Determining linear and circular forms of circulating nucleic acids

Patent metadata

Field	Value
Publication number	US-12098429-B2
Application number	US-202016829771-A
Country	US
Kind code	B2
Filing date	Mar 25, 2020
Priority date	Mar 25, 2019
Publication date	Sep 24, 2024
Grant date	Sep 24, 2024

Abstract

Techniques are provided for analyzing circular DNA in a biological sample (e.g., including cell-free DNA, such as plasma). For example, to measure circular DNA, cleaving can be performed to linearize the circular DNA so that they may be sequenced. Example cleaving techniques include restriction enzymes and transposases. Then, one or more criteria can be used to identify linearized DNA molecules, e.g., so as to differentiate from linear DNA molecules. An example criterion is mapping a pair of reversed end sequences to a reference genome. Another example criterion is identification of a cutting tag, e.g., associated with a restriction enzyme or an adapter sequence added by a transposase. Once circular DNA molecules (e.g., eccDNA and circular mitochondrial DNA) are identified, they may be analyzed (e.g., to determine a count, size profile, and/or methylation) to measure a property of the biological sample, including genetic properties and level of a disease.

First claim

What is claimed is:

1. A method comprising:
receiving a biological sample of an organism, the biological sample including a plurality of extrachromosomal circular DNA (eccDNA) molecules, wherein each of the plurality of eccDNA molecules includes a junction at which nucleotides at two separated genomic locations are immediately adjacent to one another;
cleaving the plurality of eccDNA molecules to form a set of linearized DNA molecules that each includes the junction, the set of linearized DNA molecules comprising at least 1,000 linearized DNA molecules;
for each of the set of linearized DNA molecules:
sequencing at least both ends of the linearized DNA molecule to obtain one or more sequence reads;
selecting a pair of end sequences for the linearized DNA molecule from the one or more sequence reads, the pair of end sequences not including the junction;
reversing a direction of each of the pair of end sequences to obtain a pair of reversed end sequences; and
mapping the pair of reversed end sequences to a reference genome; and

analyzing the mapped reversed end sequences to measure a property of the biological sample.

2. The method of claim 1 , further comprising:
detecting the plurality of eccDNA molecules based on the pair of reversed end sequences mapping to the reference genome; and
determining a collective value of the detected eccDNA molecules, wherein analyzing the mapped reversed end sequences to measure the property of the biological sample uses the collective value.

3. The method of claim 2 , wherein the collective value includes a count, a size, or a methylation level determined using the detected eccDNA molecules.

4. The method of claim 1 , wherein, for each of the linearized DNA molecules, the one or more sequence reads include the junction.

5. The method of claim 4 , wherein analyzing a mapped reversed end sequence includes:
comparing bases in the one or more sequence reads extending past each of the mapped reversed end sequences to the reference genome until a mismatch condition is identified; and
identifying end positions of a linearized DNA molecule from which the eccDNA molecule was formed based on a location of the mismatch condition in the reference genome.

6. The method of claim 5 , wherein analyzing the mapped reversed end sequence further includes:
determining a size of the linearized DNA molecule using the end positions.

7. The method of claim 6 , wherein analyzing the mapped reversed end sequences further includes:
determining a size distribution of the sizes measured for the plurality of eccDNA molecules; and
using the size distribution to measure the property of the biological sample.

8. The method of claim 1 , wherein analyzing the mapped reversed end sequences includes:
counting a number of the plurality of eccDNA molecules that map to a chromosomal region, where the property of the biological sample is of the chromosomal region; and
using the number to measure the property of the chromosomal region.

9. The method of claim 8 , wherein the property is a copy number aberration in the chromosomal region, the method further comprising:
measuring a methylation density in the chromosomal region using DNA molecules in the biological sample; and
using the copy number aberration and the methylation density to detect a condition with the organism.

10. The method of claim 9 , wherein the methylation density is determined to exhibit hypermethylation by comparing to a cutoff, and wherein the condition is fragile X syndrome or a triplet repeat expansion.

11. The method of claim 8 , wherein the property of the chromosomal region is that the chromosomal region carries information regarding the property of the biological sample or has an aberration including a sequence alteration, duplication, expansion, deletion or an amplification in the biological sample.

12. The method of claim 11 , wherein the biological sample is obtained from a subject being screened for cancer, further comprising:
identifying a level of cancer in the organism based on the chromosomal region having the aberration.

13. The method of claim 11 , wherein the biological sample is obtained from a female pregnant with a fetus, and wherein the aberration is in the fetus.

14. The method of claim 11 , wherein the property is sex or genotypic information.

15. The method of claim 1 , wherein the biological sample includes a first tissue type and a second tissue type, wherein the first tissue type is homozygous for a first allele at a locus, and wherein the second tissue type is heterozygous for the first allele and a second allele at the locus, the method further comprising:
determining a first number of the mapped reversed end sequences that have the first allele at the locus;
determining a second number of the mapped reversed end sequences that have the second allele at the locus; and
determining a fractional concentration of eccDNA molecules from the second tissue type using the first number and the second number.

16. The method of claim 1 , further comprising:
determining a number of sequence variants in the mapped reversed end sequences; and
determining a level of cancer using the number of sequence variants.

17. The method of claim 1 , further comprising:
prior to cleaving the plurality of eccDNA molecules, reducing linear DNA in the biological sample by exonuclease digestion.

18. The method of claim 1 , wherein the plurality of eccDNA molecules are cell-free.

19. The method of claim 1 , wherein cleaving the plurality of eccDNA molecules includes:
digesting, with a restriction enzyme, the plurality of eccDNA molecules to form the set of linearized DNA molecules.

20. The method of claim 19 , wherein the restriction enzyme cuts a particular sequence, the method further comprising:
identifying the particular sequence spanning the pair of end sequences of at least a portion of the linearized DNA molecules.

21. The method of claim 19 , wherein the restriction enzyme cuts at least a 4-bp sequence.

22. The method of claim 1 , wherein cleaving the plurality of eccDNA molecules includes:
cleaving, using a transposase, the plurality of eccDNA molecules; and
attaching, using the transposase, adapter sequences to both cleaved ends of each of the plurality of eccDNA molecules, thereby forming the set of linearized DNA molecules.

23. A method comprising:
receiving a biological sample of an organism, the biological sample including a plurality of extrachromosomal circular DNA (eccDNA) molecules, wherein each of the plurality of eccDNA molecules includes a junction at which nucleotides at two separated genomic locations are immediately adjacent to one another;
digesting, with a restriction enzyme, the plurality of eccDNA molecules to form a set of linearized DNA molecules that each includes the junction, the set of linearized DNA molecules comprising at least 1,000 linearized DNA molecules; and
for each of the set of linearized DNA molecules:
sequencing at least both ends of the set of linearized DNA molecules to obtain one or more sequence reads;
selecting a pair of end sequences for the linearized DNA molecule from the one or more sequence reads, the pair of end sequences not including the junction; and
reversing a direction of each of the pair of end sequences to obtain a pair of reversed end sequences.

24. A method comprising:
receiving a biological sample of an organism, the biological sample including a plu

Assignees

• Univ Hong Kong Chinese
• Grail Inc

Inventors

• Lo Yuk-Ming Dennis
• Chiu Rossa Wai Kwun
• Chan Kwan Chee
• Jiang Peiyong
• Ji Lu
• Sin Tsz Kwan
• Zhang Haiqiang
• Deng Jiaen

Classifications

• G16B20/10

  Ploidy or copy number detection · CPC title

• G16B20/20

  Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection · CPC title

• G16B30/10

  Sequence alignment; Homology search · CPC title

• C12Y301/21004

  Type II site-specific deoxyribonuclease (3.1.21.4) · CPC title

• C12Q2600/118

  Prognosis of disease development · CPC title