Compositions and methods for detecting circulating tumor dna
AssigneeQuest Diagnostics Investments Incorporated
InventorsSuzzette Arnal, Taraneh Angeloni
Life of this patent
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Abstract
The present technology provides polynucleotide compositions and methods of using the same to detect circulating tumor DNA (ctDNA) in a patient. Kits for use in practicing the methods are also provided.
Claims19
1-16. (canceled)
A method of preparing a DNA amplicon library, the method comprising:
- (a) ligating a plurality of Y-shaped nucleic acid adapters to both ends of double-stranded circulating tumor DNA (ctDNA) molecules in a cell-free DNA (cfDNA) sample obtained from a patient to form a library of adapter-ligated ctDNA molecules, wherein the adapters are configured to efficiently ligate to cfDNA at input concentrations as low as 5 ng, and wherein each Y-shaped adapter comprises:
- (i) a first oligonucleotide strand comprising a first unique molecular identifier (UMI) sequence and a 5′-TGACT-3′ spacer sequence (SEQ ID NO:
- 49) with a 3′ terminal phosphorothioate bond;
- and
- (ii) a second oligonucleotide strand comprising a second UMI sequence and a 5′-GTCA-3′ spacer sequence (SEQ ID NO:
- 50);
- and
- (b) amplifying the library of adapter-ligated ctDNA molecules to produce a plurality of amplicons.
The method of claim 1, wherein the cfDNA sample comprises 5 ng or less of cfDNA.
The method of claim 1, further comprising step (c) enriching the library for cancer-related gene regions using bait sequences.
The method of claim 3, wherein the cancer-related gene regions are selected from ALK, BRAF, EGFR, ERBB2, KIT, KRAS, MET, NRAS, NTRK1, PIK3CA, ROS1, and RET.
The method of claim 1, wherein the second oligonucleotide strand of the Y-shaped adapter is selected from SEQ ID NOs:
- 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, and 48.
The method of claim 1, wherein the first oligonucleotide strand comprises a nucleotide sequence selected from SEQ ID NOs:
- 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, and 47.
The method of claim 1, wherein the method achieves approximately 20% greater ligation efficiency as compared to a method using Y-shaped adapters lacking said spacer sequences and said phosphorothioate bond.
The method of claim 1, wherein the sample is obtained from whole blood, plasma, or serum.
The method of claim 1, wherein the first unique molecular identifier sequence of the first oligonucleotide strand is selected from the group consisting of SEQ ID NOs:
- 51-74.
The method of claim 1, wherein the second unique molecular identifier sequence of the second oligonucleotide strand is selected from the group consisting of SEQ ID NOs:
- 75-98.
The method of claim 1, further comprising generating a duplex consensus sequence for each unique ctDNA molecule.
The method of claim 1, wherein at least one of the first or second oligonucleotide strands further comprises a biotin label.
The method of claim 1, wherein the first oligonucleotide strand comprises a first proximal region and a first distal region, the first proximal region comprising a first unique molecular identifier sequence and a first spacer sequence having the sequence 5′-TGACT-3′ (SEQ ID NO:
- 49) located 3′ to the first unique molecular identifier, wherein the 3′ terminal thymidine of the first spacer sequence contains a phosphorothioate bond.
The method of claim 1, wherein the second oligonucleotide strand comprises a second proximal region and a second distal region, the second proximal region comprising a second unique molecular identifier sequence and a second spacer sequence having the sequence 5′-GTCA-3′ (SEQ ID NO:
- 50), wherein the second spacer sequence is located 5′ to the second unique molecular identifier.
The method of claim 13, wherein the first and second proximal regions hybridize to form a double-stranded portion, and the first and second distal regions are non-complementary, forming a Y-shaped fork.
The method of claim 14, wherein the first and second proximal regions hybridize to form a double-stranded portion, and the first and second distal regions are non-complementary, forming a Y-shaped fork.
The method of claim 1, wherein the method further comprises sequencing the plurality of amplicons.
The method of claim 1, wherein the ctDNA molecules are subject to end-repair and A-tailing prior to the ligation of step (a).
Dependencies are inferred from the claim text.