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 .
Status of the Application
Claims 1-17 are pending and under examination.
Specification
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code. For example, see para 0070, pg. 21; para 0079, pg. 23.
Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01.
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.
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 3 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.
Claim 3 recites the limitation "the method of claim 1, wherein the deducing of the sequence ..." in the first line of the claim. There is insufficient antecedent basis for this limitation in the claim because claim 1 does not recite a step of deducing a sequence. Appropriate correction is required.
However, claim 2 recites the “ method of claim 1, wherein the method further comprises deducing the sequence...”. Therefore, it is suggested that claim 3 is amended to depend from claim 2.
For the purposes of compact prosecution, claim 3 is interpreted to depend from claim 2.
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.
Drmanac et al. and Wang et al.
Claim(s) 1-4, 7 and 10-17 are rejected under 35 U.S.C. 103 as being unpatentable over Drmanac et al. (US20140323316) in view of Wang et al. ("Efficient and unique cobarcoding of second-generation sequencing reads from long DNA molecules enabling cost-effective and accurate sequencing, haplotyping, and de novo assembly." Genome research 29.5 (2019): 798-808.; as cited in IDS filed 17 September 2024).
Prior to the effective filing date of the claimed invention, Drmanac et al. teach methods of generating sequencing libraries comprising providing long DNA fragments from genomic DNA(e.g. para 0076-0079, pg. 5); combining the long DNA fragments with beads functionalized with tagged transposons and subjecting to conditions that allow incorporation by transposition of multiple copies of the same tag , amplification, sequencing, mapping and sequence assembly. Furthermore, Drmanac et al. teach fragmentation of a DNA sample to generate overlapping fragments is known (e.g. Entire Drmanac reference and especially para 0077, pg. 5; para 0121, pg. 8; para 0157, pg. 12; method uses long overlapping fragments are generated as in para 0160, pg. 13). Furthermore, Drmanac et al. teach 5kb-750kb fragments (e.g. para 0076, pg. 5).
Furthermore, Drmanac et al. teach different tags are incorporated into different fragments in some embodiments. Furthermore, Drmanac et al. teach tags are incorporated on beads by transposition (e.g. Entire Drmanac reference and especially para 0104-0105, pg. 7; claims 1-9; Fig. 1; ... FIG. 3, this approach employs beads covered with transposon sequences or a concatemer of transposon sequences created by rolling circle replication of a circular DNA that includes the transposon sequence-a transposon nano ball. As in method (1) above, the "transposon sequences" are DNA constructs that include (i) transposon ends and, at a selected location in the transposon sequences between the transposon ends, e.g., near each of the transposon ends, (ii) unique tag sequences (the same tag sequence near both ends), and (iii) a common PCR primer binding site. The transposon-containing bead or nano ball is combined with the long fragments of a target nucleic acid. Conditions are selected to promote the interaction of only a tag assembly, i.e., bead or nano ball bearing a single transposon sequence, with each long fragment... the transposon sequence or another sequence on the transposon
assembly ( e.g., an adaptor ligated to an end of the transposon sequence or concatemer; a homopolymer sequence added by a terminal transferase) Upon addition of transposase, transposition occurs... as in para 0104; beads represent clones of tagged transposons as in para 0110-0112, pg. 7-8; different tags as in para 0013, para 0017, pg. 2; para 0055, pg. 3; claim 6). Furthermore, Drmanac et al. teach spacing between tags in fragments can be 100bp to 5000bp (e.g. para 0082, pg. 5).
Furthermore, Drmanac et al. teach an embodiment wherein long DNA fragments are nicked; ligated with a 3’ common adaptor and adaptor-ligated fragments are combined with beads comprising a capture sequence complementary to the adaptor and a tag. The captured fragments are subjected to conditions that allow tagging prior to amplification, sequencing, mapping and sequence assembly (e.g. Entire Drmanac reference and especially para 0106, pg. 7).
Furthermore, Drmanac et al. teach amplification of target DNA before or after any step, wherein the preferred method is an improved protocol of MDA (e.g. Entire Drmanac reference and especially ...Amplification can be performed after fragmenting or before or after any step outlined herein as in para 0124; para 0125-0130, pg. 9).
Furthermore, Drmanac et al. teach methylation analysis wherein target DNA is fragmented, tagged, denatured prior to MDA and treated with bisulfite before sequencing analysis (e.g. Entire Drmanac reference and especially para 0161-0164, pg. 13). Drmanac et al. teach sequencing using different technologies is known, wherein the resultant reads are mapped to a reference genome to facilitate sequence assembly (e. g. Entire Drmanac reference and especially para 0051-0061, pg. 3-4). Furthermore, Drmanac et al. teach sequencing and assembly is known in methylation analysis, wherein mapping provides information on methylation status for every cytosine in a target sequence and comparative analysis involves unmutagenized sequence(e.g. Entire Drmanac reference and especially... Using MT it is possible to sequence all bases of the genome and assemble a complete diploid genome with digital information on levels of methylation for every cytosine position in the human genome (i.e., 5-base sequencing). Further, MT allow blocks of methylated sequence of 100 kb or greater to be linked to sequence haplotypes, providing methylation haplotyping, information that is impossible to achieve with any currently available method as in para 0161, pg. 13; MDA will amplify each strand of a specific fragment independently yielding for any given cytosine position 50% of the reads as unaffected by bisulfite (i.e., the base opposite of cytosine, a guanine is unaffected by bisulfate) and 50% providing methylation status. Reduced
DNA complexity helps with accurate mapping and assembly of the less informative, mostly 3-base (A, T, G) reads) as in para 0163; pg. 13).
Therefore, Drmanac et al. teach methods are known comprising preparing a sequencing library by mutagenizing (i.e. bisulfite treatment) and barcoding (i.e. tagging). However, Drmanac et al. do not expressly teach co-barcoding.
Prior to the effective filing date of the claimed invention, Wang et al. also teach methods of preparing sequencing libraries comprising transposition followed by barcoding. Specifically, Wang et al. teach methods comprising transposition of hybridization sequence into sample DNA prior to contacting with clonally barcoded beads comprising 4 X 105 capture adapters in hybridization buffer. The incubation allows capture of long DNA fragments ( >100kb). After the DNA capture, barcodes on the beads are incorporated by nick ligation into the target DNA. A second adapter is ligated and the adapter-ligated DNA is subjected to amplification, sequencing and mapping to a reference genome (e.g. Entire Wang reference and especially Results section, pg. 799-804; Fig. 1, pg. 800).
Therefore, as both Drmanac et al. and Wang et al. teach methods of preparing sequencing libraries are known comprising transposition of sequences that facilitate subsequent barcoding steps, it would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date to modify the method of Drmanac et al. comprising tag incorporation by transposition, bisulfite treatment and amplification to include incorporation of additional sequences such as hybridization sequences that allow capture to clonally barcoded beads, which ultimately facilitates co-barcoding of sample DNA, as taught by Wang et al., as a person of ordinary skill in the art would recognize that 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 of preparing a barcoded sequencing library. Furthermore, Wang et al. teach co-barcoding facilitates methylation analysis (e.g. We envision this type of cheap massive barcoding can be useful for RNA analyses, such as full-length mRNA sequencing from thousands of cells by combination with single-cell technologies or deep population sequencing of 16S RNA in microbial samples. Phased chromatin mapping by the Assay for Transposase- Accessible Chromatin (ATAC-seq) (Buenrostro et al. 2013) or methylation studies are all also possible with stLFR... as in 2nd para, pg. 805) and other merits of co-barcoding (e.g. simple to perform and can be implemented with a relatively small investment in oligonucleotides to generate barcoded beads; uses standard equipment found in most molecular biology laboratories and is sequencing technology agnostic; replaces standard second-generation sequencing library preparation methods, requires only 1 ng DNA, and does not add significantly to the cost of whole-genome or whole-exome library preparation with a total cost per sample of less than 30 dollars as in 2nd para, Results section, pg. 799).
Therefore, the combined teachings of Drmanac et al. and Wang et al. render obvious the limitations: A method of sequencing a library of target nucleic acid molecules, the method comprising:
(a) mutagenizing and co-barcoding the library of target nucleic acid molecules, thereby
producing a plurality of mutagenized barcoded fragments for each target nucleic acid molecule,
wherein each of the plurality of mutagenized barcoded fragments comprises a barcode,
where the barcode comprises a barcode sequence, wherein the plurality of mutagenized barcoded fragments produced from the same target nucleic acid share the same barcode sequence, wherein the mutagenized barcoded fragments produced from different target nucleic acid molecules have different barcode sequences, wherein the mutagenesis converts selected nucleic acid bases to different nucleic acid bases at a rate of 1% to 30% (e.g. bisulfite treatment as in para 0161-0164, pg. 13, Drmanac); (b) sequencing the mutagenized barcoded fragments to produce sequence reads, thereby producing sequence reads, wherein at least some of the sequence reads are significantly overlapping; and (c) assembling the sequence reads to generate an assembled sequence of the target nucleic acid based on the barcode sequence in the sequence reads and mutation patterns as recited in claim 1.
Furthermore, as Drmanac et al. teach methylation analysis wherein target DNA is fragmented, tagged, denatured prior to MDA and treated with bisulfite before sequencing analysis (e.g. Entire Drmanac reference and especially para 0161-0164, pg. 13); and teach amplification of target DNA before or after any step, wherein the preferred method is an improved protocol of MDA (e.g. Entire Drmanac reference and especially ...Amplification can be performed after fragmenting or before or after any step outlined herein as in para 0124; para 0125-0130, pg. 9) and Wang teach co-barcoding using beads, the combined teachings of Drmanac et al. and Wang et al. render claim 11 obvious.
As Drmanac et al. teach sequencing and assembly is known in methylation analysis, wherein mapping provides information on methylation status for every cytosine in a target sequence and comparative analysis also involves unmutagenized sequence (e.g. para 0161, MDA will amplify each strand of a specific fragment independently yielding for any given cytosine position 50% of the reads as unaffected by bisulfite (i.e., the base opposite of cytosine, a guanine is unaffected by bisulfate) and 50% providing methylation status. Reduced DNA complexity helps with accurate mapping and assembly of the less informative, mostly 3-base (A, T, G) reads) as in para 0163, pg. 13), the combined teachings of Drmanac et al. and Wang et al. render claims 2 and 3 obvious.
Furthermore, as Drmanac et al. teach tagging using beads prior to bisulfite treatment and Wang teach the co-barcoding using beads, the combined teachings of Drmanac et al. and Wang et al. render claims 4 and 7 obvious.
Furthermore, as Drmanac et al. teach 5kb-750kb fragments (e.g. para 0076, pg. 5), the combined teachings of Drmanac et al. and Wang et al. render claim 10 obvious.
Furthermore, as Drmanac et al. teach spacing between tags in fragments can be 100bp to 5000bp (e.g. para 0082, pg. 5), the combined teachings of Drmanac et al. and Wang et al. render claim 12 obvious.
Furthermore, as Drmanac et al. teach beads functionalized with barcoded transposons are known in the art and Wang et al. teach methods comprising transposition of hybridization sequence into sample DNA prior to contacting with clonally barcoded beads in hybridization buffer; DNA capture, and incorporation of barcodes on the beads by ligation into the target DNA; ligation oof a second adapter; amplification of the adapter-ligated DNA , followed by sequencing and mapping to a reference genome (e.g Results section, pg. 799-804; Fig. 1, pg. 800), the combined teachings of Drmanac et al. and Wang et al. render claims 13-15 obvious.
Furthermore, as Drmanac et al. teach bisulfite treatment which causes deamination of cytosine to uracil which is read as thymine, the combined teachings of Drmanac et al. and Wang et al. render claim 16 obvious.
Furthermore, as Drmanac et al. teach methylation analysis to determine levels of methylation across repetitive elements in a target sample is known, the combined teachings of Drmanac et al. and Wang et al. render obvious the limitation: assigning two sequence reads having distinguishable mutation patterns to two different repetitive regions in the target nucleic acid as recited in claim 17.
Yang et al. and Wang et al.
Claim(s) 1, 6 and 16 are rejected under 35 U.S.C. 103 as being unpatentable over Yang et al. (US20220056519; filed 05 October 2021) in view of Wang et al. ( "Efficient and unique cobarcoding of second-generation sequencing reads from long DNA molecules enabling cost-effective and accurate sequencing, haplotyping, and de novo assembly." Genome research 29.5 (2019): 798-808.; as cited in IDS filed 17 September 2024).
Prior to the effective filing date of the claimed invention, Yang et al. teach a method of preparing a nucleic acid sample for sequencing comprising mutagenizing sample DNA (i.e. bisulfite treatment) and barcoding (i.e. incorporating a universal sequence), wherein these steps can be rearranged (e.g. The method for constructing a sequencing library ... First, a universal sequence is introduced to at least one end of the methylated DNA template, and then a bisulfite treatment is performed; or the bisulfite treatment is first performed, and then the universal sequence is introduced... as in para 0012, pg. 2).
In one embodiment, Yang et al. teach providing a DNA sample comprising methylated DNA, incorporating by transposition a universal adapter comprising a sequencing adapter sequence, wherein the sequencing adapter comprises methylated cytosines and a 6-12 random sequence (i.e. barcode), and subsequently treating with bisulfite under conditions to transform the target sample (i.e. mutagenizing), to yield a bisulfite -treated DNA sample comprising a universal sequence.
Yang et al. teach another embodiment wherein a target DNA sample is treated with bisulfite ( i.e. mutagenizing) and then amplified with a first sequencing primer, wherein the sequencing primer comprises a 6-12 random sequence (i.e. barcode), resulting in a sample that is modified with a universal sequence by amplification with the sequencing primer, to yield a bisulfite -treated DNA sample comprising a universal sequence.
The modified and bisulfite treated DNA is then subjected to a two-step PCR process. The process comprises a first step comprising amplification with a first target specific primer and a first universal primer that anneals to the incorporated universal sequence and incorporates a sequence that is partially complementary to a tagged primer; and a second step comprising amplification with a second target specific primer, which incorporates a second universal sequence; a second universal primer, which anneals to the newly incorporated second universal sequence ; and a tagged primer, which anneals to the previously incorporated complementary sequence and includes a unique 8-12 base sequence that is present in the middle of the tagged primer. The resultant bisulfite treated DNA sample comprises a tag from the sequencing primer that is specific for a particular sequencing platform and a second tag that is unique to the sample (e.g. Entire Yang reference and especially para 0082-0089, transposition as in para 0086; para 0091,pg.6-7; Fig. 1A, 1B and 3; Examples 1 and 2, pg. 8-12) .
Yang et al. also teach high throughput sequencing of the treated and tagged DNA samples followed by mapping to a reference genome (e.g. Example 2, pg. 9-12; especially methylated adapter as in para 0140-0142;bisulfite treatment as in para 0145-0158; two-step PCR as in para 0161-0166; sequencing and mapping to genome as in para 0167-0175; “the mappability refers to a ratio of mapping to the genome;” as in para 0171).
Therefore, Yang et al. teach methods are known comprising preparing a sequencing library by mutagenizing (i.e. bisulfite treatment) and barcoding. However, Yang et al. do not expressly teach co-barcoding.
Prior to the effective filing date of the claimed invention, Wang et al. also teach methods of preparing sequencing libraries comprising transposition followed by barcoding. Specifically, Wang et al. teach methods comprising transposition of hybridization sequence into sample DNA prior to contacting with clonally barcoded beads comprising 4 X 105 capture adapters in hybridization buffer. The incubation allows capture of long DNA fragments ( >100kb). After the DNA capture, barcodes on the beads are incorporated by nick ligation into the target DNA. A second adapter is ligated and the adapter-ligated DNA is subjected to amplification, sequencing and mapping to a reference genome (e.g. Entire Wang reference and especially Results section, pg. 799-804; Fig. 1, pg. 800).
Therefore, as both Yang et al. and Wang et al. teach method of preparing sequencing libraries are known comprising transposition of sequences that facilitate subsequent barcoding steps, it would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date to modify the method of Yang comprising adapter incorporation by transposition, bisulfite treatment and amplification to include incorporation of additional sequences such as hybridization sequences that allow capture to clonally barcoded beads, which ultimately facilitates co-barcoding of sample DNA, as taught by Wang et al., as a person of ordinary skill in the art would recognize that 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 of preparing a barcoded sequencing library. Furthermore, Wang et al. teach the merits of co-barcoding (e.g. simple to perform and can be implemented with a relatively small investment in oligonucleotides to generate barcoded beads; uses standard equipment found in most molecular biology laboratories and is sequencing technology agnostic; replaces standard second-generation sequencing library preparation methods, requires only 1 ng DNA, and does not add significantly to the cost of whole-genome or whole-exome library preparation with a total cost per sample of less than 30 dollars as in 2nd para, Results section, pg. 799).
Therefore, the combined teachings of Yang and Wang render obvious the limitations: A method of sequencing a library of target nucleic acid molecules, the method comprising:
(a) mutagenizing and co-barcoding the library of target nucleic acid molecules, thereby
producing a plurality of mutagenized barcoded fragments for each target nucleic acid molecule,
wherein each of the plurality of mutagenized barcoded fragments comprises a barcode,
where the barcode comprises a barcode sequence, wherein the plurality of mutagenized barcoded fragments produced from the same target nucleic acid share the same barcode sequence, wherein the mutagenized barcoded fragments produced from different target nucleic acid molecules have different barcode sequences, wherein the mutagenesis converts selected nucleic acid bases to different nucleic acid bases at a rate of 1% to 30% (e.g. bisulfite treatment as in Examples 1 and 2, pg. 8-12, Yang); (b) sequencing the mutagenized barcoded fragments to produce sequence reads, thereby producing sequence reads, wherein at least some of the sequence reads are significantly overlapping; and (c) assembling the sequence reads to generate an assembled sequence of the target nucleic acid based on the barcode sequence in the sequence reads and mutation patterns as recited in claim 1.
Furthermore, as Yang et al. teach an embodiment wherein a target DNA sample is treated with bisulfite ( i.e. mutagenizing) and then amplified with a first sequencing primer, wherein the sequencing primer comprises a 6-12 random sequence (i.e. barcode), resulting in a sample that is modified with a universal sequence by amplification with the sequencing primer, to yield a bisulfite -treated DNA sample comprising a universal sequence, the combined teachings of Yang et al. and Wang et al. render claim 6 obvious.
Furthermore, as Yang et al. teach bisulfite treatment which causes deamination of cytosine to uracil which is read as thymine, the combined teachings of Yang et al. and Wang et al. render claim 16 obvious.
Allowable Subject Matter
Claim 5 is objected to as being dependent upon a rejected base claim, but would be allowable if rewritten in independent form including all of the limitations of the base claim and any intervening claims.
Claims 8 and 9 are allowable. The closest art, i.e. Wang et al. and Drmanac et al. (US20210115595), teach co-barcoding but do not expressly teach repeating co-barcoding on previously barcoded fragments.
Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to SAHANA S KAUP whose telephone number is (571)272-6897. The examiner can normally be reached on M-F 7am-7pm EST.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Jennifer Michener can be reached on 571-272-1424. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/SAHANA S KAUP/Supervisory Primary Examiner, Art Unit 1612
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