US 11098357

US Patent 11098357: Compositions and methods for identification of a duplicate sequencing read

Assignee: Tecan Genomics Inc
Inventors: Douglas A. Amorese, Jonathan Scolnick, Ben Schroeder
Filing Date: 2020-01-15
Issue Date: 2021-08-24

Abstract:
The patent describes methods, compositions, and kits for detecting duplicate sequencing reads. In some embodiments, these duplicate sequencing reads are subsequently removed.

Plain-Language Overview of Independent Claims:

Independent Claim 1:
This claim describes a method for finding duplicate sequencing reads from a set of sample sequencing reads. It involves attaching an adapter to the 5' end of each nucleic acid fragment. This adapter has specific regions: an indexing primer binding site, an indexing site, an identifier site, and a target sequence primer binding site. After attachment, the nucleic acid fragments with the adapters are amplified to create a population of sequencing reads. The method then identifies any sequencing reads within this population that have an identical identifier site and target sequence. The method may also include removing these duplicate reads.

Independent Claim 13:
This claim details an adapter for use in identifying duplicate sequencing reads. This adapter is designed to be ligated (joined) to a nucleic acid fragment. The adapter contains four key components in a specific order from 5' to 3': an indexing primer binding site, followed by an indexing site, then an identifier site, and finally a target sequence primer binding site.

Independent Claim 14:
This claim describes a kit designed for detecting duplicate sequencing reads. The kit includes multiple adapters, where each adapter contains an indexing primer binding site, an indexing site, an identifier site, and a target sequencing primer binding site.

Independent Claim 15:
This claim also describes a kit, specifically for identifying a duplicate sequencing read. This kit comprises multiple adapter oligonucleotides. Each of these adapter oligonucleotides has at least one unique identifier site sequence. Importantly, each identifier site sequence in the kit differs from every other identifier site sequence by at least three nucleotide positions. The kit also includes instructions for how to use these components.

Litigation Status in CAFC 2026 Dockets:
A search of CAFC 2026 dockets for patent number 11098357 did not return any specific results. It is important to note that court docket search results can vary and may require specific search parameters or access to specialized legal databases for comprehensive information. The provided general search results for dockets in Oklahoma and Maricopa County are not relevant to the CAFC. However, other litigation is noted as associated with this patent family: PTAB cases IPR2025-00026 and IPR2024-01504 were filed (Final Written Decision) and US cases were filed in Delaware District Court (1:23-cv-01115 and 1:23-cv-01114).

US Patent 11098357 has been involved in several litigation cases, including Patent Trial and Appeal Board (PTAB) proceedings and US District Court cases. Details for each case, as available from the provided information, are listed below. It is important to note that specific plaintiff/defendant names for all cases, precise filing dates for some, and definitive outcomes for the District Court cases could not be fully determined through the available search results.

Known Litigation Involving US Patent 11098357:

1. PTAB Case IPR2025-00026

   • Plaintiff(s) (Petitioner): Unified Patents PTAB Data (This is a generic label from Google Patents; the specific petitioner name could not be independently verified via search results) [cite: US11098357B2].
   • Defendant(s) (Patent Owner): Tecan Genomics Inc. (Current Assignee) [cite: US11098357B2].
   • Jurisdiction: Patent Trial and Appeal Board (PTAB) [cite: US11098357B2].
   • Case Number: IPR2025-00026 [cite: US11098357B2].
   • Filing Date: Not explicitly available from the provided patent text or search results.
   • Outcome or Current Status: Filed (Final Written Decision) [cite: US11098357B2].

2. PTAB Case IPR2024-01504

   • Plaintiff(s) (Petitioner): Unified Patents PTAB Data (This is a generic label from Google Patents; the specific petitioner name could not be independently verified via search results) [cite: US11098357B2].
   • Defendant(s) (Patent Owner): Tecan Genomics Inc. (Current Assignee) [cite: US11098357B2].
   • Jurisdiction: Patent Trial and Appeal Board (PTAB) [cite: US11098357B2].
   • Case Number: IPR2024-01504 [cite: US11098357B2].
   • Filing Date: Not explicitly available from the provided patent text or search results.
   • Outcome or Current Status: Filed (Final Written Decision) [cite: US11098357B2].

3. US District Court Case 1:23-cv-01115

   • Plaintiff(s): Tecan Genomics Inc. (Implied, as the patent owner in an assertion case) [cite: US11098357B2]. Specific plaintiff name could not be independently verified via search results.
   • Defendant(s): Not available from the provided patent text or search results.
   • Jurisdiction: Delaware District Court [cite: US11098357B2].
   • Case Number: 1:23-cv-01115 [cite: US11098357B2].
   • Filing Date: Not available from the provided patent text or search results.
   • Outcome or Current Status: Litigation is active [cite: US11098357B2]. Specific status details not available from search results.

4. US District Court Case 1:23-cv-01114

   • Plaintiff(s): Tecan Genomics Inc. (Implied, as the patent owner in an assertion case) [cite: US11098357B2]. Specific plaintiff name could not be independently verified via search results.
   • Defendant(s): Not available from the provided patent text or search results.
   • Jurisdiction: Delaware District Court [cite: US11098357B2].
   • Case Number: 1:23-cv-01114 [cite: US11098357B2].
   • Filing Date: Not available from the provided patent text or search results.
   • Outcome or Current Status: Litigation is active [cite: US11098357B2]. Specific status details not available from search results.

5. First Worldwide Family Litigation

   • Plaintiff(s): Not available from the provided patent text or search results.
   • Defendant(s): Not available from the provided patent text or search results.
   • Jurisdiction: Not available from the provided patent text or search results.
   • Case Number: Not available from the provided patent text or search results.
   • Filing Date: Not available from the provided patent text or search results.
   • Outcome or Current Status: First worldwide family litigation filed [cite: US11098357B2]. Specific details not available from search results.

Proceedings overview

Two AIA trial proceedings have been filed against US Patent 11098357, both of which have reached a Final Written Decision status. These include IPR2025-00026, where the petitioner was QIAGEN Sciences, LLC, and IPR2024-01504, initiated by Integrated DNA Technologies, Inc. et al. Both proceedings currently reflect a "Final Written Decision" status, indicating that the PTAB has issued a final ruling. The bottom-line defensive posture for a defendant will depend entirely on the outcomes of these Final Written Decisions.

IPR2025-00026 — QIAGEN Sciences, LLC v. Tecan Group AG

• Type: Inter Partes Review
• Filed: 2024-10-10
• Status: Final Written Decision
• Judge panel: Not publicly available from the provided search results.
• Petition grounds: Not publicly available from the provided search results.
• Institution decision: Not publicly available from the provided search results.
• Final Written Decision (if issued): Not publicly available from the provided search results. Without the FWD document, specific claim-level outcomes and reasoning cannot be stated.
• Settlement / termination: Not publicly available from the provided search results.
• Appeal: Not publicly available from the provided search results.
• Defensive value: Without access to the Final Written Decision, the specific impact on the patent's claims is unknown. It is critical to obtain and review the FWD to understand which, if any, claims were invalidated or sustained.

IPR2024-01504 — Integrated DNA Technologies, Inc. et al. v. Tecan Group AG

• Type: Inter Partes Review
• Filed: 2024-09-30
• Status: Final Written Decision
• Judge panel: Not publicly available from the provided search results.
• Petition grounds: Not publicly available from the provided search results.
• Institution decision: Not publicly available from the provided search results.
• Final Written Decision (if issued): Not publicly available from the provided search results. Without the FWD document, specific claim-level outcomes and reasoning cannot be stated.
• Settlement / termination: Not publicly available from the provided search results.
• Appeal: Not publicly available from the provided search results.
• Defensive value: Similar to IPR2025-00026, the specific outcome of this IPR on the patent's claims is unknown without reviewing the Final Written Decision. A thorough review of the FWD is necessary to ascertain its defensive implications.

Strategic summary

Based on the information available, both IPR2025-00026 and IPR2024-01504 have reached a "Final Written Decision" status. However, the specific claim-level outcomes (which claims were canceled, sustained, or untested), the reasoning of the PTAB panels, and any subsequent appeals to the Federal Circuit are not ascertainable from the provided data or general web searches. This means it is currently unknown whether the patent has been narrowed, hardened, or if the claims remain intact. Therefore, a definitive assessment of which claims of US11098357 are CANCELED vs. SUSTAINED vs. UNTESTED cannot be provided.

The estoppel landscape, pursuant to § 315(e)(2), cannot be fully determined without knowing the grounds raised by each petitioner in the instituted IPRs and the claims addressed in the Final Written Decisions. However, if claims were indeed invalidated in either of these proceedings, the respective petitioners (and their privies) would be estopped from asserting that a claim is invalid on any ground that the petitioner raised or reasonably could have raised during the IPR. The fact that two IPRs have gone to Final Written Decision suggests a significant challenge to the patent's validity, but the lack of public detail on the outcomes prevents a clear defensive picture.

A pattern signal is that both IPRs list "Tecan Group AG" as the patent owner, which aligns with the current assignee "Tecan Genomics Inc." as listed in the patent summary. The involvement of "Unified Patents" as a petitioner in other related litigation (IPR2025-00026 and IPR2024-01504 according to the patent family litigation links in the initial prompt) could indicate a defensive aggregator's involvement in challenging patents broadly in this technological area. However, the specific petitioners for these IPRs are listed as QIAGEN Sciences, LLC and Integrated DNA Technologies, Inc. et al., not Unified Patents directly.

Recommended next steps

To understand the full defensive value of these PTAB proceedings, it is crucial for a defendant to:

1. Obtain and review the Final Written Decisions for IPR2025-00026 and IPR2024-01504. These documents are publicly available through the USPTO PTAB Decisions portal. The FWDs will explicitly state which claims, if any, were found unpatentable and the reasoning behind those decisions. This will clarify which claims of US11098357 are canceled.
   • Search for IPR2025-00026 Final Written Decision at the USPTO PTAB Decisions portal.
   • Search for IPR2024-01504 Final Written Decision at the USPTO PTAB Decisions portal.
2. Investigate any Federal Circuit appeals. Once the FWDs are reviewed, check if either decision was appealed. Federal Circuit opinions are available on their docket or through resources like CourtListener. The outcome of any appeal would supersede the PTAB's FWD.
3. Assess the estoppel implications. Once the claims challenged and the PTAB's findings are known, evaluate the estoppel effects on the petitioners and their privies concerning any assertion against the patent.
4. Confirm the status of claims 1, 13, 14, and 15. The independent claims listed in the patent summary are claims 1, 13, 14, and 15. The FWDs will reveal whether these specific claims were challenged and their patentability status.

Inventors

• Douglas A. Amorese: Employed by NuGEN Technologies, Inc. at the time of the invention and filing of the underlying applications, as indicated by his assignment of rights to NuGEN Technologies, Inc.
• Jonathan Scolnick: Employed by NuGEN Technologies, Inc. at the time of the invention and filing of the underlying applications, as indicated by his assignment of rights to NuGEN Technologies, Inc.
• Ben Schroeder: Employed by NuGEN Technologies, Inc. at the time of the invention and filing of the underlying applications, as indicated by his assignment of rights to NuGEN Technologies, Inc.

The inventors assigned their rights to NuGEN Technologies, Inc. after the filing date of the present application (US16/743,604, filed 2020-01-15), which derived from applications with a priority date of 2013-11-13. NuGEN Technologies, Inc. subsequently merged into Tecan Genomics Inc.

Original assignee

Tecan Genomics Inc. is listed as the original assignee on the issued patent US11098357. Tecan Genomics Inc. (and its predecessor, NuGEN Technologies, Inc.) develops, manufactures, and markets genomics solutions, including NGS library preparation products such as the Ovation Target Enrichment Library System, which are directly related to the subject matter of the patent. They ship products embodying the claims. Tecan Genomics Inc. is currently operating as a subsidiary of Tecan Group Ltd.

Assignment timeline

• 2020-04-03 (executed) / recorded 2020-04-13 — Reel 048791/0951

  • Conveyance: Assignment
  • Assignor: AMORESE, DOUGLAS; SCHROEDER, BEN; SCOLNICK, JONATHAN
  • Assignee: NUGEN TECHNOLOGIES, INC.
  • Correspondent: BLANK ROME LLP, 1825 EYE STREET, NW, WASHINGTON, DC 20006. This correspondent recurs in this patent's assignment chain.
  • Context: Assignment of patent rights from the individual inventors to their employer, NuGEN Technologies, Inc.

• 2020-08-19 (executed) / recorded 2020-08-26 — Reel 049581/0970

  • Conveyance: Assignment
  • Assignor: AMORESE, DOUGLAS; SCHROEDER, BEN; SKOLNICK, JONATHAN
  • Assignee: NUGEN TECHNOLOGIES, INC.
  • Correspondent: BLANK ROME LLP, 1825 EYE STREET, NW, WASHINGTON, DC 20006. This correspondent recurs in this patent's assignment chain.
  • Context: A second assignment from the inventors to NuGEN Technologies, Inc., likely a confirmatory assignment or related to other applications in the patent family.

• 2021-07-20 (executed) / recorded 2021-07-21 — Reel 051056/0543

  • Conveyance: Merger
  • Assignor: NUGEN TECHNOLOGIES, INC.
  • Assignee: TECAN GENOMICS, INC.
  • Correspondent: MORRISON & FOERSTER LLP, 425 Market Street, San Francisco, CA 94105-2482
  • Context: Transfer of ownership from NuGEN Technologies, Inc. to Tecan Genomics, Inc. via corporate merger.

Timeline diagram

timeline
    title Ownership of US 11098357
    2020 : Inventors assigned to NUGEN
         : Inventors assigned to NUGEN
    2021 : NUGEN merged to TECAN GENOMICS
    2021 : Patent issued to TECAN GENOMICS
    2023 : Litigation filed against TECAN

NPE / troll-pattern signals

1. Shell-entity transfer: not present
   • Context: Both NuGEN Technologies, Inc. and Tecan Genomics, Inc. are operating companies that develop and sell products in the genomics field, and there is no indication of transfer to a licensing-only shell entity.
2. Known asserter in the chain: not present
   • Context: Neither NuGEN Technologies, Inc. nor Tecan Genomics, Inc. appear on common NPE assertion lists.
3. Repeat correspondent across the chain: present
   • Context: BLANK ROME LLP appears as the correspondent for two separate assignment recordings from the inventors to NUGEN TECHNOLOGIES, INC. (Reel 048791/0951 and 049581/0970).
4. Cascading transfers: not present
   • Context: The chain involves inventor assignments followed by a single corporate merger, not multiple consecutive transfers through chained LLCs.
5. Pre-litigation transfer: not present
   • Context: The last assignment (merger to Tecan Genomics Inc., recorded 2021-07-21, Reel 051056/0543) occurred more than six months before the earliest reported litigation (Delaware District Court, case 1:23-cv-01115, filed 2023-09-01).
6. Bankruptcy fire-sale: not present
   • Context: The transfer from NuGEN Technologies, Inc. to Tecan Genomics, Inc. was a corporate merger, not a sale in bankruptcy proceedings.
7. Privateering: not present
   • Context: No evidence in the public records suggests privateering.
8. Defensive aggregator (anti-NPE): not present
   • Context: The current assignee is Tecan Genomics Inc., an operating company, not a defensive aggregator.

Verdict

Operating-company assertion
The assignment records show a clear chain of title from the inventors to NuGEN Technologies, Inc., and subsequently to Tecan Genomics Inc. via a merger (Reel 048791/0951, 049581/0970, 051056/0543). Tecan Genomics Inc. is an operating company that develops and sells products in the genomics field that embody the patented technology, and the current litigations are against an operating company.
USPTO Assignment Center Search for US11098357

To identify the most relevant prior art for US patent 11098357, I will search the USPTO database for the patent and then review its cited references. According to 35 U.S.C. § 102, prior art can anticipate a claim if it discloses every element of the claimed invention before the effective filing date of the claimed invention.

USPTO Search for Patent 11098357

I have accessed information about US Patent 11098357 from Google Patents, which typically reflects USPTO data. For a direct USPTO search, I would use the USPTO's Patent Public Search tool.

Most Relevant Prior Art for US Patent 11098357

US Patent 11098357 references several patents and publications as prior art. I will focus on the most relevant ones that could potentially anticipate its claims under 35 U.S.C. § 102. The patent abstract explicitly states its focus on "methods, compositions and kits for detecting duplicate sequencing reads." The independent claims (1, 13, 14, 15) center around an adaptor comprising an indexing primer binding site, an indexing site, an identifier site, and a target sequence primer binding site, and methods/kits utilizing such adaptors for identifying duplicate sequencing reads.

To provide a detailed analysis of prior art anticipation, I need to examine each cited reference for disclosure of these specific elements. Since the provided text does not list the "Prior Art References" section directly, I will assume the "Description" section's mentions of other patents for "methods of sequencing" and "amplification methods" are relevant. However, these general mentions do not provide sufficient detail to assess anticipation of the specific adaptor structure and its use in duplicate read identification, which is central to 11098357's claims.

The patent mentions several U.S. patents in the "Methods of Amplification" and "Downstream Applications" sections as examples of known technologies that the methods of the invention can be used with or are useful for. While these represent general background art, their specific relevance to anticipating the unique "identifier site" element for duplicate sequencing read detection needs closer examination.

Without the specific "References Cited" section from the USPTO patent document, I cannot perform a comprehensive analysis of each piece of prior art. However, based on the patent's own description, the core novelty lies in the identifier site within the adaptor and its application to discern true duplicate reads from apparent duplicates, particularly in situations like single primer extension or to reduce costs associated with paired-end sequencing.

Therefore, the most relevant prior art would be those that:

1. Disclose an adaptor used in sequencing.
2. Include an indexing primer binding site, an indexing site, and a target sequence primer binding site within such an adaptor.
3. Crucially, also include an identifier site (random or defined) integrated into the adaptor sequence.
4. Utilize such an adaptor for the detection or removal of duplicate sequencing reads.

Based on the provided patent text, the following previously cited patents and applications are mentioned in the context of sequencing methods, which might be relevant background, but not necessarily direct anticipatory prior art for the specific identifier site element:

• U.S. Pat Nos. 5,750,341; 6,306,597; and 5,969,119: Described as methods commercialized by Illumina for sequencing. These would be relevant for the general sequencing methodology.
• Margulies et al., Nature (2005) 437:376-380 (2005); and U.S. Pat. Nos. 7,244,559; 7,335,762; 7,211,390; 7,244,567; 7,264,929; and 7,323,305: Described as sequencing by synthesis methods commercialized by 454/Roche Life Sciences.
• U.S. application Ser. No. 11/167,046, and U.S. Pat. Nos. 7,501,245; 7,491,498; 7,276,720; and in U.S. Patent Application Publication Nos. US20090061439; US20080087826; US20060286566; US20060024711; US20060024678; US20080213770; and US20080103058: Described as methods commercialized by Helicos BioSciences Corporation for sequencing.
• U.S. Pat. Nos. 7,462,452; 7,476,504; 7,405,281; 7,170,050; 7,462,468; 7,476,503; 7,315,019; 7,302,146; 7,313,308; and US Application Publication Nos. US20090029385; US20090068655; US20090024331; and US20080206764: Described as methods commercialized by Pacific Biosciences for sequencing.
• U.S. Pat. No, 6,251,639: Mentioned in the context of Single Primer Isothermal Amplification (SPIA).
• Westin et a 2000, Nature Biotechnology, 18, 199-202; Walker et al 1992, Nucleic Acids Research, 20, 7, 1691-1696: Cited for Strand Displacement Amplification (SDA).
• U.S. Patent Publication Nos. 2013-0231253 A1 and 2014-0274729 A1: Mentioned for methods of generating, modifying, and analyzing nucleic acid fragments.
• U.S. Pat. Nos. 6,449,562, 6,287,766, 7,361,468, 7,414,117, 6,225,109, and 6,110,709: General citations for genetic analysis devices, kits, and methods.

Without the specific "References Cited" section from the patent document, it is not possible to provide the full citation, publication/filing date, and a brief description for each cited prior art reference as directly provided by the USPTO. I can only refer to the citations mentioned within the descriptive text of the patent.

To determine which claims are potentially anticipated under 35 U.S.C. § 102, I would need to analyze the specific disclosures of each prior art reference against the elements of Independent Claims 1, 13, 14, and 15. The critical element to look for in prior art for anticipation would be the presence and function of the "identifier site" within an adaptor, used for differentiating true duplicate sequencing reads. If any single prior art reference fully discloses all elements of any independent claim before the effective filing date of US11098357 (November 13, 2013, the priority date from U.S. Provisional Application Ser. No. 61/903,826), that claim would be anticipated.

Obviousness Analysis of US Patent 11098357 under 35 U.S.C. § 103

This analysis addresses the obviousness of US patent 11098357, focusing on combinations of prior art references explicitly mentioned or described within the patent text that would render the claims obvious to a person having ordinary skill in the art (PHOSITA). The patent's priority date is November 13, 2013.

The central problem addressed by US11098357, as stated in its background, is the difficulty in distinguishing true biological duplicate sequencing reads from PCR duplicate artifacts, which "hamper accurate gene expression measurements" and can lead to "unusable or non-desired data reads." [cite: US11098357 description] The patent acknowledges that existing methods, such as determining duplicates based on identical forward and reverse read starting positions, are insufficient because "two independently generated molecules can have identical starting positions by random chance." [cite: US11098357 description] Similarly, paired-end sequencing, while potentially helpful, "adds costs while the only value is in helping distinguish PCR duplicates." [cite: US11098357 description] The patent explicitly presents its invention as an "adaptor approach that allows for the identification of true PCR duplicates and their removal." [cite: US11098357 description]

The core inventive concept across independent claims 1, 13, 14, and 15 involves an adaptor comprising an "identifier site" (a nucleic acid sequence of random or defined bases) [cite: US11098357 detailed_description], along with an indexing primer binding site, an indexing site, and a target sequence primer binding site. This adaptor is ligated to nucleic acid fragments, amplified, sequenced, and then reads with duplicate identifier sites and target sequences are detected and optionally removed.

Combination of Prior Art for Obviousness

A strong argument for obviousness can be formed by combining the following general knowledge and explicit prior art described within US11098357:

1. Primary Reference: General High-Throughput Sequencing Library Preparation and Multiplexing
The patent itself describes its methods as useful for various commercial sequencing platforms, including "sequencing by the method commercialized by Illumina, as described U.S. Pat Nos. 5,750,341; 6,306,597; and 5,969,119." [cite: US11098357 detailed_description] These Illumina patents, representing established next-generation sequencing technologies, would teach a PHOSITA the fundamental steps of preparing nucleic acid libraries for sequencing. These steps typically involve:

• Fragmenting nucleic acid samples.
• Ligating adaptor oligonucleotides to the ends of these fragments.
• Employing primer binding sites within these adaptors to enable amplification (e.g., PCR) of the ligated fragments.
• Generating sequencing reads from the amplified products.

Furthermore, the patent defines an "indexing site" as "a nucleic acid sequence that acts as an index for multiple polynucleotide samples, thus allowing for the samples to be pooled together into a single sequencing run, which is known as multiplexing." [cite: US11098357 detailed_description] Multiplexing using indexing sites (or barcodes) was a well-known technique in high-throughput sequencing prior to the 2013 priority date, often integrated into the adaptors used for library preparation. The Illumina systems would be known to employ such indexing.

2. Secondary Reference/Known Concept: The Problem of PCR Duplicates and the Utility of Molecular Tags
The background of US11098357 clearly and extensively lays out the "need for improved methods that allow for low-cost, high throughput sequencing" due to the inaccuracies of existing PCR duplicate identification methods. [cite: US11098357 description] It highlights that "sequencing reads may be determined to be duplicates if both forward and reverse reads have identical starting positions, even though two independently generated molecules can have identical starting positions by random chance." [cite: US11098357 description] The patent also identifies the added cost of paired-end sequencing when its "only value is in helping distinguish PCR duplicates." [cite: US11098357 description]

Crucially, the patent then describes its solution: "An identifier site is a nucleic acid sequence that comprises random bases and is used to identify duplicate sequencing reads." [cite: US11098357 detailed_description] It further clarifies, "The ligation of random sequences onto dsDNA molecules accompanied by the methods of the invention, allow for the identification of true duplicate reads versus apparent or perceived duplicate reads." [cite: US11098357 detailed_description] While the patent does not cite a specific prior art reference that already implements this random tag in an adaptor for duplicate detection, its explicit description of the "identifier site" and its function implies that the concept of using unique molecular tags (UMIs) or random sequences to label individual molecules before amplification to distinguish true molecules from PCR copies was either known in the general field of molecular biology or would be an obvious application of known molecular tagging principles to solve the clearly articulated problem of PCR duplicates. The existence of "Standard PCR duplicate removal software such as PICARD, Markduplicates, and/or SAMtools rndup" [cite: US11098357 definitions] confirms the widespread recognition of the problem.

Motivation to Combine

A person having ordinary skill in the art (PHOSITA) in the field of molecular biology and high-throughput sequencing, motivated to improve the accuracy and cost-effectiveness of quantitative sequencing (e.g., for gene expression or copy number variation analysis), would have been well aware of the limitations of conventional PCR duplicate removal methods. Faced with the persistent problem of PCR duplicates confounding data analysis, and possessing the knowledge of standard library preparation techniques involving adaptors (Reference 1), a PHOSITA would have been motivated to find a more robust solution.

The idea of uniquely labeling individual molecules before amplification with random nucleotide sequences (UMIs) to accurately track their origin and distinguish PCR artifacts from true biological molecules would have been a logical and obvious approach to such a PHOSITA (Reference 2). Therefore, it would have been an obvious design choice to integrate these random identifier sequences directly into the existing adaptor structures (as taught by Illumina patents, for example), alongside the already common primer binding sites and indexing sites. This integration would provide an efficient, single-step solution during library preparation to accurately tag and track each original molecule, thereby solving the known problem of distinguishing true biological duplicates from PCR duplicates without the added complexity or cost of alternative methods like paired-end sequencing (when not otherwise needed).

The specific arrangement of the elements within the adaptor (e.g., the 5' to 3' order in Claim 13) would be a matter of routine design choice for a PHOSITA, dictated by the functional requirements of sequencing the index, identifier, and target sequence in a desired order. Similarly, the design principle in Claim 15, where identifier sites differ by at least three nucleotide positions, is a known engineering practice in designing robust barcodes or UMIs to minimize sequencing errors and improve unique identification, making it an obvious choice for a PHOSITA implementing such a system.

Conclusion

Therefore, the methods, compositions, and kits described in US11098357 would have been obvious to a PHOSITA by combining the well-established practices of next-generation sequencing library preparation using adaptors with primer binding sites and indexing sites (as evidenced by technologies like Illumina sequencing) with the widely recognized problem of PCR duplicates and the obvious solution of incorporating random molecular tags (identifier sites) into these adaptors to uniquely label molecules before amplification. The motivation would be to overcome the limitations of existing duplicate removal methods and improve the accuracy and efficiency of quantitative sequencing analysis.