Methods of analyzing nucleic acid fragments
Provided herein are methods for enriching a biological sample for a target nucleic acid, and analyzing the nucleic acid. In some cases, a biological sample is enriched for target nucleic acids associated with a cancer or tumor. In some cases, a biological sample is enriched for target nucleic acids, and the target nucleic acids vary in length. In some cases, one or more probes are used to enrich the biological sample for the target nucleic acid. In some cases, one or more probes hybridize to one or more ends of a target nucleic acid.
1. A method for processing a cell-free nucleic acid molecule from a biological sample, the method comprising:
(a) ligating an adapter to each end of a cell-free nucleic acid molecule, wherein the adapter comprises a recognition sequence for a restriction endonuclease configured to cleave the adapter from the cell-free nucleic acid molecule;
(b) amplifying the cell-free nucleic acid molecule comprising the adapter at each end, thereby generating an amplified cell-free nucleic acid molecule; and
(c) separating at least one adapter from the amplified cell-free nucleic acid molecule by digesting the adaptor at the recognition sequence using a corresponding restriction endonuclease.
2. The method of claim 1 , wherein the cell-free nucleic acid molecule is double stranded.
3. The method of claim 1 , wherein the restriction endonuclease is a Type IIs nuclease.
4. The method of claim 3 , wherein the Type IIs nuclease is selected from the group consisting of BtsCI, FOKI, AP endonuclease, and Si endonuclease.
5. The method of claim 1 , wherein the concentration of a plurality of the cell-free nucleic acid molecules ranges from 10 to 10000 genomes per milliliter in the biological sample.
6. The method of claim 1 , wherein the biological sample is selected from the group consisting of whole blood, blood plasma, blood serum, urine, cerebrospinal fluid, buffy coat, and a combination of any thereof.
7. The method of claim 1 , further comprising repairing one or more ends of the cell-free nucleic acid molecule.
8. The method of claim 7 , wherein the repairing comprises blunting at least one of the one or more ends of the cell-free nucleic acid molecule to comprise at least one of a terminal 5′-phosphate group or a 3′-hydroxyl group.
9. The method of claim 8 , wherein the repairing further comprises attaching a deoxyadenosine nucleotide at the one or more ends of the cell-free nucleic acid molecule.
10. The method of claim 1 , wherein the adapter is double stranded.
11. The method of claim 1 , further comprising hybridizing at least one probe to each end of the amplified cell-free nucleic acid molecule, wherein
each probe comprises a given sequence that is complementary to at least one end of the amplified cell-free nucleic acid molecule, and wherein each nucleotide of the given sequence of each probe hybridizes with the amplified cell-free nucleic acid molecule.
12. The method of claim 1 , further comprising:
(d) subjecting the amplified cell-free nucleic acid molecule to sequencing to obtain a plurality of sequence reads corresponding to the cell-free nucleic acid molecule;
(e) aligning the plurality of sequence reads to a reference genome; and
(f) determining a set of genomic positions in the reference genome at which ends of the plurality of sequence reads occur at a rate above threshold, thereby determining a nucleic acid fragmentation pattern.
13. A method for processing a cell-free nucleic acid molecule from a biological sample, the method comprising:
(a) ligating an adapter to each end of the cell-free nucleic acid molecule from a biological sample, wherein an end of the adapter that is ligated to the cell-free nucleic acid molecule comprises an adenine base or a thymine base;
(b) amplifying the cell-free nucleic acid molecule comprising the adaptor at each end, thereby generating an amplified cell-free nucleic acid molecule comprising an amplified adapter at each end, wherein the amplified adaptor comprises one of a uracil base, a uridine base, or a deoxyuridine base in a position of the thymine base or complementary to the adenine base; and
(c) separating at least one amplified adapter from the amplified cell-free nucleic acid molecule by converting the uracil base, uridine base, or deoxyuridine base to an apurinic/apyrimidinic site (AP site) and then generating a nick at the AP site using a corresponding endonuclease while keeping the amplified cell-free nucleic acid molecule intact.
14. The method of claim 13 , wherein conversion of the uracil base, uridine base, or deoxyuridine base to an AP site is with a Uracil-DNA Glycosylase (UDG).
15. The method of claim 13 , wherein the amplifying comprises applying one or more primers comprising one of a uracil base, a uridine base, or a deoxyuridine base at a 3′ end of the one or more primers.
16. The method of claim 14 , wherein the endonuclease is an AP endonuclease.
17. The method of claim 16 , further comprising treating the amplified cell-free nucleic acid molecule with an additional nuclease specific for a single-stranded nucleic acid molecule, thereby separating the adapter sequence from the cell-free nucleic acid molecule.