Methods and compositions for preparing nucleic acids that preserve spatial-proximal contiguity information
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.
1. 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 comprising crosslinked nucleic acid molecules;
b) de-waxing the sample to produce a de-waxed sample;
c) rehydrating the de-waxed sample, thereby generating a de-waxed/rehydrated sample;
d) contacting the de-waxed/rehydrated sample with lysis buffer, thereby generating a lysed sample;
e) contacting the lysed sample with a denaturing detergent at a temperature greater than 65° C. and less than 80° C., thereby generating a solubilized and decompacted sample; and
f) generating proximity ligated nucleic acid molecules, thereby preserving spatial-proximal contiguity information in the nucleic acids of the solubilized and decompacted sample.
2. The method of claim 1 , wherein the formalin-fixed paraffin-embedded (FFPE) sample is a tissue sample.
3. The method of claim 1 , wherein the de-waxed/rehydrated sample is contacted with an extracellular matrix protease prior to contact with lysis buffer.
4. The method of claim 1 , wherein the temperature is 74° C.
5. The method of claim 1 , wherein contact with the denaturing detergent is 15 to 80 minutes.
6. The method of claim 5 , wherein contact with the denaturing detergent is 40 minutes.
7. The method of claim 1 , wherein the detergent is sodium dodecyl sulfate (SDS).
8. The method of claim 1 , wherein the formalin-fixed paraffin-embedded sample is provided as a tissue section of about 5 um to about 10 um in thickness.
9. The method of claim 1 , wherein the nucleic acids with preserved spatial-proximal contiguity information prepared from the sample are sequenced and produce >40% of mapped de-duplicated reads representing long-range readouts.
10. The method of claim 1 , wherein the proximity ligated nucleic acid molecules are generated in situ.
11. The method of claim 9 , wherein the long-range readouts are utilized for identification of genomic variants, to inform genome assemblies de novo, for deconvolution of haplotype phase information and/or for genomic rearrangement detection.
12. The method of claim 1 , wherein the solubilized and decompacted sample generated in (e) comprises crosslinked nucleic acid molecules.