IP Library › Granted Patent US 12,404,550
Granted Patent B2
US 12,404,550 · App. 18/807,061 · Granted Sep 2, 2025

Dark cycle sequencing

Inventors: Eli N. Glezer (Del Mar, CA); Abrehet Abdu (San Diego, CA); Timothy Looney (Austin, TX)
Assignee: Singular Genomics Systems, Inc.
C12Q1/6869
View Patent ↗
Loading inventors, assignments & file history…
Monitor This Case
Get email alerts when status or documents change.
Order Certified Copies
Most orders are placed with the USPTO same day — all within 24 business hours.
Order via The Patent Place →
Pre-filled with this patent's details
Quick Facts
Patent No.
US 12,404,550
App. No.
18/807,061
Granted
Sep 2, 2025
Kind
B2
Abstract

Provided herein are methods including alternating series of sequencing cycles and dark extension cycles allowing longer read lengths and addressing disadvantages of traditional nucleic acid sequencing protocols.

Claims (31)

1. A method of sequencing a nucleic acid molecule, said method comprising:

extending a primer hybridized to the nucleic acid molecule in a plurality of consecutive dark cycles; followed by extending the primer in a plurality of identifying cycles;

wherein each dark cycle comprises:

contacting the primer with a plurality of unlabeled nucleotides wherein an unlabeled nucleotide comprises a reversible terminator moiety, and incorporating with a polymerase the unlabeled nucleotide into the primer, and removing the reversible terminator moiety, wherein the unlabeled nucleotide comprising the reversible terminator moiety comprises the same nucleobase type in the plurality of consecutive dark cycles;

wherein each identifying cycle comprises:

contacting the primer with a plurality of sequencing nucleotides, and binding one sequencing nucleotide associated with a detectable label to the nucleic acid molecule, and detecting the label.

2. The method of claim 1 , wherein each dark cycle does not comprise a detection process to identify the nucleotide incorporated during the dark cycle.

3. The method of claim 1 , wherein each reversible terminator moiety comprises a disulfide, azido, or allyl moiety.

4. The method of claim 1 , wherein each reversible terminator moiety has the formula:

5. The method of claim 1 , wherein the sequencing nucleotide comprises a cyanine moiety.

6. The method of claim 1 , wherein the sequencing nucleotide is covalently attached to a fluorophore.

7. The method of claim 1 , wherein the sequencing nucleotide comprises a nucleobase and the identity of the nucleobase is associated with the detectable label.

8. The method of claim 1 , wherein the detectable label is a fluorophore.

9. The method of claim 1 , comprising 2 consecutive dark cycles.

10. The method of claim 1 , comprising 5 consecutive dark cycles.

11. The method of claim 1 , comprising 10 consecutive dark cycles.

12. The method of claim 1 , wherein each dark cycle comprises contacting the primer with a plurality of unlabeled nucleotides, wherein each unlabeled nucleotide comprises a reversible terminator moiety.

13. The method of claim 12 , wherein each reversible terminator moiety comprises a disulfide, azido, or allyl moiety.

14. The method of claim 1 , wherein each dark cycle comprises contacting the primer with a plurality of unlabeled nucleotides, the plurality comprises:

a. a plurality of adenine nucleotides, or analogs thereof;

b. (i) a plurality of thymine nucleotides, or analogs thereof, or (ii) a plurality of uracil nucleotides, or analogs thereof;

c. a plurality of cytosine nucleotides, or analogs thereof; and

d. a plurality of guanine nucleotides, or analogs thereof.

15. The method of claim 1 , wherein the nucleic acid molecule is in a sample comprising cells.

16. The method of claim 1 , wherein the nucleic acid molecule is in a sample comprising a tissue.

17. The method of claim 1 , wherein the nucleic acid molecule is immobilized to a surface within a flow cell.

18. The method of claim 1 , wherein four different sequencing nucleotides are present during each identifying cycle and each sequencing nucleotide comprises a different label.

19. The method of claim 14 , wherein the adenine nucleotides, or analogs thereof, comprise the reversible terminator moiety.

20. The method of claim 14 , wherein the thymine nucleotides, or analogs thereof, comprise the reversible terminator moiety.

21. The method of claim 14 , wherein the cytosine nucleotides, or analogs thereof, comprise the reversible terminator moiety.

22. The method of claim 14 , wherein the guanine nucleotides, or analogs thereof, comprise the reversible terminator moiety.

Assignments (2)
SECURITY INTEREST Recorded Mar 7, 2025
From: SINGULAR GENOMICS SYSTEMS, INC.
To: FIRST-CITIZENS BANK & TRUST COMPANY
Reel/Frame 070440/0465 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Aug 22, 2024
From: GLEZER, ELI N.; ABDU, ABREHET; LOONEY, TIMOTHY
To: SINGULAR GENOMICS SYSTEMS, INC.
Reel/Frame 068371/0088 →
Continuity (4)
Continuation 17545226 · Dec 8, 2021
Continuation 17127308 · Dec 18, 2020
Provisional Application 62953028 · Dec 23, 2019
Related Publication 20240401125A1 · Dec 5, 2024
References Cited (78)
US 5034506A · Summerton et al. · 1991 [cited by applicant]
US 5235033A · Summerton et al. · 1993 [cited by applicant]
US 5302509A · Cheeseman · 1994 [cited by applicant]
US 5599675A · Brenner · 1997 [cited by applicant]
US 5750341A · Macevicz · 1998 [cited by applicant]
US 5763594A · Hiatt et al. · 1998 [cited by applicant]
US 5808045A · Hiatt et al. · 1998 [cited by applicant]
US 5872244A · Hiatt et al. · 1999 [cited by applicant]
US 6013445A · Albrecht et al. · 2000 [cited by applicant]
US 6232465B1 · Hiatt et al. · 2001 [cited by applicant]
US 6255475B1 · Kwiatkowski · 2001 [cited by applicant]
US 6664079B2 · Ju et al. · 2003 [cited by applicant]
US 7057026B2 · Barnes et al. · 2006 [cited by applicant]
US 7211390B2 · Rothberg et al. · 2007 [cited by applicant]
US 7244559B2 · Rothberg et al. · 2007 [cited by applicant]
US 7264929B2 · Rothberg et al. · 2007 [cited by applicant]
US 7541444B2 · Milton et al. · 2009 [cited by applicant]
US 8143030B2 · Maxham et al. · 2012 [cited by applicant]
US 8703461B2 · Peris et al. · 2014 [cited by applicant]
US 8882980B2 · Ling et al. · 2014 [cited by applicant]
US 9416409B2 · Hayden · 2016 [cited by applicant]
US 10738072B1 · Graham et al. · 2020 [cited by applicant]
US 11225688B2 · Glezer et al. · 2022 [cited by applicant]
US 12123054B2 · Glezer et al. · 2024 [cited by applicant]
US 20050191698A1 · Chee et al. · 2005 [cited by applicant]
US 20100279882A1 · Ronaghi et al. · 2010 [cited by applicant]
US 20100323350A1 · Gordon et al. · 2010 [cited by applicant]
US 20130281306A1 · Rigatti et al. · 2013 [cited by applicant]
US 20170029883A1 · Zhao et al. · 2017 [cited by applicant]
US 20170044601A1 · Crnogorac · 2017 [cited by applicant]
US 20200131484A1 · Golynskiy et al. · 2020 [cited by applicant]
US 20200181587A1 · Klausing et al. · 2020 [cited by applicant]
US 20200199666A1 · Gatti-Lafranconi et al. · 2020 [cited by applicant]
US 20200232024A1 · Esfandyarpour et al. · 2020 [cited by applicant]
US 20200377937A1 · Pratt et al. · 2020 [cited by applicant]
US 20210054442A1 · Pratt et al. · 2021 [cited by applicant]
US 20210189481A1 · Glezer et al. · 2021 [cited by applicant]
US 20220170092A1 · Glezer et al. · 2022 [cited by applicant]
US 20230265501A1 · Looney et al. · 2023 [cited by applicant]
EP 4081653B1 · 2024 [cited by applicant]
WO WO1996007669A1 · 1996 [cited by applicant]
WO WO2004018497A2 · 2004 [cited by applicant]
WO WO2009097626A2 · 2009 [cited by applicant]
WO WO2010127304A2 · 2010 [cited by applicant]
WO WO20180148723A1 · 2018 [cited by applicant]
WO WO2020056044A1 · 2020 [cited by applicant]
WO WO2020227161A1 · 2020 [cited by applicant]
WO WO2022197942A1 · 2022 [cited by applicant]
Kruglyak, S. et al. (Feb. 5, 2024). “Characterizing and addressing error modes to improve sequencing accuracy,” [cited by applicant]
Bains, I. et al. (May 28, 2009). “Quantifying the development of the peripheral naive CD4 T-cell pool in humans,” [cited by applicant]
Baker, G. C. et al. (Dec. 2003, e-published Nov. 3, 2003). “Review and re-analysis of domain-specific 16S primers,” [cited by applicant]
Bentley, D. R. et al. (Nov. 6, 2008). “Accurate whole human genome sequencing using reversible terminator chemistry,” [cited by applicant]
Bergen, K. et al. (Jun. 17, 2013, e-published Jun. 3, 2013). “Structures of KOD and 9°N DNA polymerases complexed with primer template duplex,” [cited by applicant]
Campbell, M. A. et al. (Dec. 28, 2006). “Comprehensive analysis of alternative splicing in rice and comparative analyses with [cited by applicant]
Chakravorty, S. et al. (May 2007, e-published Feb. 22, 2007). “A detailed analysis of 16S ribosomal RNA gene segments for the diagnosis of pathogenic bacteria,” [cited by applicant]
De Koning, A. P. J. et al. (Dec. 1, 2011). “Repetitive elements may comprise over two-thirds of the human genome,” [cited by applicant]
Deininger, P. (Dec. 28, 2011). “Alu elements: know the SINEs,” [cited by applicant]
Dubrovina, A. S. et al. (Jan. 2013, e-published Dec. 26, 2012). “The Role of Canonical and Noncanonical Pre-mRNA Splicing in Plant Stress Responses,” [cited by applicant]
Extended European Search Report mailed on Jun. 27, 2023, for EP Application No. 20907550.6, 6 pages. [cited by applicant]
Foltz, S. M. et al. (May 29, 2020). “Evolution and structure of clinically relevant gene fusions in multiple myeloma,” [cited by applicant]
Fuller, C.W. et al. (May 10, 2016, e-published Apr. 18, 2016). “Real-time single-molecule electronic DNA sequencing by synthesis using polymer-tagged nucleotides on a nanopore array,” [cited by applicant]
Ganusov, V. V. et al. (Dec. 2007, e-published Oct. 26, 2007). “Do most lymphocytes in humans really reside in the gut?,” [cited by applicant]
Guo, J. et al. (Jul. 8, 2008, e-published Jun. 30, 2008). “Four-color DNA sequencing with 3′-O-modified nucleotide reversible terminators and chemically cleavable fluorescent dideoxynucleotides,” [cited by applicant]
International Search Report and Written Opinion mailed on Aug. 12, 2022, for PCT Application No. PCT/US2022/020776, filed Mar. 17, 2022, 12 pages. [cited by applicant]
Ju, J. et al. (Dec. 26, 2006). “Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators,” [cited by applicant]
Kumar, S. et al. (Sep. 21, 2012). “PEG-labeled nucleotides and nanopore detection for single molecule DNA sequencing by synthesis,” [cited by applicant]
Li, Y. et al. (Apr. 2017. e-published Dec. 20, 2016). “Global identification of alternative splicing via comparative analysis of SMRT-and Illumina-based RNA-seq in strawberry,” [cited by applicant]
Liao, X. et al. (Jun. 1, 2019) “Current challenges and solutions of de novo assembly,” [cited by applicant]
Liao, Y-C. et al. (Sep. 3, 2019). “Completing circular bacterial genomes with assembly complexity by using a sampling strategy from a single MinION run with barcoding,” [cited by applicant]
Sawada, J. et al. (Jan. 6, 2016, e-published Aug. 27, 2015). “A surprisingly simple de Bruijn sequence construction,” [cited by applicant]
Shendure, J. et al. (Sep. 9, 2005, e-published Aug. 4, 2005). “Accurate multiplex polony sequencing of an evolved bacterial genome,” [cited by applicant]
Southworth, M. W. et al. (May 28, 1996). “Cloning of thermostable DNA polymerases from hyperthermophilic marine Archaea with emphasis on [cited by applicant]
Wahl, M. C. (Feb. 20, 2009). “The spliceosome: design principles of a dynamic RNP machine,” [cited by applicant]
Wang, Q. et al. (2018). “JUM is a computational method for comprehensive annotation-free analysis of alternative pre-mRNA splicing patterns,” [cited by applicant]
Wang, Y. et al. (Mar. 3, 2014). “Optimal eukaryotic 18S and universal 16S/18S ribosomal RNA primers and their application in a study of symbiosis,” [cited by applicant]
Yaari, G. et al. (Nov. 20, 2015). “Practical guidelines for B-cell receptor repertoire sequencing analysis,” [cited by applicant]
Zhao, S. et al. (Mar. 19, 2018). “Evaluation of two main RNA-seq approaches for gene quantification in clinical RNA sequencing: polyA+ selection versus rRNA depletion,” [cited by applicant]
Hu et al. (Feb. 7, 2020) “Analysis of Alternative Splicing and Alternative Polyadenylation in Populus alba var. pyramidalis by Single-Molecular Long-Read Sequencing”, Frontiers in Genetics, 11:48, 10 pages. [cited by applicant]