Methods of determining tissues and/or cell types giving rise to cell-free dna, and methods of identifying a disease or disorder using same

Patent metadata

Field	Value
Publication number	US-2017211143-A1
Application number	US-201515329228-A
Country	US
Kind code	A1
Filing date	Jul 27, 2015
Priority date	Jul 25, 2014
Publication date	Jul 27, 2017
Grant date	—

Abstract

The present disclosure provides methods of determining one or more tissues and/or cell-types contributing to cell-free DNA (“cfDNA”) in a biological sample of a subject. In some embodiments, the present disclosure provides a method of identifying a disease or disorder in a subject as a function of one or more determined more tissues and/or cell-types contributing to cfDNA in a biological sample from the subject.

First claim

1 . A method of determining tissues and/or cell types giving rise to cell free DNA (cfDNA) in a subject, the method comprising:
isolating cfDNA from a biological sample from the subject, the isolated cfDNA comprising a plurality of cfDNA fragments; determining a sequence associated with at least a portion of the plurality of cfDNA fragments; determining a genomic location within a reference genome for at least some cfDNA fragment endpoints of the plurality of cfDNA fragments as a function of the cfDNA fragment sequences; and determining at least some of the tissues and/or cell types giving rise to the cfDNA fragments as a function of the genomic locations of at least some of the cfDNA fragment endpoints.

2 . The method of claim 1 wherein the step of determining at least some of the tissues and/or cell types giving rise to the cfDNA fragments comprises comparing the genomic locations of at least some of the cfDNA fragment endpoints to one or more reference maps.

3 . The method of claim 1 or claim 2 wherein the step of determining at least some of the tissues and/or cell types giving rise to the cfDNA fragments comprises performing a mathematical transformation on a distribution of the genomic locations of at least some of the cfDNA fragment endpoints.

4 . The method of claim 3 wherein the mathematical transformation includes a Fourier transformation.

5 . The method of any one of claims 1 to 4 further comprising determining a score for each of at least some coordinates of the reference genome, wherein the score is determined as a function of at least the plurality of cfDNA fragment endpoints and their genomic locations, and wherein the step of determining at least some of the tissues and/or cell types giving rise to the observed cfDNA fragments comprises comparing the scores to one or more reference map.

6 . The method of claim 5 , wherein the score for a coordinate represents or is related to the probability that the coordinate is a location of a cfDNA fragment endpoint.

7 . The method of any one of claims 2 to 6 wherein the reference map comprises a DNase I hypersensitive site dataset generated from at least one cell-type or tissue; or wherein the reference map comprises an RNA expression dataset generated from at least one cell-type or tissue; or wherein the reference map comprises a chromosome conformation map generated from at least one cell-type or tissue; or wherein the reference map comprises a chromatin accessibility map generated from at least one cell-type or tissue.

8 . (canceled)

9 . The method of any one of claims 2 to 7 wherein the reference map is generated from cfDNA from an animal to which human tissues or cells that have been xenografted.

10 - 11 . (canceled)

12 . The method of any one of claims 2 to 7 and 9 wherein the reference map comprises sequence data obtained from samples obtained from at least one reference subject.

13 . The method of any one of claims 2 to 7 , 9 and 12 wherein the reference map corresponds to at least one cell-type or tissue that is associated with a disease or a disorder or condition.

14 . The method of any one of claims 2 to 7 , 9 , and 12 to 13 wherein the reference map comprises positions or spacing of nucleosomes and/or chromatosomes in a tissue or cell type.

15 . The method of any one of claims 2 to 7 , 9 , and 12 to 14 wherein the reference map is generated by digesting chromatin obtained from at least one cell-type or tissue with an exogenous nuclease (e.g., micrococcal nuclease).

16 . The method of any one of claims 2 to 7 , 9 and 12 to 15 , wherein the reference maps comprise chromatin accessibility data determined by a transposition-based method (e.g., ATAC-seq).

17 . The method of any one of claims 2 to 7 , 9 and 12 to 16 wherein the reference maps comprise data associated with positions of a DNA binding and/or DNA occupying protein for a tissue or cell type.

18 . The method of claim 17 wherein the DNA binding and/or DNA occupying protein is a transcription factor.

19 . The method of claim 17 or claim 18 wherein the positions are determined by chromatin immunoprecipitation of a crosslinked DNA-protein complex.

20 . The method of claim 17 or claim 18 wherein the positions are determined by treating DNA associated with the tissue or cell type with a nuclease (e.g., DNase-I).

21 . The method of any one of claims 2 to 7 , 9 and 12 to 20 wherein the reference map comprises a biological feature related to the positions or spacing of nucleosomes, chromatosomes, or other DNA binding or DNA occupying proteins within a tissue or cell type.

22 . The method of claim 21 wherein the biological feature is quantitative expression of one or more genes; or wherein the biological feature is presence or absence of one or more histone marks; or wherein the biological feature is hypersensitivity to nuclease cleavage.

23 - 24 . (canceled)

25 . The method of any one of claims 2 to 7 , 9 , and 12 to 22 wherein the tissue or cell type used to generate a reference map is a primary tissue from a subject having a disease or disorder or condition.

26 . The method of claim 25 wherein the disease or disorder or condition is selected from the group consisting of: cancer, normal pregnancy, a complication of pregnancy (e.g., aneuploid pregnancy), myocardial infarction, inflammatory bowel disease, systemic autoimmune disease, localized autoimmune disease, allotransplantation with rejection, allotransplantation without rejection, stroke, and localized tissue damage.

27 . The method of any one of claims 2 to 7 , 9 and 12 to 22 wherein the tissue or cell type used to generate a reference map is a primary tissue from a healthy subject; or wherein the tissue or cell type used to generate a reference map is an immortalized cell line; or wherein the tissue or cell type used to generate a reference map is a biopsy from a tumor.

28 - 29 . (canceled)

30 . The method of claim 12 wherein the sequence data comprises positions of cfDNA fragment endpoints.

31 . The method of claim 30 wherein the reference subject is healthy; or wherein the reference subject has a disease or disorder or condition.

32 . (canceled)

33 . The method of claim 31 wherein the disease or disorder or condition is selected from the group consisting of: cancer, normal pregnancy, a complication of pregnancy (e.g., aneuploid pregnancy), myocardial infarction, inflammatory bowel disease, systemic autoimmune disease, localized autoimmune disease, allotransplantation with rejection, allotransplantation without rejection, stroke, and localized tissue damage.

34 . The method of any one of claims 13 to 22 , 25 to 27 , 30 to 31 and 33 wherein the reference map comprises reference scores for at least a portion of coordinates of the reference genome associated with the tissue or cell type.

35 . The method of claim 34 wherein the reference map comprises a mathematical transformation of the scores.

36 . The method of claim 34 wherein the scores represent a subset of all reference genomic coordinates associated with the tissue or cell type.

37 . The method of claim 36 wherein the subset is associated with positions or spacing of nucleoso

Assignees

• Univ Washington

Inventors

• Shendure Jay
• Snyder Matthew
• Kircher Martin

Classifications

• C12Q1/6881 Primary

  for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes · CPC title

• G16H50/20

  for computer-aided diagnosis, e.g. based on medical expert systems · CPC title

• G16B45/00

  ICT specially adapted for bioinformatics-related data visualisation, e.g. displaying of maps or networks · CPC title

• C12Q1/6883

  for diseases caused by alterations of genetic material · CPC title

• G16B40/00

  ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding · CPC title