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 analyzing cell-free DNA (cfDNA), said method comprising:
(a) obtaining a cfDNA sample;
(b) enriching said cfDNA sample for large fragments to generate an enriched cfDNA sample, wherein said large fragments have a length greater than about 200 bp;
(c) identifying cfDNA molecules within said enriched cfDNA sample; and
(d) evaluating said cfDNA molecules, wherein said evaluating comprises comparing an amount of said cfDNA molecules derived from a first genomic region with an additional amount of cfDNA molecules derived from a second genomic region, and wherein said first genomic region and second genomic region are non-overlapping.
2. The method of claim 1 , wherein (a) comprises extracting cfDNA from a blood draw.
3. The method of claim 1 , wherein (b) comprises solid phase reversible immobilization.
4. The method of claim 1 , wherein (b) comprises PCR-amplification of target regions of interest using pre-determined specific primers.
5. The method of claim 1 , wherein said large fragments are greater than about 250 bp in length.
6. The method of claim 1 , wherein said large fragments are greater than about 300 bp in length.
7. The method of claim 1 , wherein said large fragments are greater than about 200 bp and smaller than about 1000 bp in length.
8. The method of claim 1 , wherein (c) comprises sequencing said cfDNA molecules.
9. The method of claim 8 , wherein sequencing said cfDNA molecules comprises next-generation sequencing.
10. The method of claim 8 , wherein sequencing said cfDNA molecules comprises DNA nanoball sequencing.
11. The method of claim 1 , wherein, in (c), said cfDNA molecules are within a droplet reaction environment.
12. The method of claim 1 , wherein (c) comprises performing qPCR.
13. The method of claim 1 , wherein (c) comprises performing rtPCR.
14. The method of claim 1 , wherein (d) comprises evaluating mobilities of said cfDNA molecules with a known standard.
15. The method of claim 14 , wherein said known standard comprises a reference library based on clinical data.
16. The method of claim 14 , wherein said known standard comprises a molecular weight reference set.
17. The method of claim 1 , wherein (d) comprises comparing an amount of said cfDNA molecules to an amount of reference molecules.
18. The method of claim 17 , wherein said amount of reference molecules comprises an amount of cfDNA molecules less than about 190 bp.
19. The method of claim 1 , wherein (d) comprises sequencing said cfDNA molecules and 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.
20. The method of claim 19 , wherein said first set of reference sequences and second set of reference sequences represent a first fragmentation pattern and second fragmentation pattern, respectively.
21. The method of claim 20 , further comprising calculating a transcriptional activity score using said first fragmentation pattern and second fragmentation pattern.
22. The method of claim 20 , wherein said first fragmentation pattern and second fragmentation pattern reflect histone-protected DNA fragments.
23. The method of claim 21 , wherein said second fragmentation pattern is indicative of a pathological condition.
24. The method of claim 23 , wherein said second fragmentation pattern comprises regions having one or more epigenetic states indicative of said pathological condition.
25. The method of claim 23 , wherein said second fragmentation pattern comprises regions having one or more genetic states indicative of said pathological condition.