DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 April 07, 2026 has been entered.
Information Disclosure Statement
The information disclosure statements (IDS) submitted on 04/07/2026 is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Status of Claims / Response to Amendment
This office action is in response to an amendment filed on April 07, 2026.
Claims 1-2, 4-13 and 15-21 were previously pending. Applicant amended claim 1; cancelled claim 21; claim 22 is newly added.
Claims 1-2, 4-13, 15-20 and 22 are currently pending, with claim 20 withdrawn.
Claims 1-2, 4-13, 15-19 and 22 are under consideration.
All of the previously presented rejections have been withdrawn as being obviated by the amendment of the claims, which added new limitations to the claims 1, that were not considered in the previous rejections.
Applicant' s amendments and arguments have been thoroughly reviewed, but are not persuasive to place the claims in condition for allowance for the reasons that follow.
This office action contains new grounds for rejection necessitated by amendment.
Priority
The priority date of the instant claims 1-2, 4-13, 15-19 and 22 is July 13, 2020, filling date of the US provisional application NO. 63/051,149.
Claim Interpretation
In evaluating the patentability of the claims presented in this application, claim terms have been given their broadest reasonable interpretation (BRI) consistent with the specification, as understood by one of ordinary skill in the art, as outlined in MPEP§ 2111.
For the purpose of applying prior art, regarding all claims, terms such as "first" and "second" are interpreted as labels for identification purposes and do not imply any specific priority or sequential order of steps. This interpretation aligns with the claim language, as evidenced by claim 7/6/1, which recites only a "second solid support" is mentioned, without reference to a first solid support. This interpretation is made so the claim can be examined.
For the purpose of applying prior art, claim 1 recites a term "molecular label," which is not defined in the specification.
Because the application's disclosure does not define the term "molecular label" with any required structural features, under BRI this term is interpreted to encompass any type of molecular label, including any detectable label known in the field of molecular biology such as nucleic acid barcode sequences, fluorescent moieties, or other functional groups such as biotin, methylation, or antibodies.
Claim Rejections - 35 USC § 112(d)
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claim 15 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 15 is an improper dependent claim because it refers back to the cancelled claim 14. A dependent claim must refer back to and further limit a preceding claim, see MPEP§608.01(n). In this instant case, because claim 14 has been cancelled, claim 15 does not properly depend from an existing claim and therefore is incomplete.
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-2, 4-11 and 15-19 are rejected under 35 U.S.C. 103 as being unpatentable over
Chang (US20180346970A1, Published on 2018-12-06, cited in IDS US Patent Documents #26, filed on 12/11/23), in view of
Buschmann (Buschmann et al., error-correcting barcodes for multiplexed DNA sequencing. BMC Bioinformatics. 2013 Sep 11;14:272. doi: 10.1186/1471-2105-14-272. PMID: 24021088; PMCID: PMC3853030.);
Willey (US20180216163A1 - Methods for standardized sequencing of nucleic acids and uses thereof; Published 2018-08-02) ;
Bent (US20190367997A1- Systems and methods for quality control in single cell processing; published on 2019-12-05);
as evidenced by Illumina Marketing Document (Optimizing Cluster Density on Illumina Sequencing Systems (2016), Pub. No. 770-2014-038, Published on 11 April 2016).
A) Chang teaches methods for labeling nucleic acids in samples using molecular barcodes with barcoded control nucleic acids, and generating control library data using next-generation sequencing (Fig.10; [0669]).
Regarding claim 1, Chang teaches a method for labeling nucleic acid targets in a sample, comprising:
(a) barcoding copies of a nucleic acid target (Fig.10; [0669]”mRNA”) with a first plurality of oligonucleotide barcodes to generate a plurality of barcoded nucleic acid molecules each comprising a sequence complementary to at least a portion of the nucleic acid target([0669] reverse transcription of mRNA ‘1030’ generates extension products using probe of beads ‘1020’comprising barcode and mRNA as templates),
wherein at least 10 oligonucleotide barcodes of the first plurality of oligonucleotide barcodes comprise different molecular label sequences from each other([0013] lines 19-24, the barcodes of the bead can comprise molecular label sequences selected from at least 1000 or 10000 different molecular label sequences), and
wherein each barcoded nucleic acid molecule of the plurality of barcoded nucleic acid molecules comprises a first universal sequence and a molecular label (see FIGs. 1-2);
(b) after step (a), providing one or more barcoded control nucleic acids (FIG 10A; [0669-0670] barcoded extension products of control particle oligonucleotides 1025 in 1045 b are pooled with barcoded cDNA of mRNA in 1045 a),
wherein the number of copies of each of the one or more barcoded control nucleic acids is predetermined ([0668] lines 4-6).
Chang teaches separating target nucleic acids and control nucleic acids into different droplets ([0668]lines 9-13), performing reverse transcription (RT) with barcodes for target and control nucleic acid separately (FIG. 10; [0669]), and subsequently pooling the RT extension products together ([0670]lines1-3), thereby providing the barcoded control nucleic acid to the barcoded target nucleic acids. Therefore, Change fully teaches the claimed method steps with the specified order for steps a and b.
Chang further teaches (c) generating a sequencing library comprising
a plurality of nucleic acid target library members and
a plurality of control nucleic acid library members ([0670]line 3; [0046]; [0494]; [0503]lines 3-8),
wherein generating a sequencing library comprises:
attaching sequencing adaptors to the plurality of barcoded nucleic acid molecules generated in step (a), or products thereof, to generate the plurality of nucleic acid target library members ([0670]line 3; [0046]; [0494]; [0503]lines 3-8; [0513-0514]); and
attaching sequencing adaptors to the one or more barcoded control nucleic acids provided in step (b), or products thereof, to generate the plurality of control nucleic acid library members([0670]line 3; [0046]; [0494]; [0503]lines 3-8; [0513-0514]);
(d) obtaining sequencing data comprising a plurality of sequencing reads of one or more nucleic acid target library members and a plurality of sequencing reads of one or more control nucleic acid library members ([0670]).
Claim 1 recites a step of “(e) determining the presence of a workflow failure.” Claim 1 has been amended to further describe the workflow failure as “a failure in sequencing library generation downstream of barcoding copies of the nucleic acid target in step (a) and after providing the one or more barcoded control nucleic acids in step (b).”
This feature is suggested by Chang and is obvious in view of the knowledge in the prior art.
The concept of specifically determining library generation failure in part of the workflow, after a target nucleic acid barcoding step and a step providing barcoded control nucleic acids, is suggested by Chang.
Chang teaches performing amplification and sequencing after pooling the barcoded target nucleic acids and control nucleic acids, and the barcodes (e.g., molecular label and control particle oligonucleotide sequence) can be used to determine the efficiency of the entire or part of the workflow ([0670]).
“ [0670] After breaking up the droplets 1045 a, 1045 b, the extension products produced by the reverse transcriptase can be pooled and subject to amplification and sequencing. Sequencing reads can be subject to demultiplexing of cell label, molecular label, gene identity, control particle oligonucleotide sequence, etc. to determine single cell gene expression profiles and quantity efficiency of the entire or part of the workflow.”
Chang further teaches that the barcodes in its teaching “can be used to assess amplification or sequencing errors.” ([0393] lines 8-9).
“[0393]As used herein, the term “stochastic barcode” can refer to a polynucleotide sequence comprising labels of the present disclosure. A stochastic barcode can be a polynucleotide sequence that can be used for stochastic barcoding. Stochastic barcodes can be used to quantify targets within a sample. Stochastic barcodes can be used to control for errors which may occur after a label is associated with a target. For example, a stochastic barcode can be used to assess amplification or sequencing errors.”
Accordingly, in view of the specific workflow taught by Chang, which performs pooled amplification of barcoded target nucleic acids and control nucleic acids, together with Chang’s teaching that barcodes can be used to determine the efficiency of a part of the workflow, specifically to assess amplification errors ꟷ a person of ordinary skill in the art before the effective filing date of the claimed invention would have found it prima facie obvious to apply, in Chang’s method, a step of determining the presence of amplification error. Such amplification error is a failure in the sequencing library generation workflow downstream of the target nucleic acid barcoding step and the step providing barcoded control nucleic acids, because Chang teaches performing amplification after pooling the barcoded target nucleic acids and control nucleic acids ([0670]).
A skilled artisan would have been motivated to assess the presence and degree of amplification error in library generation workflow, because amplification error is a well-known issue in sequencing library generation, as supported by Buschmann 2(see page 2, left-hand col, para 1, lines 1-4), Willey3 (see [0009]-[0012]), and Bent ([0100]). Accordingly, a person of ordinary skill in the art would have sought to evaluate this error rate in order to gain insight into the data quality and the success or failure of the library generation.
The person of ordinary skill would have had a reasonable expectation of success because detecting amplification errors in library construction, using barcoded control nucleic acid is a known quality control approach in the art, this is supported by Bent.
Bent teaches a similar approach to Chang, where control nucleic acids are barcoded (e.g., with UMI) in partitions, amplified then sequenced ([0084]; [0008]) in order to determine the efficiency of the library preparation process. Bent specifically explains that nucleic acid barcodes are useful in providing analyte-specific information, such as the original copy number of an analyte. However, errors that occur in PCR amplification alter the barcode sequence and impedes accurate barcode assignment ([0100]). Bent notes that UMI purity, which indicates degrees of chimerism in the PCR (amplification errors) which contributes to significant technical noise in sequencing data ([0084] lines 26-28), can be measured via comparing the experimental UMI count of a control nucleic acid to an expected UMI count based on the know number of inputted controls ([0100] lines 27-30).
Accordingly, a person of ordinary skill in the art would have possessed sufficient knowledge and skill to apply a step of determining amplification error as a failure in sequencing library generation in the method of Chang, using the barcoded nucleic acid controls.
B) Regarding claim 2, Chang teaches barcoding copies of a nucleic acid target with the first plurality of oligonucleotide barcodes comprises: contacting copies of the nucleic acid target with the first plurality of oligonucleotide barcodes (FIG. 2, 216), wherein each oligonucleotide barcode of the first plurality of oligonucleotide barcodes comprises a first universal sequence, a molecular label, and a target-binding region capable of hybridizing to the nucleic acid targe (FIG.2); and extending the first plurality of oligonucleotide barcodes hybridized to the copies of the nucleic acid target to generate a plurality of barcoded nucleic acid molecules each comprising a sequence complementary to the at least a portion of the nucleic acid target (FIG. 2, 224).
Regarding claim 4, Chang teaches a sample comprises of a plurality of single cells, comprising, prior to contacting copies of the nucleic acid target with the first plurality of oligonucleotide barcodes: partitioning the plurality of single cells to a plurality of partitions ([0455]lines 2-4 ; FIG. 2), wherein a partition of the plurality of partitions comprises a single cell from the plurality of single cells; lysing the single cell after the partitioning step (FIG2, 216); and in the partition comprising the single cell, contacting copies of the nucleic acid target with the first plurality of oligonucleotide barcodes (FIG. 2, 216; [0455]).
Regarding claim 5, Chang teaches a first plurality of oligonucleotide barcodes associated with a first solid support, the method comprising associating the first solid support with the single cell in the sample, and wherein a partition of the plurality of partitions comprises a single first solid support ([0455]; FIG. 2).
Regarding claim 6, Chang teaches one or more barcoded control nucleic acids are generated by: contacting a predetermined number of copies of one or more control nucleic acids with a second plurality of oligonucleotide barcodes (FIG 8A1), wherein each oligonucleotide barcode of the second plurality of oligonucleotide barcodes comprises a first universal sequence (FIG 8A1, 815u ; [0055]), a control label (FIG 8A1, 815c; [0055]), and a target-binding region (FIG 8A1, 815t; [0055]) capable of hybridizing to the one or more control nucleic acids; and extending the second plurality of labeled oligonucleotides hybridized to the one or more control nucleic acids to generate a predetermined number of copies of one or more barcoded control nucleic acids each comprising a sequence complementary to the at least a portion of the one or more barcoded control nucleic acids (FIG 8A1, RT).
Regarding claim 7, Chang teaches a second plurality of oligonucleotide barcodes associated with a second solid support and/or one or more barcoded control nucleic acids are associated with a second solid support (FIG 8A1).
Regarding claim 8, Chang teaches each of the barcoded control nucleic acids comprise one or more of a first universal sequence (FIG 8A1, 815u; [0055]), a control label (FIG 8A1, 815c; [0055]), and a target-binding region (FIG 8A1, 815t; [0055]).
Regarding claim 9, Chang teaches one or more barcoded control nucleic acids ([0622] lines 18-20; [0628]; [0055]) comprises at least 2 different barcoded control nucleic acids.
Regarding claim 10, Chang teaches one or more barcoded control nucleic acids is homologous to genomic sequences of a species, wherein the species is a nonmammalian species, and wherein the non-mammalian species is a phage species ([630]).
Regarding claim 11, Chang teaches each of the plurality of sequencing reads of the plurality of barcoded nucleic acid molecules, or products thereof, comprise (1) a molecular label sequence, and (2) a subsequence of the nucleic acid target; and each of the plurality of sequencing reads of the plurality of barcoded control nucleic acid molecules, or products thereof, comprise (1) a control label sequence (FIG 13 C is the analysis from sequencing data, which indicates molecular label, subsequence of nucleic acid target, and control label sequence are present in the sequence as they are required by this figure).
Regarding claim 15, Chang teaches presence of a failure in barcoding copies of the nucleic acid target is determined by the ratio of sequencing reads of the one or more control nucleic acid library members to sequencing reads of the one or more nucleic acid target library members exceeding a predetermined barcoding threshold, and wherein the predetermined barcoding threshold is at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 ([0658], lines4-6, predetermined barcoding threshold is 5).
Regarding claim 16, Chang teaches determining the copy number of the nucleic acid target ([0655]; [0659] ;Table 4; FIGS. 53A-D; FIGS. 52A-C) in the sample based on the plurality of sequencing reads of one or more nucleic acid target library members.
Regarding claim 17, Chang teaches determining the copy number of the nucleic acid target in the sample comprises determining the copy number of the nucleic acid target in the sample based on the number of molecular labels ([0655]lines15-17, lines36-18; [0423]) with distinct sequences, complements thereof, or a combination thereof, associated with the one or more nucleic acid target library members, or products thereof.
Regarding claim 18, Chang teaches presence of a failure in barcoding copies of the nucleic acid target is determined by the ratio of the predetermined number of copies of the one or more barcoded control nucleic acids to the copy number of the nucleic acid target in the sample exceeding a predetermined barcoding threshold, and wherein the predetermined barcoding threshold is at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 ([0658], lines4-6, predetermined barcoding threshold is 5).
Regarding claim 19, Chang teaches obtaining sequencing data comprising a plurality of sequencing reads of a predetermined number of one or more spike-in library members, wherein the presence of a failure in sequencing library generation is determined by the ratio of sequencing reads of the predetermined number of the one or more spike-in library members to sequencing reads of the one or more control nucleic acid library members exceeding a predetermined library generation threshold, and wherein the predetermined library generation threshold is at least 1 ([501] Illumina sequencing). While Chang does not explicitly disclose the use of spike-in library members with a pre-determined library generation threshold, Chang teaches the sequencing method is Illumina sequencing. It is well-known in the art that Illumina sequencing method utilizes the PhiX library spike-in as sequencing control with a pre-determined library generation threshold, as evidenced by Illumina Marketing Document ( Page 7, c. the summary Tab, % Aligned; page 9: recommended spike in threshold is 1 percent).
Claims 12-13 are rejected under 35 U.S.C. 103 as being unpatentable over Chang,
in view of Buschmann, Willey and Bent, as applied to claim 1 above and further in view of
Fu (Fu et al. Molecular indexing enables quantitative targeted RNA sequencing and reveals poor efficiencies in standard library preparations. Proc Natl Acad Sci U S A. 2014 Feb 4;111(5):1891-6. PMID: 24449890; PMCID: PMC3918775.),
as evidenced by Illumina Technical Note (Illumina Technical Note: Sequencing; Pub. No. 770-2011-022, Published on 12/01/14 by Illumina, Inc).
A) The teachings of Chang, Buschmann, Willey and Bent are recited above and applied as for base claim 1.
Regarding claim 12, Chang teaches determining a sequencing status of the one or more control nucleic acid library members in the sequencing data, wherein the sequencing status of the one or more control nucleic acid library members in the sequencing data is saturated sequencing or under sequencing ([0656]; [0657];[0658]), and wherein the under sequencing status ([0657] example of under sequencing with predetermined threshold) is determined by the one or more control nucleic acid library members having a number of sequencing reads less than a predetermined saturation threshold.
While the teachings of Chang does not specifically teach in detail a case of saturated sequencing, and how the saturated sequencing status is determined using a pre-determined threshold, this feature is obvious in view of Fu.
Fu teaches a method for molecular indexing for quantitative target RNA sequencing, using a set of barcoded control nucleic acid library to monitor the efficiency of library construction (abstract).
Fu teaches a specific case of saturated sequencing, wherein the saturated sequencing status (Table 3; Page 1894, right hand col, lines13-15) is determined by the one or more control nucleic acid library members having a number of sequencing reads at or greater than a predetermined saturation threshold; and the predetermined saturation threshold (Page 1894, right hand col, lines13-15) is a number at least 1.1-fold greater than the predetermined number of copies of the one or more barcoded control nucleic acids (Page 1894, right hand col, lines13-15, 20 fold saturation).
Fu also suggests that the 20 fold saturation threshold should be implemented because : "At this sequencing depth, effectively all unique molecules in the library have been sampled, and very little new information will be obtained by additional sequencing." (page 1894, right hand col, line 15-7 to page 1895, left hand col, line 1)
Accordingly, it would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the method for labeling nucleic acids using molecular barcodes taught by Chang with the teachings of determining a saturated sequencing status using a saturation threshold disclosed by Fu, because both references are in the overlapping field of nucleic acid sequencing and library construction. A skilled artisan in the field of molecular biology, specifically in sequencing technologies, would have naturally considered these references together when seeking to optimize sequencing processes.
The skilled artisan would have been motivated to do so because a saturation threshold of 20 fold is optimal for ensuring that all unique molecules in the library have been sampled, providing a clear benchmark for sequencing efforts, as suggested by Fu. This is beneficial for enhancing the reliability and efficacy of the sequencing process described in Chang.
The person of ordinary skill would have had a reasonable expectation of success in applying Fu's specific saturation threshold concept to the method of Chang because these teachings are technically compatible: Chang teaches the similar concept of under sequencing but only lacks specificity in the case of saturated sequencing. Doing so would yield the predictable result of effectively determining the sequencing status of nucleic acid libraries, particularly in case of saturated sequencing.
B) Regarding claim 13, which depends from claim 12. All limitations in claim 13 are taught by the combination of Chang and Fu, as evidenced by Illumina Technical Note.
The combination of Chang and Fu teach the steps to determine sequencing status and motivation to reach saturated sequencing status, as discussed above for claim 12.
While the combined teachings of Chang and Fu does not explicitly disclose that if sequencing status is the under sequencing status, the step of obtaining sequencing data should be repeated until the sequencing status is the saturated sequencing status, both the methods of Chang ([501] Illumina sequencing) and Fu (page 1895, materials and methods) teach Illumina sequencing as the sequencing method. The approach of repeated sequencing is well-known in the art especially in the application of Illumina sequencing, as evidenced by Illumina Technical Note (page 2, left hand col, when to sequence more). Therefore, the combination of Chang and Fu teach and suggest all elements of claim 13.
Claim 22 is rejected under 35 U.S.C. 103 as being unpatentable over Chang,
in view of Buschmann, Willey and Bent, as applied to claim 1 above and further in view of Saxonov (US20120316074A1 - Methods and compositions for nucleic acid analysis; published on 2012-12-13).
Regarding claim 22, Chang teaches, in a partition (e.g., droplet), performing reverse transcription to barcode nucleic acid targets ([0669]mRNA), comprising hybridizing the target with oligonucleotides comprising barcode sequences, and extending the barcode sequence to generate barcoded cDNAs (Fig.10; [0669])
Although Chang does not explicitly teach that its barcoding reverse transcription reaction is performed in the presence of acetamide, this feature is obvious because acetamide is a known additive to be included in barcoding reverse transcription reaction, especially in partitions, this is supported by Saxonov.
Saxonov, similar to Chang, teaches performing barcoding reverse transcription reaction in partitions ([0016]). Saxonov further teaches a partition can comprise one or more additives, including acetamide ([0093]; [0095] line 1).
Accordingly, a skilled artisan would have found it prima facie obvious that acetamide, as taught in Saxonov, is a suitable additive to be included in the partition of Chang, as Chang and Saxonov both teach performing barcoding reverse transcription reaction in partitions, and Saxonov teaches acetamide as an additive that can be included in the partition.
There would have been a reasonable expectation of success because both references teach performing reverse transcription in partitions, and the modification merely involves applying a known additive in the same technological context to achieve the same result of generating barcoded cDNA. This modification would have been obvious as it represents the KSR principle of predictable use of prior art element according to a known method to yield predictable results. (See MPEP §2143).
Conclusion
No claims are allowed.
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/TIAN NMN YU/Examiner , Art Unit 1681
1 Specifically, claim 1 has been amended to require “a failure in sequencing library generation downstream of barcoding copies of the nucleic acid target in step (a) and after providing the one or more barcoded control nucleic acids in step (b).”
2 Buschmann, see page 2, left-hand col, para 1, lines 1-4:
“It is known, however, that multiple errors can occur with DNA sequencing due to defects in primer synthesis, the ligation process, sample pre-amplification, and finally sequencing. These errors can be either nucleotide substitutions or small insertions and deletions. “
3 Willey, see [0009]-[0012]:
[0009] One concern with NGS is that these quantitative sequencing methods have high intra-lab and inter-lab variation. This problem thus reduces the value of any results, and has prevented the use of these sequencing methods in molecular diagnostics.
[0010] For example, non-systematic (i.e., non-reproducible) biases (i.e., errors), are often inadvertently introduced during preparation of the sequencing library. These non-systemic biases are a major roadblock to implementing NGS as a reliable and efficient routine measurement of nucleic acid abundance (quantification) in the clinical setting.
[0011] The most likely source of non-systematic bias (thus preventing inter-laboratory comparison, and hence routine clinical use, of quantitative NGS data) stems from issues arising from nucleic acid fragmentation, adaptor ligation and PCR.
[0012] Also, although not explicitly required, the FDA has issued guidance and industry recommendations that PCR-based in vitro diagnostic (IVD) devices should contain internal amplification controls (IAC) to control for interfering substances and verify that a negative result for a sample is not caused by inhibitors.
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