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US-20260125408-A1 - METHODS AND COMPOSITIONS FOR PREPARING NUCLEIC ACIDS THAT PRESERVE SPATIAL-PROXIMAL CONTIGUITY INFORMATION

US20260125408A1US 20260125408 A1US20260125408 A1US 20260125408A1US-20260125408-A1

Abstract

Provided herein are methods and compositions for preparing nucleic acids in samples that preserve spatial-proximal contiguity information. Samples include, but are not limited to, formalin-fixed paraffin-embedded (FFPE) samples, deeply formalin-fixed samples and samples that comprise protein:cfDNA complexes.

Inventors

  • Anthony Schmitt
  • Catherine Tan
  • Derek REID
  • Chris DE LA TORRE
  • Siddarth SELVARAJ

Assignees

  • ARIMA GENOMICS, INC.

Dates

Publication Date
20260507
Application Date
20251219

Claims (20)

  1. 1 - 20 . (canceled)
  2. 21 . A method for preparing nucleic acids from a formalin-fixed paraffin-embedded (FFPE) sample that preserves spatial-proximal contiguity information comprising: a) providing a formalin-fixed paraffin-embedded (FFPE) sample, b) treating the formalin-fixed paraffin-embedded (FFPE) sample at an elevated temperature, and; c) contacting the formalin-fixed paraffin-embedded (FFPE) sample with one or more reagents that preserve spatial-proximal contiguity information in the nucleic acids of the formalin-fixed paraffin-embedded (FFPE) sample.
  3. 22 . The method of claim 21 , wherein the elevated temperature is equal to or greater than about 65° C.
  4. 23 . The method of claim 22 , wherein the elevated temperature is equal to or greater than about 74° C.
  5. 24 . The method of claim 22 , wherein the elevated temperature is equal to or greater than about 65° C. to about 90° C.
  6. 25 . The method of claim 21 , wherein the formalin-fixed paraffin-embedded (FFPE) sample is selected from the group consisting of: a dewaxed/rehydrated formalin-fixed paraffin-embedded (FFPE) sample, a lysed formalin-fixed paraffin-embedded (FFPE) sample, and a dewaxed/rehydrated and lysed formalin-fixed paraffin-embedded (FFPE) sample.
  7. 26 . The method of claim 21 , wherein the formalin-fixed paraffin-embedded (FFPE) sample is contacted with a denaturing detergent.
  8. 27 . The method of claim 26 , wherein contact with the denaturing detergent is greater than about 10 minutes.
  9. 28 . The method of claim 27 , wherein the contact with the denaturing detergent is (i) greater than about 10 minutes to about 80 minutes, (ii) about 120 minutes, or (iii) about 180 minutes.
  10. 29 . The method of claim 27 , wherein the contact with the denaturing detergent is about 35 minutes to about 40 minutes.
  11. 30 . The method of claim 27 , wherein the contact with the denaturing detergent is greater than about 20 minutes.
  12. 31 . The method of claim 30 , wherein the elevated temperature is greater than or equal to about 62° C.
  13. 32 . The method of claim 31 , wherein the elevated temperature is about 62° C.
  14. 33 . The method of claim 21 , wherein the formalin-fixed paraffin-embedded (FFPE) sample comprises mammalian cells or mammalian tissue.
  15. 34 . The method of claim 33 , wherein the mammalian cells or mammalian tissue are human cells or human tissue.
  16. 35 . A method for preparing nucleic acids from a formalin-fixed paraffin-embedded (FFPE) sample that preserves spatial-proximal contiguity information comprising: a) providing a formalin-fixed paraffin-embedded (FFPE) sample, b) contacting the formalin-fixed paraffin-embedded (FFPE) sample with a denaturing detergent for greater than about 10 minutes, and c) contacting the formalin-fixed paraffin-embedded (FFPE) sample with one or more reagents that preserve spatial-proximal contiguity information in the nucleic acids of the formalin-fixed paraffin-embedded (FFPE) sample.
  17. 36 . The method of claim 35 , wherein contact with the denaturing detergent is (i) greater than about 10 minutes to about 80 minutes, (ii) about 120 minutes, or (iii) about 180 minutes.
  18. 37 . The method of claim 35 , wherein the contact with the denaturing detergent is about 35 minutes to about 40 minutes.
  19. 38 . The method of claim 35 , wherein the contact with the denaturing detergent is greater than about 20 minutes.
  20. 39 . The method of claim 35 , wherein the formalin-fixed paraffin-embedded (FFPE) sample is selected from the group consisting of: a dewaxed/rehydrated formalin-fixed paraffin-embedded (FFPE) sample, a lysed formalin-fixed paraffin-embedded (FFPE) sample, and a dewaxed/rehydrated and lysed formalin-fixed paraffin-embedded (FFPE) sample.

Description

RELATED PATENT APPLICATION(S) This application is a continuation of U.S. patent application Ser. No. 16/689,002, filed Nov. 19, 2019, entitled METHODS AND COMPOSITIONS FOR PREPARING NUCLEIC ACIDS THAT PRESERVE SPATIAL-PROXIMAL CONTIGUITY INFORMATION, naming Anthony Schmitt, Catherine Tan, Derek Reid, Chris De La Torre and Siddarth Selvaraj as inventors and assigned attorney docket no. AMG-1003-UT, which claims the benefit of U.S. Provisional Ser. No. 62/785,643, filed Dec. 27, 2018, entitled METHODS AND COMPOSITIONS FOR PREPARING NUCLEIC ACIDS THAT PRESERVE SPATIAL-PROXIMAL CONTIGUITY INFORMATION, naming Anthony Schmitt, Catherine Tan, Derek Reid, Chris De La Torre and Siddarth Selvaraj as inventors and assigned attorney docket no. AMG-1003-PV2, and also claims the benefit of U.S. Provisional Patent Application No. 62/770,135, filed Nov. 20, 2018, entitled METHODS FOR PREPARING NUCLEIC ACIDS THAT PRESERVE SPATIAL-PROXIMAL CONTIGUITY INFORMATION, naming Anthony Schmitt, Catherine Tan, Derek Reid, Chris De La Torre and Siddarth Selvaraj as inventors and assigned attorney docket no. AMG-1003-PV. This application is also related to U.S. Provisional Ser. No. 62/589,505, filed Nov. 21, 2017, entitled PRESERVING SPATIAL-PROXIMAL CONTIGUITY AND MOLECULAR CONTIGUITY IN NUCLEIC ACID TEMPLATES, naming Siddarth Selvaraj, Anthony Schmitt and Bret Reid as inventors and assigned attorney docket no. AMG-1002-PV. This patent application is also related to PCT Application No. PCT/US18/62005, filed Nov. 20, 2018, entitled PRESERVING SPATIAL-PROXIMAL CONTIGUITY AND MOLECULAR CONTIGUITY IN NUCLEIC ACID TEMPLATES naming Siddarth Selvaraj, Anthony Schmitt and Bret Reid as inventors and assigned attorney docket no. AMG-1002-PC. The entire content of the foregoing patent applications is incorporated herein by reference, including all text, tables and drawings. FIELD This technology relates to sequencing nucleic acids. BACKGROUND Next-generation sequencing (NGS) has emerged as the predominant set of methods for determining nucleic acid sequence for a plethora of research and clinical applications. The typical NGS workflow is as follows: the native genomic DNA, often organized as chromosome(s), is isolated from the nucleic acid source leading to its fragmentation, to produce nucleic acid templates which are subsequently read by a sequencing instrument to generate sequence data. SUMMARY The technology pertains to methods for preparing nucleic acids in such a way that preserves DNA spatial-proximal contiguity sequence information enabling the detection of spatially proximal nucleic acids (e.g. HiC), serving applications that benefit from long-range sequence contiguity information (e.g. haplotype phasing, genomic rearrangement detection and other applications that are enabled by long-range sequence contiguity information). Preserving spatial-proximal contiguity information during the preparation of DNA from a sample of interest allows preserving contiguity in sequencing data obtained therefrom. Contiguity-preserved sequencing data enables comprehensive determination of nucleic acid sequence, as manifested in the contiguity-preserved nucleic acid template, by enabling identification of genomic variants, determination of contiguity information to inform genome assemblies de novo, deconvolution of haplotype phase information, genomic rearrangement detection, which together are fundamental to understand the role of genetics in living systems. Formalin-fixed paraffin-embedded samples are typically not successfully prepared using the initial steps of standard protocols designed for cells, i.e., cells that are not formalin-fixed paraffin-embedded (see Example 18). Provided in certain aspects is a method for preparing nucleic acids from a formalin-fixed paraffin-embedded (FFPE) sample, that preserves spatial-proximal contiguity information, comprising: a) providing a formalin-fixed paraffin-embedded sample; b) de-waxing the sample to produce a dewaxed sample; c) rehydrating the dewaxed sample, thereby generating a dewaxed/rehydrated sample; d) contacting the dewaxed/rehydrated sample with lysis buffer, thereby generating a lysed sample; e) contacting the lysed sample with denaturing detergent at a temperature greater than 65° C., thereby generating a solubilized and decompacted sample; and f) contacting the solubilized and decompacted sample with one or more reagents that preserve spatial-proximal contiguity information in the nucleic acids of the solubilized and decompacted sample. Also provided in certain aspects is a method wherein the dewaxed/rehydrated sample is contacted with an extracellular matrix protease prior to contact with lysis buffer. Also provided in certain aspects is a method for preparing nucleic acids from a formalin-fixed paraffin-embedded (FFPE) sample, that preserves spatial-proximal contiguity information, comprising: a) providing a formalin-fixed paraffin-embedded sample; b) de-waxing the sample to produce a dewaxed sam