Methods and compositions for nucleic acid analysis
The present invention is directed to methods, compositions and systems for analyzing sequence information while retaining structural and molecular context of that sequence information.
1. A method of analyzing nucleic acids from a formalin fixed paraffin embedded (FFPE) sample while maintaining structural context of nucleic acids within the sample, the method comprising:
(a) forming linked nucleic acids within the FFPE sample such that spatially adjacent nucleic acid segments are linked;
(b) processing the linked nucleic acids to produce a plurality of ligation products, wherein the ligation products contain portions of the spatially adjacent nucleic acid segments;
(c) depositing individual portions of the FFPE sample into discrete partitions, wherein each individual portion of the FFPE sample comprises one or more of the ligation products;
(d) barcoding the ligation products within the discrete partitions to form a plurality of barcoded fragments, wherein fragments within a given discrete partition each comprise a common barcode, thereby associating each fragment with the linked nucleic acid from which it is derived;
(e) obtaining sequence information from the plurality of barcoded fragments, thereby analyzing nucleic acids from the sample while maintaining structural context.
2. The method of claim 1 , wherein the processing step (b) includes blunt-end ligation under conditions favoring intramolecular ligation, such that the spatially adjacent nucleic acid segments are ligated within the same molecule.
3. The method of claim 2 , wherein the conditions favoring intramolecular ligation comprise diluting the sample to reduce concentration of the nucleic acids under 10 ng/μL.
4. The method of claim 1 , wherein the sample comprises a nuclear matrix.
5. The method of claim 1 , wherein the nucleic acids comprise RNA.
6. The method of claim 1 , wherein the amount of nucleic acids in the sample is less than 50 ng/ml.
7. The method of claim 1 , wherein the partitions comprise beads.
8. The method of claim 7 , wherein the beads are gel beads.
9. The method of claim 1 , wherein the sample comprises a tumor sample.
10. The method of claim 1 , wherein the sample comprises a mixture of tumor and normal cells.
11. The method of claim 1 , wherein the processing step includes reversal of the linking subsequent to forming the ligation products.
12. The method of claim 1 , wherein the obtaining step (e) provides information on intrachromosomal and/or interchromosomal interactions between genomic loci.
13. The method of claim 1 , wherein the obtaining step (e) provides information on chromosome conformations.
14. The method of claim 13 , wherein the chromosome conformations are associated with disease states.
15. The method of claim 1 , wherein the processing step results in ligation products comprising nucleic acids that were originally in close spatial proximity in the sample.
16. The method of claim 1 , wherein the obtaining step (e) comprises a sequencing reaction selected from the group consisting of: short read-length sequencing reactions and long read-length sequencing reactions.
17. The method of claim 16 , wherein the sequencing reaction is a short read, high accuracy sequencing reaction.
18. The method of claim 1 , wherein the forming step (a) comprises cross-linking nucleic acids in the sample.
19. The method of claim 1 , wherein the forming step (a) results in covalent links between spatially adjacent nucleic acid segments.
20. A method of analyzing nucleic acids from a formalin fixed paraffin embedded (FFPE) sample while maintaining structural context of nucleic acids within the sample, the method comprising:
(a) forming linked nucleic acids within the FFPE sample such that spatially adjacent nucleic acid segments are linked;
(b) depositing individual portions of the FFPE sample into discrete partitions, wherein each individual portion of the FFPE sample comprises one or more linked nucleic acids;
(c) processing the linked nucleic acids to produce a plurality of ligation products, wherein the ligation products contain portions of the spatially adjacent nucleic acid segments;
(d) barcoding the ligation products within the discrete partitions to form a plurality of barcoded fragments, wherein fragments within a given discrete partition each comprise a common barcode, thereby associating each fragment with the linked nucleic acid from which it is derived;
(e) obtaining sequence information from the plurality of barcoded fragments, thereby analyzing nucleic acids from the sample while maintaining structural context.
21. The method of claim 20 , wherein the processing step (c) includes blunt-end ligation under conditions favoring intramolecular ligation, such that the spatially adjacent nucleic acid segments are ligated within the same molecule.
22. The method of claim 20 , wherein the sample comprises a nuclear matrix.
23. The method of claim 20 , wherein the nucleic acids comprise RNA.
24. The method of claim 20 , wherein the partitions comprise beads.
25. The method of claim 24 , wherein the beads are gel beads.
26. The method of claim 20 , wherein the sample comprises a tumor sample.
27. The method of claim 20 , wherein the sample comprises a mixture of tumor and normal cells.
28. The method of claim 20 , wherein the processing step (c) results in ligation products comprising nucleic acids that were originally in close spatial proximity in the sample.
29. The method of claim 20 , wherein the obtaining step (e) provides information on intrachromosomal and/or interchromosomal interactions between genomic loci.
30. The method of claim 20 , wherein the obtaining step (e) comprises a sequencing reaction selected from the group consisting of: short read-length sequencing reactions and long read-length sequencing reactions.
31. The method of claim 30 , wherein the sequencing reaction is a short read, high accuracy sequencing reaction.