Recovering long-range linkage information from preserved samples
The disclosure provides methods to isolate genome or chromosome level structural information from preserved samples. In some cases, samples preserved under conditions where long-range nucleic acid information is believed to be irreparably lost, such as FFPE samples, are treated to recover nucleic acid-protein complexes stabilized as part of the sample preservation process. The complexes are processed so as to recover information regarding which nucleic acids are bound to a common complex, and the information is used to recover genomic structural information.
1. A method, comprising
obtaining a preserved sample from a subject, the sample comprising protein-DNA complexes;
treating the preserved sample with a proteinase at a temperature not greater than 40° C. to isolate nucleic acids such that protein-DNA complexes are not destroyed, such that a first double-stranded segment and a second double-stranded segment are held together independent of a phosphodiester backbone, and such that the first double-stranded segment and the second double-stranded segment each have at least one exposed nucleic acid end; and
deriving genomic structural information by analyzing nucleic acids of the protein-DNA complexes in the sample, wherein the preserved sample is a formalin fixed paraffin-embedded (FFPE) sample.
2. The method of claim 1 , wherein the preserved sample is crosslinked.
3. The method of claim 2 , wherein the preserved sample is crosslinked using at least one of a formaldehyde, a formalin, UV light, mitomycin C, nitrogen mustard, melphalan, 1,3-butadiene diepoxide, cis diaminedichloroplatinum(II) and cyclophosphamide.
4. The method of claim 1 , wherein the preserved sample maintains positional information as to nucleic acids within it.
5. The method of claim 1 , wherein the genomic structural information is indicative of a structural variant comprising at least one of an inversion, an insertion, a deletion, a chromosomal translocation, a copy number variant, a loss of heterozygosity, or a gene fusion relative to a reference genome.
6. The method of claim 1 , comprising deriving information indicative of phase status for the first double-stranded segment and the second double-stranded segment of the nucleic acids.
7. The method of claim 1 , comprising tagging an exposed nucleic acid end of the first double-stranded segment and an exposed nucleic acid end of the second double-stranded segment so as to convey physical linkage information.
8. The method of claim 7 , wherein the tagging comprises ligating an oligonucleotide to an exposed end of the first double-stranded segment of the preserved sample such that the oligonucleotide conveys information indicative of genomic structural information.
9. The method of claim 7 , wherein the tagging comprises ligating an exposed end of the first double-stranded segment to an exposed end of the second double-stranded segment to form a paired end molecule.
10. The method of claim 1 , wherein the preserved sample is treated by contacting the preserved tissue sample to at least one of xylene and ethanol.
11. The method of claim 1 , wherein the preserved sample is treated by contacting the preserved tissue sample to at least one of an anthranilate and a phosphanilate.
12. The method of claim 1 , wherein the preserved sample preserves positional information reflective of its configuration in a tissue.
13. The method of claim 1 , wherein the preserved sample is not homogenized prior to isolating nucleic acids.
14. The method of claim 1 , wherein the preserved sample is stored for at least one week prior to isolating nucleic acids.
15. The method of claim 1 , wherein the preserved sample is stored for at least 6 months prior to isolating nucleic acids.
16. The method of claim 1 , wherein the preserved sample is transported from a collection point prior to isolating nucleic acids.