Methods for characterizing cell-free nucleic acid fragments
The present disclosure provides methods and systems for various uses of cell-free nucleic acid (cfNA). Functional typing of cfNA fragmentation patterns may be utilized in the non-invasive detection, diagnosis, and monitoring of disease. One embodiment may determine a stage of cancer in a subject, the progression of cancer in a subject, or the responsiveness to treatment of a cancer in a subject. Another embodiment disclosed herein may include sequencing-free diagnostic methods.
1. A method of characterizing cell-free (cfDNA) fragments, comprising:
(a) obtaining a set of cfDNA fragments;
(b) identifying cfDNA fragments of said set of cfDNA fragments, wherein said cfDNA fragments of said set of cfDNA fragments:
(i) have a length greater than about 200 bp; and
(ii) are derived from a genomic region, wherein said genomic region comprises at least one nucleotide of a promoter, and wherein said at least one nucleotide of said promoter is not derived from a transcriptional start site; and
(c) evaluating said cfDNA fragments against a reference sequence associated with a pathological condition.
2. The method of claim 1 , wherein (b) comprises sequencing said cfDNA fragments.
3. The method of claim 2 , wherein sequencing said cfDNA fragments comprises next-generation sequencing.
4. The method of claim 2 , wherein sequencing said cfDNA fragments comprises nanoball sequencing.
5. The method of claim 1 , wherein, in (b), said cfDNA fragments are within a droplet reaction environment.
6. The method of claim 1 , wherein (b) comprises performing quantitative polymerase chain reaction (qPCR).
7. The method of claim 1 , wherein (b) comprises performing real-time polymerase chain reaction (rtPCR).
8. The method of claim 1 , wherein said cfDNA fragments of said set of cfDNA fragments are greater than 250 bp in length.
9. The method of claim 1 , wherein said cfDNA fragments of said set of cfDNA fragments are greater than 300 bp in length.
10. The method of claim 1 , wherein said cfDNA fragments of said set of cfDNA fragments are greater than 200 bp and smaller than 1000 bp in length.
11. The method of claim 1 , wherein said pathological condition is an autoimmune disease.
12. The method of claim 11 , wherein said autoimmune disease is rheumatoid arthritis, multiple sclerosis, or lupus.
13. The method of claim 1 , wherein (c) comprises comparing mobilities of said cfDNA fragments to a known standard.
14. The method of claim 13 , wherein said known standard comprises a reference library based on clinical data.
15. The method of claim 13 , wherein said known standard comprises a molecular weight reference set.
16. The method of claim 1 , wherein (c) comprises comparing an amount of cfDNA sequences matching a first set of reference sequences to an additional amount of cfDNA sequences matching a second set of reference sequences.
17. The method of claim 16 , wherein said first set of reference sequences and second set of reference sequences represents a first fragmentation pattern and second fragmentation pattern, respectively.
18. The method of claim 17 , further comprising calculating a transcriptional activity score using said first fragmentation pattern and said second fragmentation pattern.
19. The method of claim 17 , wherein said first fragmentation pattern and second fragmentation pattern reflects histone-protected DNA fragments.
20. The method of claim 19 , wherein said second fragmentation pattern comprises regions having one or more epigenetic states indicative of said pathological condition.
21. The method of claim 19 , wherein said second fragmentation pattern comprises regions having one or more genetic states indicative of said pathological condition.