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 nucleic acid (cfNA) fragments, comprising:
(a) providing a sample comprising said cfNA fragments;
(b) sequencing said cfNA fragments to obtain a set of sequences of said cfNA fragments; and
(c) identifying sequences of said set of sequences of said cfNA fragments that:
(i) have a length of at least about 255 nucleotides; and
(ii) comprise a sequence that regulates expression of a gene associated with an inflammatory condition.
2 . The method of claim 1 , wherein said cfNA fragments are derived from serum, saliva, urine, blood components, cerebrospinal fluid, pleural fluid, amniotic fluid, peritoneal fluid, ascitic fluid, abdominopelvic washings/lavage, serous effusions, tracheobronchial lavage or bronchoalveolar lavage.
3 . The method of claim 1 , wherein said plurality of cfNA fragments are derived from plasma.
4 . The method of claim 1 , wherein (c) further comprises identifying sequences of said set of sequences of said cfNA fragments that comprise at least ten consecutive nucleotides of a promoter of said gene associated with said inflammatory condition.
5 . The method of claim 1 , wherein (c) further comprises identifying sequences of said set of sequences of said cfNA fragments that comprise at least ten consecutive nucleotides of an active transcriptional start site of said gene associated with said inflammatory condition.
6 . The method of claim 1 , wherein said gene associated with said inflammatory condition is a steroid responsive gene.
7 . The method of claim 6 , wherein said steroid responsive gene is a glucocorticoid responsive gene, an anti-inflammatory gene, or a neutrophil activation signature gene.
8 . The method of claim 1 , wherein said cfNA fragments are from a cell that has undergone necrosis or apoptosis.
9 . The method of claim 1 , further comprising obtaining said sample from a subject that is suspected of having an autoimmune disease.
10 . The method of claim 1 , further comprising obtaining said sample from a subject that has at least one symptom of an autoimmune disease.
11 . The method of claim 1 , wherein (c) further comprises identifying sequences of said set of sequences of said cfNA fragments that comprise at least ten consecutive nucleotides of a transcription enhancer of said gene associated with said inflammatory condition.
12 . The method of claim 1 , wherein (c) further comprises identifying sequences of said set of sequences of said cfNA fragments that comprise at least ten consecutive nucleotides of a transcription insulator of said gene associated with said inflammatory condition.
13 . The method of claim 1 , wherein (c) further comprises identifying sequences of said set of sequences of said cfNA fragments that comprise at least ten consecutive nucleotides of a promoter of said gene associated with said inflammatory condition.
14 . The method of claim 1 , wherein (c) further comprises identifying sequences of said set of sequences of said cfNA fragments that comprise a transcription factor binding site of said gene associated with said inflammatory condition.
15 . The method of claim 1 , wherein said inflammatory condition is an autoimmune disease.
16 . The method of claim 15 , wherein said autoimmune disease is rheumatoid arthritis, multiple sclerosis, psoriasis, or scleroderma.
17 . The method of claim 1 , wherein (b) comprises performing next-generation sequencing on said cfNA fragments.
18 . The method of claim 1 , further comprising before (b), enriching said sample for cfNA fragments having a length greater than 255 nucleotides.
19 . The method of claim 1 , wherein (c) comprises identifying sequences of said set of sequences of said cfNA fragments that match at least ten consecutive nucleotides of said sequence that regulates expression of said gene associated with said inflammatory condition.
20 . The method of claim 1 , wherein (c) further comprises identifying sequences of said set of sequences of said cfNA fragments that have a length of at least 400 nucleotides.
21 . The method of claim 20 , wherein said sequences having a length of at least 400 nucleotides have a length of at least about 500 nucleotides.
22 . The method of claim 1 , further comprising prior to (a) obtaining said cfNA fragments from a subject and amplifying said cfNA fragments obtained from the subject to provide said sample in (a).
23 . The method of claim 22 , wherein said subject is suspected of having an autoimmune disease.
24 . The method of claim 23 , wherein said autoimmune disease is rheumatoid arthritis.
25 . The method of claim 23 , wherein said autoimmune disease is multiple sclerosis, psoriasis, or scleroderma.
26 . The method of claim 22 , wherein said subject has at least one symptom of an autoimmune disease.