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 DNA (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:
extending, using a strand-displacing polymerase, a nicked dsDNA molecule to produce an ssDNA molecule;
ligating a universal adapter comprising a unique sequence tag to the ssDNA molecule;
extending the universal adapter to generate a nicked-ssDNA-derived dsDNA molecule; and
generating a cfDNA sequencing library from the nicked-ssDNA-derived dsDNA molecule.
2. The method according to claim 1 , further comprising ligating a sequencing Y-adapter to the nicked-ssDNA-derived dsDNA molecule before generating the cfDNA sequencing library.
3. The method according to claim 2 , wherein the sequencing Y-adapter comprises a unique sequence tag.
4. The method according to claim 1 , further comprising:
ligating a first sequencing Y-adapter to a first end of an intact dsDNA molecule in the cfDNA sample; and
ligating a second sequencing Y-adapter to a second end of the intact dsDNA molecule;
generating a cfDNA sequencing library from the intact dsDNA molecule.
5. The method according to claim 4 , wherein the first sequencing Y-adapter comprises a first unique sequence tag, and the second sequencing Y-adapter comprises a second unique sequence tag.
6. The method according to claim 5 , wherein the first and the second unique sequence tags are the same.
7. The method according to claim 5 , wherein the first and the second unique sequence tags are different.
8. The method according to claim 1 , wherein one or more of the unique sequence tags comprises a universal primer and at least one of a molecular barcode sequence, a unique molecular identifier (UMI), or an index sequence.
9. The method according to claim 1 , wherein one or more of the universal adapters comprises an adenylated 5′ end.
10. The method according to claim 1 , wherein one or more of the universal adapters comprises a blocked or a phosphorylated 3′ end.
11. The method according to claim 1 , further comprising performing an end repair reaction on a dsDNA molecule.
12. The method according to claim 1 , wherein generating the cfDNA sequencing library comprises performing a PCR amplification reaction.
13. The method according to claim 1 , wherein the cfDNA sample is isolated from a plasma fraction of a blood sample.