steps (Pratt., abstract), and further that “One advantage of the methods described herein is that the sequence of the sequenced nucleic acid molecule does not need to be aligned with each candidate sequence using an alignment algorithm in some cases, which is generally computationally expensive. Instead, a match score can be determined for each of the candidate sequences using the sequencing data in flowspace, a more computationally efficient operation.” (Pratt et al., paragraph 0181).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have modified the methods comprising single cell RNA sequencing with multiple barcodes separated by spacer sequences on the same end of a polynucleotide sequence of interest, taught by Belgrader et al., with the teachings of Pratt et al. comprising “fast forward” “dark sequencing” through the non-informative spacer sequences between the barcodes. The ordinary artisan would have been motivated to modify the methods of Belgrader et al. with the teachings of Pratt et al. because of Pratt et al. teaches that “fast forward” “dark sequencing” beneficially increases the speed of sequencing and provides for candidate sequence matching without the use of more computationally expensive alignment algorithms (Pratt et al., Abstract and paragraph 0181).
Regarding claim 2, Belgrader et al. teach that the barcode and intervening sequences are known (Belgrader et al., paragraphs 0551-0557, 0608, and Figure 51A).
Regarding claim 3, Pratt et al. teach the dark flow steps extend the primer through the intervening region (Pratt et al., paragraph 0128).
Regarding claim 5, Belgrader et al. also teach sequencing the region of interest (Belgrader et al., paragraph 0274)
Regarding claim 6, Belgrader et al. teach sequencing the two barcodes (i.e. “BC” and “UMI” from the “Read 1 primer” prior to sequencing the region of interest (i.e. V D J C) (Belgrader et al., figure 84B).
Regarding claim 12, Belgrader et al. teach associating each sequencing read (i.e. region of interest) with its barcode sequences (Belgrader et al., paragraph 0064).
Regarding claims 13 and 14, Pratt et al. teach the nucleotides used in the light (and dark) sequencing steps can comprise non-terminating nucleotides (Pratt et al., paragraph 0099 and 0146).
Regarding claim 15, Pratt et al. teach the first and second plurality of sequencing flow steps comprise labeled and unlabeled nucleotides (Pratt et al., paragraph 0103).
Regarding claim 16, Pratt et al. teach that the nucleotides used during primer extension through the second region (see figure above) (i.e. the dark or fast forward steps) comprise unlabeled nucleotides (Pratt et al., paragraph 0078).
Regarding claim 18, Belgrader et al. teach that the polynucleotide may further comprise a unique molecular identifier (Belgrader et al., paragraph 0011 and 0029).
Regarding claim 20, Belgrader et al. teach the polynucleotide can be a cDNA molecule (Belgrader et al., paragraph 0031).
Regarding claim 21, Pratt et al. teach the nucleotides used in the first and second plurality of sequencing steps can comprise a single type of nucleotide base (Pratt et al., paragraph 0098-0099).
Regarding claim 22, Pratt et al. teach the nucleotides used in the first and second plurality of sequencing steps can comprise two or three different types of nucleotide bases (Pratt et al., paragraphs 0081-0082 and 0100).
Regarding claim 26, Pratt et al. teach that the nucleotides used during primer extension through the second region (see figure above) (i.e. the dark or fast forward steps) comprise a mixture of at least two different types of nucleotide bases (Pratt et al., paragraph 0081).
Regarding claim 27, Belgrader et al. teach the barcode regions, in combination, uniquely identify a cell of origin for the polynucleotide (Belgrader et al., paragraph 0317).
Regarding claim 36, Belgrader et al. teach the sequence of each of a plurality of polynucleotides each having different sequences are determined in parallel (Belgrader et al., paragraph 0317 and 0406).
Regarding claim 37, Belgrader et al. teach that cDNAs (i.e. polynucleotides) derived from the same cell will include common barcode sequences as well as a unique molecular identifier enabling quantitation of the different mRNA molecules present in that particular single cell (Belgrader et al., paragraph 0317).
Regarding claim 38, Belgrader et al. teach associating polynucleotides having the same barcode sequences to the same cell of origin (Belgrader et al., paragraph 0317).
This rejection under 35 U.S.C. 103 might be overcome by: (1) a showing under 37 CFR 1.130(a) that the subject matter disclosed in the reference was obtained directly or indirectly from the inventor or a joint inventor of this application and is thus not prior art in accordance with 35 U.S.C.102(b)(2)(A); (2) a showing under 37 CFR 1.130(b) of a prior public disclosure under 35 U.S.C. 102(b)(2)(B); or (3) a statement pursuant to 35 U.S.C. 102(b)(2)(C) establishing that, not later than the effective filing date of the claimed invention, the subject matter disclosed and the claimed invention were either owned by the same person or subject to an obligation of assignment to the same person or subject to a joint research agreement. See generally MPEP § 717.02.
Conclusion
No claim is allowed.
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/Z.M.T./Examiner, Art Unit 1682
/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682