Methods of preparing and analyzing cell-free nucleic acid sequencing libraries
Aspects of the invention relate to methods for preparing and analyzing a sequencing library from a mixed cell-free DNA (cfDNA) sample, wherein the mixed sample includes double-stranded DNA (dsDNA), damaged dsDNA (e.g., nicked dsDNA), and single-stranded DNA (ssDNA) molecules. The subject methods facilitate the collection of information from dsDNA, ssDNA and damaged DNA (e.g., nicked DNA) molecules in a sample, thereby providing enhanced diagnostic information as compared to sequencing libraries that are prepared from dsDNA alone.
1. A method for preparing a cell-free (cfDNA) sequencing library from a cfDNA sample comprising a plurality of double-stranded DNA (dsDNA), nicked dsDNA, and single-stranded DNA (ssDNA) molecules, the method comprising:
ligating a first sequencing Y-adapter to a first end of a nicked dsDNA molecule in the cfDNA sample, wherein the nicked dsDNA molecule comprises a nicked strand and an unnicked strand;
ligating a second sequencing Y-adapter to a second end of the nicked dsDNA molecule in the cfDNA sample;
denaturing the sequencing Y-adapter-ligated nicked dsDNA molecule to generate a first ssDNA molecule derived from the unnicked strand, a second ssDNA molecule derived from the nicked strand, and a third ssDNA molecule derived from the nicked strand;
converting the second ssDNA molecule derived from the nicked strand to a first nick-derived dsDNA molecule in a primer extension reaction;
ligating a third sequencing Y-adapter to the first nick-derived dsDNA molecule; and
generating a cfDNA sequencing library from the first nick-derived dsDNA molecule.
2. The method of claim 1 , wherein the first sequencing Y-adapter comprises a first unique sequence tag, the second sequencing Y-adapter comprises a second unique sequence tag, and the third sequencing Y-adapter comprises a third unique sequence tag.
3. The method according to claim 2 , wherein the first, second and third unique sequence tags are the same.
4. The method according to claim 2 , wherein the first, second and third unique sequence tags are different.
5. The method according to claim 2 , wherein the first and second unique sequence tags are the same, and wherein the third unique sequence tag is different.
6. The method according to claim 2 , wherein the first and third unique sequence tags are the same, and wherein the second unique sequence tag is different.
7. The method according to claim 2 , wherein the second and third unique sequence tags are the same, and wherein the first unique sequence tag is different.
8. The method of claim 1 , further comprising:
ligating a first sequencing Y-adapter to a first end of an intact dsDNA molecule in the cfDNA sample;
ligating a second sequencing Y-adapter to a second end of the intact dsDNA molecule; and
generating a cfDNA sequencing library from the intact dsDNA molecule.
9. The method according to claim 8 , wherein the first sequencing Y-adapter comprises a first unique sequence tag, and the second sequencing Y-adapter comprises a second unique sequence tag.
10. The method according to claim 9 , wherein the first and the second unique sequence tags are the same.
11. The method according to claim 9 , wherein the first and the second unique sequence tags are different.
12. The method of claim 1 , wherein one or more of the unique sequence tags comprises a molecular barcode sequence, a unique molecular identifier (UMI), an index sequence, a universal primer region, or any combination thereof.
13. The method of claim 1 , wherein one or more of the universal adapters comprises an adenylated 5′ end.
14. The method of claim 1 , wherein one or more of the universal adapters comprises a blocked or a phosphorylated 3′ end.
15. The method of claim 1 , further comprising performing an end repair reaction on a dsDNA molecule.
16. The method of claim 1 , wherein generating the cfDNA sequencing library comprises performing a PCR amplification reaction.
17. The method of claim 1 , wherein the cfDNA sample is isolated from a plasma fraction of a blood sample.