DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application is being examined under the pre-AIA first to invent provisions.
The Applicants’ response to the office action filed on 24 October 2025 has been considered and acknowledged.
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 24 October 2025 has been entered.
Status of the Application
Claims 1,5,7,8,18, 19 and 21-25 and 28-31 are under examination.
Priority
This application filed on 21 March 2022 is a CON of 16/735, 348 (US Patent No. 11,319, 534) filed on 06 January 2020 which is a CON of 14/766,089 (US Patent No. 10,557,133) filed on 05 August 2015 which is a CON of PCT/US2013/031023 filed on 13 March 2013.
Applicant’s claim for the benefit of a prior-filed application under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged. Applicant has not complied with one or more conditions for receiving the benefit of an earlier filing date under 35 U.S.C. 119(e) as follows: The later-filed application must be an application for a patent for an invention which is also disclosed in the prior application (the parent or original nonprovisional application or provisional application). The disclosure of the invention in the parent application and in the later-filed application must be sufficient to comply with the requirements of 35 U.S.C. 112(a) or the first paragraph of pre-AIA 35 U.S.C. 112, except for the best mode requirement. See Transco Products, Inc. v. Performance Contracting, Inc., 38 F.3d 551, 32 USPQ2d 1077 (Fed. Cir. 1994).
The disclosure of the prior-filed application, Application No. 16/735, 348, Application No. 14/766,089 and Application No. PCT/US2013/031023, fails to provide adequate support or enablement in the manner provided by 35 U.S.C. 112(a) or pre-AIA 35 U.S.C. 112, first paragraph for one or more claims of this application.
In the instant case, claim 1 is amended to recite: a method comprising:
generating indexed template nucleic acid fragments derived from template nucleic acid of single cells or organelles via in-cell transposition using transposomes comprising a first index to tagment the template nucleic acid of each cell or organelle with different index sequences;
lysing the single cells or organelles to release the indexed template nucleic acid fragments;
diluting a concentration of the released indexed template nucleic acid fragments: removing a transposase from the indexed template nucleic acid fragments subsequent to diluting the concentration of the indexed template nucleic acid fragments in the plurality of vessels.
The disclosures of the prior-filed applications and the instant specification disclose “Transposition can also be performed directly in cells, with population of cells, lysates, and
non-purified DNA”( para 0130, instant specification and Application No. PCT/US2013/031023 );
“In some embodiments, a transposase can be removed from a template nucleic acid subsequent to distributing the template nucleic in several vessels. A transposase can be removed from the site of an insertion by various methods well known in the art, including the addition of a detergent, such as SDS...”( para 0190, instant specification and Application No. PCT/US2013/031023);
“Transposomes comprising Mu were used to obtain haplotype information. 1 ng of genomic DNA was targeted with Mu-TSM in a 50 μl reaction volume with 1 X TA buffer and 1, 2, 4, or 8 μl of 25 μM Mu-TSM complexes. Reactions were incubated at 37 °C for 2 hours. Samples were diluted to 1 pg/μl. For Mu inactivation, 10μ1 of each sample containing either 1 pg or 5 pg total genomic DNA were prepared. SDS was added to final concentration of 0.05%....”(para 0213,instant specification and Application No. PCT/US2013/031023).
Therefore, the prior-filed applications and the instant specification provide support for conducting transposition directly in cells and conducting transposition in cell lysates. Furthermore, the prior-filed applications and the instant specification provide support for removing transposase from complexes of isolated DNA and transposomes comprising transposase, wherein indexes are attached through an in vitro transposition event.
However, the disclosures of the prior-filed applications and the instant specification do not provide support for the claimed embodiment of:
generating indexed template nucleic acid by in cell transposition and then lysing the transposed cells to release the indexed nucleic acid; diluting the concentration of indexed nucleic acid that are released from the transposed cells and removing transposase from the indexed nucleic acid that are released from the transposed cells.
However, the claims filed 24 October 2025 provide support for this embodiment.
Therefore, the effective filing date of the instant application is 24 October 2025.
Claim Rejections - 35 USC § 112
Written Description and New Matter
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1,5,7,8,18, 19 and 21-25 and 28-31 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
In the instant case, the instant specification discloses:
“Transposition can also be performed directly in cells, with population of cells, lysates, and non-purified DNA”( para 0130, instant specification);
“In some embodiments, a transposase can be removed from a template nucleic acid subsequent to distributing the template nucleic in several vessels. A transposase can be removed from the site of an insertion by various methods well known in the art, including the addition of a detergent, such as SDS...”( para 0190, instant specification);
“Transposomes comprising Mu were used to obtain haplotype information. 1 ng of genomic DNA was targeted with Mu-TSM in a 50 μl reaction volume with 1 X TA buffer and 1, 2, 4, or 8 μl of 25 μM Mu-TSM complexes. Reactions were incubated at 37 °C for 2 hours. Samples were diluted to 1 pg/μl. For Mu inactivation, 10μ1 of each sample containing either 1 pg or 5 pg total genomic DNA were prepared. SDS was added to final concentration of 0.05%....”(para 0213,instant specification).
Therefore, the instant specification provides support for conducting transposition directly in cells and conducting transposition in cell lysates. Furthermore, the instant specification provides support for removing transposase from complexes of isolated DNA and transposomes comprising transposase, wherein indexes are attached through an in vitro transposition event.
However, the instant specification does not adequately describe the claimed embodiment of generating indexed template nucleic acid by in cell transposition and then lysing the transposed cells to release the indexed nucleic acid; diluting the concentration of indexed nucleic acid that are released from the transposed cells and removing transposase from the indexed nucleic acid that are released from the transposed cells.
The instant specification does not offer sufficient description of the common structural elements or identifying characteristics that constitute the technique of lysis of transposed cells or organelles to release indexed nucleic acids. A person of ordinary skill in the art would appreciate that there are many different techniques for lysis of transposed cells to facilitate the functions recited in the instant claims. However, the instant specification is silent as to which techniques the inventor has determined to be sufficient to release indexed nucleic acids from transposed cells to implement the subsequent steps of the claimed method.
Therefore, the instant specification does not provide a disclosure of corresponding structure in sufficient detail to demonstrate to one of ordinary skill in the art that the inventor possessed the invention including how the inventor intended to perform the step of lysing of transposed cells to release indexed nucleic acids and to allow the function recited in the instant claims.
Indefiniteness
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.
Claim 31 is 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.
In the instant case, claim 31 recites a step wherein removing the transposase from the indexed template nucleic acid fragments subsequent to diluting the concentration of the indexed template nucleic acid fragments increases an amount of immediate neighbors of the indexed template nucleic acid fragments with the plurality of vessels as compared to removing the transposase after diluting the concentration.
This claim language is confusing because it requires assessing a difference in the amount of immediate neighbors based on comparing two conditions that are the same, i.e. removing a transposase after dilution.
Therefore, as the metes and bounds of this limitation are not clear, claim 31 is considered indefinite.
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 pre-AIA 35 U.S.C. 103(a) which forms the basis for all obviousness rejections set forth in this Office action:
(a) A patent may not be obtained though the invention is not identically disclosed or described as set forth in section 102, if the differences between the subject matter sought to be patented and the prior art are such that the subject matter as a whole would have been obvious at the time the invention was made to a person having ordinary skill in the art to which said subject matter 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 under pre-AIA 35 U.S.C. 103(a), the examiner presumes that the subject matter of the various claims was commonly owned at the time any inventions covered therein were made absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and invention dates of each claim that was not commonly owned at the time a later invention was made in order for the examiner to consider the applicability of pre-AIA 35 U.S.C. 103(c) and potential pre-AIA 35 U.S.C. 102(e), (f) or (g) prior art under pre-AIA 35 U.S.C. 103(a).
Steemers et al., Jendrisak et al., Goryshin et al., Brenner and Shendure et al.
Claims 1, 5,7, 8 and 28-31 are rejected under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Steemers et al. (WO2012061832; published 10 May 2012; cited in IDS filed 21 March 2022) in view of Jendrisak et al. (WO2010048605); Goryshin et al. ("Insertional transposon mutagenesis by electroporation of released Tn5 transposition complexes." Nature biotechnology 18.1 (2000): 97-100.);Brenner (US20060177833) and Shendure et al. (WO2012106546; published 09 August 2012; cited in IDS filed 21 March 2022).
Steemers et al. teach methods for tagging target nucleic acids comprising: providing target nucleic acids and providing transposome complexes (e.g. lines 5-14, pg. 2; transposome section, pg. 19-20; Fig. 8, 12, 19 and 26), each complex comprising a transposase and associated transposons, each transposon comprising barcodes and other structural features. Furthermore, Steemers et al. teach the barcode of each transposon is different(e.g. Entire Steemers reference and especially lines 5-33, pg. 2- lines 1-10,pg. 4; lines 3-11, pg. 6; lines 22-30, pg. 9; lines 11-29, pg. 14; lines 6-10; pg. 20; Fragmentation sites section, pg. 27-29, especially lines 6-22, pg. 28; Fig. 1 , 10 and 11).
Furthermore, Steemers teaches tagmented nucleic acids are subject to PCR to add additional sequences, such as sequencing adapters (e.g. lines 27-32, pg. 38; Fig. 19).
Furthermore, Steemers et al. teach embodiments wherein sequencing adapters comprising barcodes are ligated to tagged nucleic acids (e.g. lines 29-31, pg. 10; lines 25-26, pg. 23; Fig. 15). Furthermore, Steemers teaches tagged target DNA is subjected to a subsequent PCR to generate a di-tagged sequencing library(e.g. Fig. 27).
Furthermore, Steemers teaches target nucleic acids are genomic DNA and mitochondrial DNA. Furthermore, Steemers teaches target nucleic acids may come from any source, including organelles and cells ( e.g. Entire Steemers reference and especially lines 1-8; lines 29-33, pg. 34).
Furthermore, Steemers et al. teach their methods are used for single cell analysis (e.g. Entire Steemers reference and especially compartmentalization of single molecules section, pg. 49-50).
Furthermore, Steemers et al. teach an embodiment comprising a two-step tagging process wherein target nucleic acid is subjected to tagging with one type of transposition, such as HyperMu-mediated transposition and amplified. The resultant products are then subjected to a second round of tagging by a second transposition, e.g. Tn-5 mediated transposition, yielding products comprising two sets of tags (e.g. … a two-step tagging method is provided using one or more transposition reactions with different transposomes… lines 15-30, pg. 48; Fig. 33).
Steemers teaches a droplet- based method for generating a nucleic acid library wherein the contents of a single cell are released within the droplet comprising a bead and target nucleic acid is contacted with multiple complexes of transposase and transposons (e.g. lines 1-24, pg. 49; Fig. 34). Furthermore, Steemers teaches that each bead has a unique index sequence for barcoding the cell of the droplet and that the transposition reaction within the droplet results in a target DNA comprising the unique index sequence. The resultant products are pooled and can be amplified with adapter-primers to create a sequencing library (e.g. lines 10-33, pg. 49).
Furthermore, Steemers et al. teach an embodiment comprising compartmentalization of individual cells and reagents for in vitro transposition, i.e. tagmentation, including a transposon comprising a unique identifier sequence and attached by a photocleavable linker to a bead, into each well of a multi-well plate. The transposon is cleaved from the bead by UV light exposure and is available to complex with transposases in the tagmentation reagent mix. The cell is also lysed and released nucleic acid is subject to tagmentation, resulting in target nucleic acids comprising unique identifier sequences. The resultant wells are extracted and the eluate is pooled for further analysis, such as amplification (e.g. lines 2-33 pg. 49- lines 8, pg. 50; Fig. 34 and 35).
Furthermore, Steemers et al. teach the resultant tagged library is subjected to different sequencing technologies, including haplotype sequencing (e.g. Entire Steemers reference and especially lines 11-33, pg. 14-lines 1-8,pg. 15; lines 18-20, pg. 25; lines 5-11, pg. 41; lines 7-33,pg. 46-lines 1-12,pg. 48; lines 10-33, pg. 50- lines 1-15, pg. 55;claim 14; Fig. 32).
Furthermore, Steemers et al. teach their methods comprise generating barcoded nucleic acids, subjecting these nucleic acid to sequencing; obtaining sequencing data and assembling a representation from the sequencing data (e.g. Entire Steemers reference and especially lines 3-31,pg. 6; lines 10-25,pg. 13; lines 7-32,pg. 46- lines 1-24,pg. 47).
Therefore, Steemers et al. teach droplet-based methods comprising attaching identifiers to target nucleic acids by transposition, wherein a single cell is distributed to each droplet, wherein the target nucleic acid is derived from organelles, such as genomic DNA and mitochondrial DNA. Furthermore, Steemers et al. teach embodiments wherein a unique index is associated with a single cell, i.e. a bead comprising a unique index is associated with a single cell. Furthermore, Steemers et al. teach embodiments comprising a two-step tagging process.
However, Steemers et al. do not expressly teach methods comprising transposition within a cell as required by claim 1.
Furthermore, Steemers et al. do not expressly teach combining and distributing the cells containing the indexed template nucleic acid fragments into a plurality of vessels or introducing a second tag in a vessel of a plurality of vessels.
Like Steemers et al., Jendrisak et al. teach methods comprising inserting nucleic acid sequences of interest using Tn-5 and Mu- mediated transposition.
Furthermore, at the time the invention was made, Jendrisak et al. teach methods are known to introduce transposase systems into compatible cells and to subject the transformed cells to conditions that facilitate in vivo transposition events. Furthermore, Jendrisak et al. teach Tn-5 and/or Mu transposases are introduced into cells by electroporation as an expressed and functional component of a synaptic complex comprising a transposable polynucleotide comprising compatible transposase interacting sequences. Furthermore, Jendrisak et al. teach transposons comprising different tags. Furthermore, Jendrisak et al. teach the resultant transposase-modified targets are used for further analysis, including amplification and sequencing (e.g. Entire Jendrisak reference and especially method comprising tagging by transposition, amplification and sequencing as in lines 16-33,pg. 7- lines 1-22,pg. 15; Tn5 transposase and Mu transposases as in lines 5-32, pg. 15- lines 1-18,pg. 16; lines 31-32, pg. 17- lines 1-6,pg. 18; in vivo transposition by electroporation of synaptic complexes as in lines 24-34,pg. 54- lines 1-9,p g. 55; transposons comprising different tags as in Example 1, especially lines 23-32,pg. 141- lines 1-3,pg. 142; Fig. 4).
Furthermore, Jendrisak et al. teach target nucleic acid are from a biological source and include genomic DNA and mitochondrial DNA (e.g. lines 1-19, pg. 48).
Furthermore, Jendrisak et al. teach that methods are known for inactivating transposome complexes using a stop solution comprising SDS and Proteinase K and heating at 50°C (e.g. Entire Jendrisak reference and especially … After mixing, the reaction was incubated for 1 hour at 37°C. The reaction was stopped with 10 microliters of stop solution (15% sucrose, 66 mM EDTA, 20 mM TRIS, pH 8.0, 0.1 % SDS, 0.9% Orange G [Sigma 0-7252], and Proteinase Kat 100 micrograms per ml), mixed, and heated at 50°C for 10 minutes… as in lines 4-7,pg. 142, Example 1; also in Examples 2-6, 14-17).
Furthermore, Jendrisak et al. teach the resultant tagged fragments are subsequently amplified and or sequenced (e.g. Entire Jendrisak reference and especially… using fragment library of Example 3 for amplification in Examples 6 and 7, pg. 147-150; using fragment library of Example 2 for amplification and sequencing in Example 12, pg. 158-159).
Regarding the requirement of generating indexed template nucleic acid by in cell transposition and then lysing the transposed cells to release the indexed nucleic acid; diluting the concentration of indexed nucleic acid that are released from the transposed cells and removing transposase from the indexed nucleic acid that are released from the transposed cells as recited in claim 1:
As discussed above, Jendrisak et al. teach methods are known to introduce transposase systems into compatible cells and to subject the transformed cells to conditions that facilitate in vivo transposition events. Furthermore, Jendrisak et al. teach Tn-5 and/or Mu transposases are introduced into cells by electroporation. Furthermore, Jendrisak et al. teach that methods are known for inactivating transposome complexes using a stop solution comprising SDS and Proteinase K and heating at 50°C (e.g. Entire Jendrisak reference and especially … The following reaction mixture was assembled: ...50 microliters... as in lines 25-33, pg. 141; After mixing, the reaction was incubated for 1 hour at 37°C. The reaction was stopped with 10 microliters of stop solution (15% sucrose, 66 mM EDTA, 20 mM TRIS, pH 8.0, 0.1 % SDS, 0.9% Orange G [Sigma 0-7252], and Proteinase Kat 100 micrograms per ml), mixed, and heated at 50°C for 10 minutes… lines 4-7,pg. 142, Example 1; also in Examples 2-6, 14-17).
It is noted that Jendrisak et al. teach adding 10 microliters stop solution to a 50 microliters reaction mixture, thereby diluting the concentration of the reaction mixture by adding 20% of the original volume.
Furthermore, at the time that the invention was made, Goryshin et al. teach methods are known comprising performing in vivo transposition in whole cells, i.e. in cell transposition, and subsequently purifying the transposed DNA, i.e. cell lysis, for further analysis , including sequencing analysis( e.g. Entire Goryshin reference and especially 5th para, pg. 98- 1st para, pg. 99; ... The transposon target boundaries of 11 representative KanR products from the MG1655 electroporation experiment were sequenced in order to ensure that they represented bona fide transposition events (Fig. 2). All 11 DNAs contained unique Tn5-like inserts with 9 bp target duplications at the boundaries... as in 2nd para, pg. 99; ...Seven presumed transposition products in P. vulgaris and seven presumed transposition products in S. typhimurium were also studied by Southern blot analysis. As shown in Figure 3, all seven KanR isolates of P. vulgaris contained single unique transposon inserts... as in 4th para, pg. 99; Transposome formation and transposition mutagenesis. Section, Experimental Protocol section, pg. 99; Sequence analysis section, Southern Blot analysis section, Experimental Protocol section, pg. 100).
Furthermore, at the time the claimed invention was made, Shendure et al. teach a method comprising treating DNA with a transposase, which tags and fragments the sample DNA simultaneously, and with identical "recognition sequences “ ( i.e. barcodes). The tagged fragments are subsequently subjected to amplification and sequencing. Analysis after sequencing involves identifying the tagged fragment that have shared property i.e. the identical barcodes. Shendure teaches integration of recognition sequences, such as barcodes, by a transposase-mediated reaction. Shendure et al. also teach further amplification after transposition using barcoded primers wherein barcode sequences are incorporated into target nucleic acid. Additionally, Shendure et al. teach a droplet-based method wherein target nucleic acid are subjected to transposition to incorporate recognition sequences which are barcodes and a further amplification to incorporate one or more barcodes (e.g. para 0007-0009, pg. 2-3; para 0061, pg. 13; emulsion –based PCR using barcoded beads as in para 0131, pg. 37; para 0133-0134, pg. 38).
Furthermore, Shendure et al. teach their methods comprise additional steps including removing transposase after reaction using an SDS solution(e.g. para 00192, 60-61).
Therefore, as Steemers et al., Jendrisak et al. , Goryshin et al. and Shendure et al. all teach tagmentation of target nucleic acid, it would have been prima facie obvious to a person of ordinary skill in the art at the time the invention was made to modify the method of Steemers comprising providing a first unique index to a single cell within a first vessel, i.e. droplet, yielding a set of first indexed nucleic acids that is subjected to a second tagging process by transposition or PCR to add a second set of indexes and then sequencing to obtain single cell sequence information and to include introducing, by electroporation, transposase-associated compositions into individual cells and to subject the transformed cells to conditions that facilitate in vivo transposition, wherein the compositions comprise Tn5 or Mu transposase-associated synaptic complexes comprising a transposable polynucleotide comprising compatible transposase interacting sequences and the products of the in vivo transposition reactions are further analyzed, such as by amplification and sequencing as taught by Jendrisak et al. and to include purification of tagmented target DNA from tagmentation reagents using a stop solution containing 0.1% SDS, prior to further analysis, including amplification and sequencing , wherein addition of the stop solution dilutes the concentration of the original reaction volume as taught by Jendrisak et al. and to include methods comprising performing in vivo transposition in whole cells, i.e. in cell transposition, and subsequently purifying the transposed DNA, i.e. cell lysis for further analysis , including sequencing analysis as taught by Goryshin et al. and to include removal of transposase using an SDS solution as taught by Shendure et al. because these claim elements were known in the art and one of skill in the art could have combined these elements by known methods with no change in their respective functions, and the combination would have yielded the predictable outcome of a method for obtaining sequencing information.
Therefore, the combined teachings of Steemers et al., Jendrisak et al., Goryshin et al. and Shendure et al. render obvious a method comprising providing single cells comprising target nucleic acids that are subjected to reaction with transposase through in vivo transposition, yielding indexed target nucleic acid within each single cell, wherein the cells are subsequently lysed to release the indexed nucleic acids; the concentration of the original reaction volume is diluted and the transposase is removed by the addition of a stop solution containing SDS, prior to further analysis , wherein the target nucleic acid is derived from different organelles, such as genomic DNA and mitochondrial DNA.
Regarding introducing a second tag in a vessel of a plurality of vessels:
Like Steemers, Brenner teaches methods for attaching multiple tags to target nucleic acid. Furthermore, Brenner teaches a tagging method comprising a multi-vessel format wherein samples are attached by ligation with a first set of tags in a first set of wells; the tagged samples are then pooled and combined in a second set of wells with a different set of tags and subjected to ligation with the second set of tags, resulting in combinatorial attachment of tags to target nucleic acid (e.g. Entire Brenner reference and especially … single-word adaptors (206) are ligated(204) as in para 0038,pg. 6-7; attach tags by ligation in multiple rounds as in para 0089-0103, pg. 14-16; Fig. 5). Furthermore, Brenner teaches the vessel is a well or a tube (e.g. para 0089, pg. 14).
Therefore, as at least Steemers et al. and Brenner both teach methods for attaching multiple tags to target nucleic acids, it would have been prima facie obvious to a person of ordinary skill in the art at the time the invention was made to modify the method of Steemers et al., Jendrisak et al., Goryshin et al. and Shendure et al. and to include a multi-vessel format that allows redistribution of tagged target nucleic acid for combination with different sets of tags as taught by Brenner because these claim elements were known in the art and one of skill in the art could have combined these elements by known methods with no change in their respective functions, and the combination would have yielded the predictable outcome of a device for preparing a nucleic acid library.
The combined teachings of Steemers et al., Jendrisak et al., Goryshin et al., Shendure et al. and Brenner render obvious a method comprising providing indexed template nucleic acid fragments, wherein the target nucleic acid is derived from different organelles, such as genomic DNA and mitochondrial DNA; subjecting target nucleic acids to in vivo transposition, yielding indexed target nucleic acid within each single cell prior to further analysis, wherein the indexed template nucleic acid fragments comprise a first index; distributing indexed nucleic acid among a plurality of vessels; providing a second index to at least a portion of the indexed template nucleic acid fragments in a vessel of the plurality of vessels to generate second indexed template nucleic acid fragments comprising the first index and the second index in the vessel; obtaining sequence data from the indexed template nucleic acid fragments; and assembling a sequence representation of the template nucleic acid from the sequence data.
Regarding the limitation : wherein the transposomes contact the template nucleic acids such that the physical contiguity of the template nucleic acid is retained, wherein the physical contiguity indicates the relative arrangement of adjacent sequences to the template nucleic acid fragments and the limitation: assembling a sequence representation of the template nucleic acid from the sequence data based on a relative distance between indexed template nucleic acid fragments in the vessel that corresponds to the relative arrangement of the adjacent sequences as recited in claim 1:
As discussed above, Steemers et al. teach methods for tagging target nucleic acids comprising: providing target nucleic acids and providing transposome complexes (e.g. lines 5-14, pg. 2; transposome section, pg. 19-20; Fig. 8, 12, 19 and 26), each complex comprising a transposase and associated transposons, each transposon comprising barcodes and other structural features. Furthermore, Steemers et al. teach the barcode of each transposon is different(e.g. Entire Steemers reference and especially lines 5-33, pg. 2- lines 1-10,pg. 4; lines 3-11, pg. 6; lines 22-30, pg. 9; lines 11-29, pg. 14; lines 6-10; pg. 20; Fragmentation sites section, pg. 27-29, especially lines 6-22, pg. 28; Fig. 1 , 10 and 11).
Furthermore, Jendrisak et al. teach methods for tagging target nucleic acid from cells by transposase-mediated reactions.
However, regarding sequence contiguity as recited by the limitation above:
As discussed above, Shendure et al. teach a method comprising treating DNA with a transposase, which tags and fragments the sample DNA simultaneously, and with identical "recognition sequences “ ( i.e. barcodes). The tagged fragments are subsequently subjected to amplification and sequencing. Analysis after sequencing involves identifying the tagged fragment that have shared property i.e. the identical barcodes. Shendure teaches integration of recognition sequences, such as barcodes, by a transposase-mediated reaction. Shendure et al. also teach further amplification after transposition using barcoded primers wherein barcode sequences are incorporated into target nucleic acid. Additionally, Shendure et al. teach a droplet-based method wherein target nucleic acid are subjected to transposition to incorporate recognition sequences which are barcodes and a further amplification to incorporate one or more barcodes (e.g. para 0007-0009, pg. 2-3; para 0061, pg. 13; emulsion –based PCR using barcoded beads as in para 0131, pg. 37; para 0133-0134, pg. 38).
Furthermore, considering the Shendure reference as a whole, Shendure et al. teach their methods for determining sequence contiguity using the relative proximity, i.e. relative distance, of two target nucleic acid fragments.
For example, Shendure et al. teach methods for the analysis of two target fragments that are directly adjacent to each other, i.e. adjacent spatial relationship, analysis of two target fragments that are on the same segment of target DNA but are not necessarily adjacent to each other, i.e. compartmental spatial relationship; and analysis of two target fragments that are separated by a particular distance or sequence length between each other, i.e. distance spatial relationship. Furthermore, Shendure et al. teach methods of capturing contiguity information are designed according to one of these three relationships, i.e. short-range contiguity methods to determine adjacent spatial relationships; mid-range contiguity methods to determine compartmental spatial relationships; and long-range contiguity methods to determine distance spatial relationships. As taught by Shendure et al., short range contiguity analysis comprises using transposons comprising degenerate barcodes to symmetrically and uniquely tag shotgun library molecules originating from each flank of any given fragmentation event, such that sequencing analysis comprises assigning in silico “joins” between separate, adjacent-in-origin read pairs and identifying shared barcodes. Furthermore, Shendure et al. teach long-range contiguity analysis comprises determining distance between genomic segments of interest using in situ transposition of barcoded adaptors and optical sequencing ( e.g. para 0056-0077, pg. 12-20; short range contiguity analysis of adjacent target sequences as in para 0057, pg. 12-13; para 0063, pg. 14; long range contiguity as in para 0067-0071, pg. 16-18; …The basic premise is to exploit the physical properties of DNA (by random coiling or stretching of high-molecular weight (HMW) DNA), in situ library construction (via in vitro transposition of adaptors to HMW DNA within a flow-cell), and the fully developed aspects of an operationally realized next-generation sequencing instrument (polony amplification, sequencing-by synthesis, imaging and data-processing), to generate multiple spatially related reads whose physical separation is either known or can be inferred from the relative coordinates at which the reads originate on the flow-cell. In one approach, the random coil configuration adopted by DNA in solution is exploited to spatially confine the ends and generate two reads within a confined surface area…optical sequencing on stretched DNA molecules within a native flowcell may also be performed…. As in para 0069; para 0072-0073, pg. 18-19; para 00100- 00130, Example 1, pg. 29-37). Furthermore, Shendure et al. teach each type of method, short-range, mid-range and long range contiguity analysis, can be integrated for a more cost-effective and comprehensive analysis (e.g. para 00230-00235 long-range contiguity as in Example 3, pg. 45-64; Example 5, pg. 70-72; cost-effective and comprehensive analysis as in Example 6, pg. 73-75).
Therefore, as Shendure et al. also teach tagmentation of target nucleic acid comprising attaching multiple tags to target nucleic acids, it would have been prima facie obvious to a person of ordinary skill in the art at the time the invention was made to modify the method of Steemers et al., Jendrisak et al., Goryshin et al., Shendure et al. and Brenner and to include short range contiguity analysis for determining the relative sequence assembly of adjacent target fragments and long range contiguity which facilitates determining distance between segments of interest as well as integrating short range contiguity methods with method for analysis of mid-range contiguity and long range contiguity to determine relative distances between indexed segments of interest as taught by Shendure et al. because these claim elements were known in the art and one of skill in the art could have combined these elements by known methods with no change in their respective functions, and the combination would have yielded the predictable outcome of a device for preparing a nucleic acid library.
Furthermore, Shendure et al. teach such integration of contiguity analysis results in a more cost-effective and comprehensive analysis.
Therefore, as at least Steemers and Jendrisak et al. teach analysis of nucleic acid from different organelles, i.e. genomic DNA and mitochondrial DNA, the combined teachings of Steemers et al., Jendrisak et al., Goryshin et al., Brenner and Shendure et al. render obvious claim 1.
Furthermore, as Steemers et al. teach indexed nucleic acids are subjected to PCR amplification (e.g. lines 27-32, pg. 38; Fig. 19;Fig. 27; lines 2-33 pg. 49- lines 8, pg. 50; Fig. 34 and 35), the combined teachings of Steemers et al., Jendrisak et al., Goryshin et al., Brenner and Shendure et al. render obvious claim 5.
Furthermore, as Steemers et al. teach methods are known comprising sequencing, such as haplotype sequencing; obtaining sequencing data and assembling a representation from the sequencing data, the combined teachings of Steemers et al., Jendrisak et al., Goryshin et al., Brenner and Shendure et al. render obvious claim 7.
Furthermore, as Steemers et al. teach embodiments comprising a two-step tagging process and Brenner teaches multiple rounds of tagging in a multi-vessel format, the combined teachings of Steemers et al., Jendrisak et al., Goryshin et al., Brenner and Shendure et al. render obvious claim 8.
Furthermore, as Jendrisak et al. teach purification of tagmented target DNA from tagmentation reagents using a stop solution containing 0.1% SDS, prior to further analysis, including amplification and sequencing and Shendure et al. teach removal of transposase using an SDS solution, the combined teachings of Steemers et al., Jendrisak et al., Goryshin et al., Brenner and Shendure et al. render obvious claims 28-30.
Regarding claim 31:
As discussed above, the language of claim 31 is not clear. However, as claim 31 requires the method of claim 1, the combined teachings of Steemers et al., Jendrisak et al., Goryshin et al., Brenner and Shendure et al. render obvious claim 31.
Steemers et al., Brenner, Shendure et al. and Jendrisak et al.
Claim 18 is rejected under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Steemers et al. (WO2012061832; published 10 May 2012) in view of Brenner (US20060177833), Shendure et al. (WO2012106546; published 09 August 2012; cited in IDS filed 21 March 2022) and Jendrisak et al. (WO2010048605).
Steemers et al. teach methods for tagging target nucleic acids comprising: providing target nucleic acids and providing transposome complexes (e.g. lines 5-14, pg. 2; transposome section, pg. 19-20; Fig. 8, 12, 19 and 26), each complex comprising a transposase and associated transposons, each transposon comprising barcodes. Furthermore, Steemers et al. teach the barcode of each transposon is different(e.g. Entire Steemers reference and especially lines 5-33, pg. 2- lines 1-10,pg. 4; lines 3-11, pg. 6; lines 22-30, pg. 9; lines 11-29, pg. 14; lines 6-10; pg. 20; Fragmentation sites section, pg. 27-29, especially lines 6-22, pg. 28; Fig. 1 , 10 and 11).
Furthermore, Steemers et al. teach embodiments wherein universal primer sites are incorporated into template nucleic acid by transposition (e.g. lines 30-34,pg. 39- lines 1-4, pg. 41).
Furthermore, Steemers et al. teach an embodiment comprising a two-step tagging process wherein target nucleic acid is subjected to tagging with one type of transposition, such as HyperMu-mediated transposition and amplified. The resultant products are then subjected to a second round of tagging by a second transposition, e.g. Tn-5 mediated transposition, yielding products comprising two sets of tags (e.g. … a two-step tagging method is provided using one or more transposition reactions with different transposomes… lines 15-30, pg. 48; Fig. 33).
Furthermore, Steemers teaches tagmented nucleic acids are subject to PCR to add additional sequences, such as sequencing adapters (e.g. lines 27-32, pg. 38; Fig. 19). Furthermore, Steemers teaches tagged target DNA is subjected to a subsequent PCR to generate a di-tagged sequencing library(e.g. Fig. 27).
Furthermore, Steemers et al. teach embodiments wherein sequencing adapters comprising barcodes are ligated to tagged nucleic acids (e.g. lines 29-31, pg. 10; lines 25-26, pg. 23; Fig. 15).
Furthermore, Steemers et al. teach their methods are used for single cell analysis (e.g. Entire Steemers reference and especially compartmentalization of single molecules section, pg. 49-50).
Steemers teaches a droplet- based method for generating a nucleic acid library wherein the contents of a single cell are released within the droplet comprising a bead and target nucleic acid is contacted with multiple complexes of transposase and transposons (e.g. lines 1-24, pg. 49; Fig. 34). Furthermore, Steemers teaches that each bead has a unique index sequence for barcoding the cell of the droplet and that the transposition reaction within the droplet results in a target DNA comprising the unique index sequence. The resultant products are pooled and can be amplified with adapter-primers to create a sequencing library (e.g. lines 10-33, pg. 49).
Steemers et al. also teach an embodiment comprising compartmentalization of individual cells and reagents for in vitro transposition, i.e. tagmentation, including a transposon comprising a unique identifier sequence and attached by a photocleavable linker to a bead, into each well of a multi-well plate. The transposon is cleaved from the bead by UV light exposure and is available to complex with transposases in the tagmentation reagent mix. The cell is also lysed and released nucleic acid is subject to tagmentation, resulting in target nucleic acids comprising unique identifier sequences. The resultant wells are extracted and the eluate is pooled for further analysis, such as amplification (e.g. lines 2-33 pg. 49- lines 8, pg. 50; Fig. 34 and 35).
Furthermore, Steemers teaches the resultant tagged library is subjected to different sequencing technologies, including haplotype sequencing (e.g. Entire Steemers reference and especially lines 11-33, pg. 14-lines 1-8,pg. 15; lines 18-20, pg. 25; lines 5-11, pg. 41; lines 7-33,pg. 46-lines 1-12,pg. 48; lines 10-33, pg. 50- lines 1-15, pg. 55;claim 14; Fig. 32).
Furthermore, Steemers et al. teach their methods comprise generating barcoded nucleic acids, subjecting these nucleic acid to sequencing; obtaining sequencing data and assembling a representation from the sequencing data (e.g. Entire Steemers reference and especially lines 3-31,pg. 6; lines 10-25,pg. 13; lines 7-32,pg. 46- lines 1-24,pg. 47).
Therefore, Steemers et al. teach droplet-based methods comprising attaching identifiers to target nucleic acids by transposition, wherein a single cell is distributed to each droplet. Furthermore, Steemers et al. teach embodiments wherein a unique index is associated with a single cell, i.e. a bead comprising a unique index is associated with a single cell.
Furthermore, Steemers et al. teach embodiments comprising a two-step tagging process.
Steemers et al. do not expressly teach introducing tagged nucleic acid in a vessel of a plurality of vessels.
Like Steemers, Brenner teaches methods for attaching multiple tags to target nucleic acid.
Furthermore, Brenner teaches a tagging method comprising a multi-vessel format wherein samples are attached by ligation with a first set of tags in a first set of wells; the tagged samples are then pooled and combined in a second set of wells with a different set of tags and subjected to ligation with the second set of tags, resulting in combinatorial attachment of tags to target nucleic acid (e.g. Entire Brenner reference and especially … single-word adaptors (206) are ligated(204) as in para 0038,pg. 6-7; attach tags by ligation in multiple rounds as in para 0089-0103, pg. 14-16; Fig. 5). Furthermore, Brenner teaches the vessel is a well or a tube (e.g. para 0089, pg. 14).
Therefore, as Steemers et al. teach tagmentation of target nucleic acid wherein the resultant indexed products are pooled for further analysis, including amplification, and Steemers et al. and Brenner both teach methods for attaching multiple tags to target nucleic acids, it would have been prima facie obvious to a person of ordinary skill in the art at the time the invention was made to modify the method of Steemers comprising providing a first unique index to a single cell within a first vessel, i.e. droplet, yielding a set of first indexed nucleic acids that is subjected to a second tagging process by transposition or PCR to add a second set of indexes and then sequencing to obtain single cell sequence information and to include a multi-vessel format that allows redistribution of tagged target nucleic acid for combination with different sets of tags as taught by Brenner because these claim elements were known in the art and one of skill in the art could have combined these elements by known methods with no change in their respective functions, and the combination would have yielded the predictable outcome of a method for obtaining sequencing information.
The combined teachings of Steemers et al. and Brenner render obvious a method comprising providing indexed template nucleic acid fragments, wherein the indexed template nucleic acid fragments comprise a first index ; distributing indexed nucleic acid among a plurality of vessels; providing a second index to at least a portion of the indexed template nucleic acid fragments in a vessel of the plurality of vessels to generate second indexed template nucleic acid fragments comprising the first index and the second index in the vessel; obtaining sequence data from the indexed template nucleic acid fragments; and assembling a sequence representation of the template nucleic acid from the sequence data.
Regarding the limitation: inserting a plurality of transposons into the template nucleic acid such that the physical contiguity of the template nucleic acid is retained, wherein the physical contiguity indicates the relative arrangement of adjacent sequences of the template nucleic acid and the limitation: assembling a sequence representation of the template nucleic acid from the sequence data based on a relative distance between indexed template nucleic acid fragments in the vessel that corresponds to the relative arrangement of the adjacent sequences as recited in claim 18:
As discussed above, Steemers et al. teach methods for tagging target nucleic acids comprising: providing target nucleic acids and providing transposome complexes (e.g. lines 5-14, pg. 2; transposome section, pg. 19-20; Fig. 8, 12, 19 and 26), each complex comprising a transposase and associated transposons, each transposon comprising barcodes and other structural features. Furthermore, Steemers et al. teach the barcode of each transposon is different(e.g. Entire Steemers reference and especially lines 5-33, pg. 2- lines 1-10,pg. 4; lines 3-11, pg. 6; lines 22-30, pg. 9; lines 11-29, pg. 14; lines 6-10; pg. 20; Fragmentation sites section, pg. 27-29, especially lines 6-22, pg. 28; Fig. 1 , 10 and 11).
However, the combined teachings of Steemers et al. and Brenner do not expressly teach sequence contiguity as recited by the limitations above.
At the time the claimed invention was made, Shendure et al. teach a method comprising treating DNA with a transposase, which tags and fragments the sample DNA simultaneously, and with identical "recognition sequences “ ( i.e. barcodes). The tagged fragments are subsequently subjected to amplification and sequencing. Analysis after sequencing involves identifying the tagged fragment that have shared property i.e. the identical barcodes. Shendure teaches integration of recognition sequences, such as barcodes, by a transposase-mediated reaction. Shendure et al. also teach further amplification after transposition using barcoded primers wherein barcode sequences are incorporated into target nucleic acid. Additionally, Shendure et al. teach a droplet-based method wherein target nucleic acid are subjected to transposition to incorporate recognition sequences which are barcodes and a further amplification to incorporate on