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 .
DETAILED ACTION
Status of the Claims
Claims 1-20 are pending and the subject of this NON-FINAL Office Action. This is the first action on the merits.
Large IDS
Applicants filed two IDSs with a combined 50 pages and hundreds of references listed. Many seem irrelevant to the claimed invention (e.g. a 1978 reference US 4124638, regarding polyacrylamide gels) and numerous other references that have nothing to do with index hopping, much less sequencing library preparation using indexes/barcodes. In fact, the IDSs fail to cite any of the references in this Office Action, raising questions about why the references cited in the IDSs were cited in this application. Yet, many of these references are easily found through a simple Google search of “detecting index hopping,” raising questions whether Applicants actually performed any search, and if the IDSs were automatically filed without any regard whatsoever to the actually-claimed invention. The Examiner is not allotted enough time to review every one of the hundreds of references in detail. However, presumably, Applicants reviewed each reference, and filed each for a reason. Thus, the Examiner encourages applicant to specifically point out any particular references in the IDS that they may believe to be particularly relevant to the claimed invention.
Note on Claim Breadth
Applicants’ claim 1 encompass any “index hopping,” that is when an index/barcode/identifier moves from one molecule to another. It is variously referred to throughout the art as index switching, index hopping, misindexing, index misidentification, etc. It is not new. Nor is it’s detection. Thus, when the claims simply state to “determine[e] that the first nucleic acid molecule, prior to or during (b), was subject to index hopping from the sequencing reads obtained from the first nucleic acid molecule,” this encompasses any determination using any detection.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. § 102 that form the basis for the rejections under this section made in this Office action:
(A) A person shall be entitled to a patent unless –
(1)the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention; or
(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 1-10, 16-17 and 20 are rejected under 35 U.S.C. § 102(a)(1) as being anticipated by BERGER (US20210155992).
As to claim 1, BERGER teaches a method for detecting nucleic acid index hopping, comprising:
(a) appending index sequences to a plurality of nucleic acid molecules, wherein: (i) a first nucleic acid molecule of the plurality of nucleic acid molecules comprises a first index sequence and a second index sequence; and (ii) the first index sequence and the second index sequence are different index sequences (Fig. 2);
(b) obtaining sequence reads from the first nucleic acid molecule, wherein sequencing reads obtained from the first nucleic acid molecule comprise an index sequence different from the first index sequence and the second index sequence (Fig. 2); and
(c) determining that the first nucleic acid molecule, prior to or during (b), was subject to index hopping from the sequencing reads obtained from the first nucleic acid molecule (“By using both sample barcode and UMIs, errors that may arise due to index-hopping can be reduced”; para. 0119; see also para. 0115).
As to claim 2, BERGER teaches in (a), a second nucleic acid molecule of the plurality of nucleic acid molecules comprises a third index sequence and a fourth index sequence that are different from each other and each different from the first index sequence and the second index sequence (Fig. 2 and para. 0119).
As to claim 3, BERGER teaches obtaining sequence reads from the second nucleic acid molecule, wherein sequence reads obtained from the second nucleic acid molecule comprise one of the first index sequence and the second index sequence (id.)
As to claim 4, BERGER teaches determining that the second nucleic acid molecule, prior to or during (b), was subject to index hopping from the sequencing reads obtained from the second nucleic acid molecule (id.)
As to claim 5, BERGER teaches in (a), a second nucleic acid molecule of the plurality of nucleic acid molecules comprises the first index sequence and the second index sequence (id.)
As to claim 6, BERGER teaches comprising: (i) obtaining sequencing reads from the second nucleic acid molecule; (ii) further processing the sequencing reads obtained from the second nucleic acid molecule; and (iii) withholding the sequencing reads from the first nucleic acid molecule from further processing (id.)
As to claim 7, BERGER teaches the first nucleic acid molecule or the second nucleic acid molecule comprises a barcode sequence, a unique molecular identifier, or both (id.)
As to claim 8, BERGER teaches generating the first nucleic acid molecule by contacting a molecule comprising a tagged deoxyribonucleic acid (DNA) fragment sequence from a cell or cell nucleus of a sample of a plurality of samples, or complement thereof, and a first tag sequence separate from the index sequences, with a nucleic acid barcode molecule comprising: (i) a first primer binding sequence, (ii) a barcode sequence, and (iii) a first tag binding sequence complementary to the first tag sequence, to generate a first barcoded molecule (Fig. 2).
As to claim 9, BERGER teaches comprising coupling the first index sequence and the second index sequence to the first barcoded molecule, or derivative thereof, to generate the first nucleic acid molecule (Fig. 2).
As to claim 10, BERGER teaches the coupling comprises a nucleic acid extension reaction (Fig. 2)
As to claim 16, BERGER teaches the molecule comprising the tagged DNA fragment is flanked at one end with the first tag sequence and flanked at its other end with a second tag sequence (Fig. 2).
As to claim 17, BERGER teaches appending the index sequences comprises providing a first primer comprising (i) the first index sequence and (ii) a priming sequence complementary to the first primer binding sequence.
As to claim 20, BERGER teaches deep sequencing the first nucleic acid molecule (e.g. para. 0139, 0148).
Claims 1-6 are rejected under 35 U.S.C. § 102(a)(2) as being anticipated by SALATHIA (US20220081685).
As to claim 1, SALATHIA teaches a method for detecting nucleic acid index hopping, comprising:
(a) appending index sequences to a plurality of nucleic acid molecules, wherein: (i) a first nucleic acid molecule of the plurality of nucleic acid molecules comprises a first index sequence and a second index sequence; and (ii) the first index sequence and the second index sequence are different index sequences (“The method may comprise employing reagents from IDT technologies with unique dual indexes (UDI) to prevent misalignment of sequencing reeds. When standard indexes were used, sequencing reads were misassigned to a negative control (NTC).”- para. 0060; “A library was prepared with reagents from the Accel-NGS 2S Plus DNA Library Kit (Swift Biosciences cat. no. SP-2014-96) and Unique Dual Indexes (UDI) (Integrated DNA Technologies).”- para. 0084; “During PCR and before enrichment, each library prep was uniquely labeled with UDIs on both ends. UDIs were selected instead of standard indexes to minimize index hopping, which was especially important for cf-RNA libraries given the low number of copies of the input material. When standard indexes were used, sequencing reads were misassigned to a negative control (NTC). This contamination is alleviated by the use of UDIs (FIGS. 16A-16B).”- para. 0106; “FIGS. 16A-16B are graphs showing misassigned sequences using unique dual indexes (FIG. 16A) compared to standard indexes (FIG. 16B).”- para. 0042);
(b) obtaining sequence reads from the first nucleic acid molecule, wherein sequencing reads obtained from the first nucleic acid molecule comprise an index sequence different from the first index sequence and the second index sequence (id.); and
(c) determining that the first nucleic acid molecule, prior to or during (b), was subject to index hopping from the sequencing reads obtained from the first nucleic acid molecule (id.).
As to claim 2, SALATHIA teaches in (a), a second nucleic acid molecule of the plurality of nucleic acid molecules comprises a third index sequence and a fourth index sequence that are different from each other and each different from the first index sequence and the second index sequence (id.).
As to claim 3, SALATHIA teaches obtaining sequence reads from the second nucleic acid molecule, wherein sequence reads obtained from the second nucleic acid molecule comprise one of the first index sequence and the second index sequence (id.)
As to claim 4, SALATHIA teaches determining that the second nucleic acid molecule, prior to or during (b), was subject to index hopping from the sequencing reads obtained from the second nucleic acid molecule (id.)
As to claim 5, SALATHIA teaches in (a), a second nucleic acid molecule of the plurality of nucleic acid molecules comprises the first index sequence and the second index sequence (id.)
As to claim 6, SALATHIA teaches comprising: (i) obtaining sequencing reads from the second nucleic acid molecule; (ii) further processing the sequencing reads obtained from the second nucleic acid molecule; and (iii) withholding the sequencing reads from the first nucleic acid molecule from further processing (id.)
Claims 1-6 are rejected under 35 U.S.C. § 102(a)(2) as being anticipated by ROBISON (US 20240093287).
As to claim 1, ROBISON teaches a method for detecting nucleic acid index hopping, comprising:
(a) appending index sequences to a plurality of nucleic acid molecules, wherein: (i) a first nucleic acid molecule of the plurality of nucleic acid molecules comprises a first index sequence and a second index sequence; and (ii) the first index sequence and the second index sequence are different index sequences (Title, Abstract, Figs. 4 & 12-16);
(b) obtaining sequence reads from the first nucleic acid molecule, wherein sequencing reads obtained from the first nucleic acid molecule comprise an index sequence different from the first index sequence and the second index sequence (id.); and
(c) determining that the first nucleic acid molecule, prior to or during (b), was subject to index hopping from the sequencing reads obtained from the first nucleic acid molecule (id.).
As to claim 2, ROBISON teaches in (a), a second nucleic acid molecule of the plurality of nucleic acid molecules comprises a third index sequence and a fourth index sequence that are different from each other and each different from the first index sequence and the second index sequence (id.).
As to claim 3, ROBISON teaches obtaining sequence reads from the second nucleic acid molecule, wherein sequence reads obtained from the second nucleic acid molecule comprise one of the first index sequence and the second index sequence (id.)
As to claim 4, ROBISON teaches determining that the second nucleic acid molecule, prior to or during (b), was subject to index hopping from the sequencing reads obtained from the second nucleic acid molecule (id.)
As to claim 5, ROBISON teaches in (a), a second nucleic acid molecule of the plurality of nucleic acid molecules comprises the first index sequence and the second index sequence (id.)
As to claim 6, ROBISON teaches comprising: (i) obtaining sequencing reads from the second nucleic acid molecule; (ii) further processing the sequencing reads obtained from the second nucleic acid molecule; and (iii) withholding the sequencing reads from the first nucleic acid molecule from further processing (id.)
Claims 1-6 are rejected under 35 U.S.C. § 102(a)(2) as being anticipated by Costello et al, Characterization and remediation of sample index swaps by non-redundant dual indexing on massively parallel sequencing platforms, BMC Genomics 19, 332 (2018). https://doi.org/10.1186/s12864-018-4703-0.
As to claim 1, Costello teaches a method for detecting nucleic acid index hopping, comprising:
(a) appending index sequences to a plurality of nucleic acid molecules, wherein: (i) a first nucleic acid molecule of the plurality of nucleic acid molecules comprises a first index sequence and a second index sequence; and (ii) the first index sequence and the second index sequence are different index sequences (dual indexed libraries; Methods- Preparation of sequencing libraries, pg. 9);
(b) obtaining sequence reads from the first nucleic acid molecule, wherein sequencing reads obtained from the first nucleic acid molecule comprise an index sequence different from the first index sequence and the second index sequence (Methods- Cluster amplification and sequencing & Sequencing data analysis, pg. 9); and
(c) determining that the first nucleic acid molecule, prior to or during (b), was subject to index hopping from the sequencing reads obtained from the first nucleic acid molecule (id.).
As to claim 2, Costello teaches in (a), a second nucleic acid molecule of the plurality of nucleic acid molecules comprises a third index sequence and a fourth index sequence that are different from each other and each different from the first index sequence and the second index sequence (id.).
As to claim 3, Costello teaches obtaining sequence reads from the second nucleic acid molecule, wherein sequence reads obtained from the second nucleic acid molecule comprise one of the first index sequence and the second index sequence (id.)
As to claim 4, Costello teaches determining that the second nucleic acid molecule, prior to or during (b), was subject to index hopping from the sequencing reads obtained from the second nucleic acid molecule (id.)
As to claim 5, Costello teaches in (a), a second nucleic acid molecule of the plurality of nucleic acid molecules comprises the first index sequence and the second index sequence (id.)
As to claim 6, Costello teaches comprising: (i) obtaining sequencing reads from the second nucleic acid molecule; (ii) further processing the sequencing reads obtained from the second nucleic acid molecule; and (iii) withholding the sequencing reads from the first nucleic acid molecule from further processing (id.)
Claim Rejection - 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.
Claims 11 and 17-18 are rejected under 35 U.S.C. § 103 as being unpatentable over BERGER (US20210155992), SALATHIA (US20220081685) or ROBISON (US 20240093287), in view of WO2018235938A, in further view of WO2018218226.
It would have been prima facie obvious to a person of ordinary skill in the sequencing art to use other familiar indexing library preparation methods to detect index hopping with a reasonable expectation of success.
None of BERGER, SALATHIA nor ROBISON explicitly teach the indexing library preparation method of claims 11-15, 18 and 19.
However, this indexing library preparation was a well-known option in the sequencing art. For example, WO2018218226 teaches the following:
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This library preparation “allow amplification (e.g., whole genome amplification) and sequencing of chromatin accessible regions of single cells” (Abstract). Thus, the application of this indexing technique to detect index hopping was the simple application of a familiar library preparation technique to a familiar detection technique to achieve familiar index hopping detection results.
Additional prior art in the form of WO2018235938A is cited here to show that the deliberate detection of index hopping as in BERGER, SALATHIA and ROBISON, was useful on its own. Although WO2018235938A only uses one index (Figures), yet the same principle as claimed here and used in BERGER, SALATHIA and ROBISON is also used in WO2018235938A. That is,
According to the present invention, in the method for digitally quantifying a target nucleic acid molecule using an index and a barcode, when a plurality of samples are mixed and the target nucleic acid molecule is quantified, a nucleic acid derived from a sample having an unexpected index It has become apparent that problems may arise that are added to (see FIG. 6E, FIG. 7B). This problem may occur when using an index, and is referred to as index switching, index hopping, misindexing, and the like. The existence of the index switching problem has already been pointed out (Sinha, R. et al. Index switching causes “spreading-of-signal” among multiplexed samples in Illumina HiSeq 4000 DNA sequencing. Biorxiv, 10.1101 / 125724 (2017))
(Detailed Description of Invention). In other words, WO2018235938A is directed to detecting index switching, index hopping, misindexing, and the like by detecting indexes linked to a first sample nucleic acid that are from a second nucleic acid sample. And WO2018235938A makes clear that doing so is useful to affect quality control in sequencing reactions. Thus, it is abundantly clear that detecting index hopping is itself useful and obvious, including doing so using familiar indexing library preparation techniques.
Prior Art
The following prior art also teaches methods to detect misindexing: US 20190211395; US20230059086; US 20210230689; US 20180334711; US 20180334712; US 20220145286; US 20230151356; US20220298545.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to MELODY TSUI whose telephone number is (571)272-1846. The examiner can normally be reached Monday - Friday, 9am - 5pm.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Heather Calamita can be reached at 571-272-2876. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/YUNG-SHENG M TSUI/ Primary Examiner, Art Unit 1684
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