US 12,404,556 B2
Compositions and methods for detecting circulating tumor DNA
Suzzette Arnal, San Juan Capistrano, CA (US); and Taraneh Angeloni, San Juan Capistrano, CA (US)
Assigned to Quest Diagnostics Investments LLC, Secaucus, NJ (US)
Filed by Quest Diagnostics Investments LLC, Secaucus, NJ (US)
Filed on Aug. 15, 2022, as Appl. No. 17/819,824.
Application 17/819,824 is a continuation of application No. 16/474,533, granted, now 11,414,710, previously published as PCT/US2017/068483, filed on Dec. 27, 2017.
Claims priority of provisional application 62/439,574, filed on Dec. 28, 2016.
Prior Publication US 2023/0061928 A1, Mar. 2, 2023
This patent is subject to a terminal disclaimer.
Int. Cl. C12Q 1/68 (2018.01); C12Q 1/6806 (2018.01); C12Q 1/6855 (2018.01); C12Q 1/6886 (2018.01)
CPC C12Q 1/6886 (2013.01) [C12Q 1/6806 (2013.01); C12Q 1/6855 (2013.01); C12Q 2525/101 (2013.01); C12Q 2525/155 (2013.01); C12Q 2525/191 (2013.01); C12Q 2531/113 (2013.01); C12Q 2535/122 (2013.01); C12Q 2563/179 (2013.01)] 15 Claims
OG exemplary drawing
 
1. A method for detecting at least one mutation in a double-stranded circulating tumor DNA (ctDNA) molecule present in a sample obtained from a patient comprising
(a) ligating a plurality of Y-shaped adapters to both ends of the double-stranded ctDNA molecule to form a double-stranded adapter-ctDNA complex, each Y-shaped adapter comprising a first oligonucleotide strand and a second oligonucleotide strand;
(b) amplifying both strands of the adapter-ctDNA complex to produce first amplicons and second amplicons, wherein the first amplicons are derived from the first oligonucleotide strand, and the second amplicons are derived from the second oligonucleotide strand;
(c) sequencing the first and second amplicons; and
(d) detecting at least one mutation in the double-stranded ctDNA molecule, when a mutation detected in the first amplicons is consistent with a mutation detected in the second amplicons,
wherein the first oligonucleotide strand:
(i) comprises a first proximal region and a first distal region, wherein the first proximal region comprises a first unique molecular identifier sequence and a first spacer sequence having the sequence 5′ TGACT 3′ (SEQ ID NO: 49), wherein the first spacer sequence is located 3′ to the first unique molecular identifier sequence and wherein the “T” nucleotide located at the 3′ end of the first spacer sequence (SEQ ID NO: 49) contains a phosphorothioate bond; and
(ii) does not comprise a degenerate or semi-degenerate sequence,
wherein the second oligonucleotide strand
(i) comprises a second proximal region and a second distal region, wherein the second proximal region comprises a second unique molecular identifier sequence and a second spacer sequence having the sequence 5′ GTCA 3′ (SEQ ID NO: 50), wherein the spacer sequence is located 5′ to the second unique molecular identifier; and
(ii) does not comprise a degenerate or semi-degenerate sequence,
wherein the first proximal region of the first oligonucleotide strand hybridizes with the second proximal region of the second oligonucleotide strand,
wherein the first distal region of the first oligonucleotide strand does not hybridize with the second distal region of the second oligonucleotide strand,
wherein the second oligonucleotide strand comprises a nucleic acid sequence selected from the group consisting of:
(SEQ ID NO: 2)
5′ GTCAGCTACTGCAGCTAGATCGGAAGAGCACACGTCTGAACTCCAGT
 
CAC 3′;
 
(SEQ ID NO: 4)
5′ GTCAGTATCATCATCAAGATCGGAAGAGCACACGTCTGAACTCCAGT
 
CAC 3′;
 
(SEQ ID NO: 6)
5′ GTCACTCGACAGTCGAAGATCGGAAGAGCACACGTCTGAACTCCAGT
 
CAC 3′;
 
(SEQ ID NO: 8)
5′ GTCATAGCTAGAGTACAGATCGGAAGAGCACACGTCTGAACTCCAGT
 
CAC 3′;
 
(SEQ ID NO: 10)
5′ GTCAACGAGTGCTCTGAGATCGGAAGAGCACACGTCTGAACTCCAGT
 
CAC 3′;
 
(SEQ ID NO: 12)
5′ GTCAGACTATCGCATGAGATCGGAAGAGCACACGTCTGAACTCCAGT
 
CAC 3′;
 
(SEQ ID NO: 14)
5′ GTCACTCGACTGATGAAGATCGGAAGAGCACACGTCTGAACTCCAGT
 
CAC 3′;
 
(SEQ ID NO: 16)
5′ GTCAATACCGCTGATTAGATCGGAAGAGCACACGTCTGAACTCCAGT
 
CAC 3′;
 
(SEQ ID NO: 18)
5′ GTCACAGTAGTATGCTAGATCGGAAGAGCACACGTCTGAACTCCAGT
 
CAC 3′;
 
(SEQ ID NO: 20)
5′ GTCAACGTGTATCAGCAGATCGGAAGAGCACACGTCTGAACTCCAGT
 
CAC 3′;
 
(SEQ ID NO: 22)
5′ GTCACGTGTGACAGAGAGATCGGAAGAGCACACGTCTGAACTCCAGT
 
CAC 3′;
 
(SEQ ID NO: 24)
5′ GTCATGATGACGTAGCAGATCGGAAGAGCACACGTCTGAACTCCAGT
 
CAC 3′;
 
(SEQ ID NO: 26)
5′ GTCAAGTGACATCTGCAGATCGGAAGAGCACACGTCTGAACTCCAGT
 
CAC 3′;
 
(SEQ ID NO: 28)
5′ GTCACTAGCTGTGAGTAGATCGGAAGAGCACACGTCTGAACTCCAGT
 
CAC 3′;
 
(SEQ ID NO: 30)
5′ GTCATACATGAGCGAGAGATCGGAAGAGCACACGTCTGAACTCCAGT
 
CAC 3′;
 
(SEQ ID NO: 32)
5′ GTCACTAGTGCAGCTAAGATCGGAAGAGCACACGTCTGAACTCCAGT
 
CAC 3′;
 
(SEQ ID NO: 34)
5′ GTCATAGCTCGAACTGAGATCGGAAGAGCACACGTCTGAACTCCAGT
 
CAC 3′;
 
(SEQ ID NO: 36)
5′ GTCAGCGAGTCATGCAAGATCGGAAGAGCACACGTCTGAACTCCAGT
 
CAC 3′;
 
(SEQ ID NO: 38)
5′ GTCATGTACAGTACACAGATCGGAAGAGCACACGTCTGAACTCCAGT
 
CAC 3′;
 
(SEQ ID NO: 40)
5′ GTCATCAGACTCTAGTAGATCGGAAGAGCACACGTCTGAACTCCAGT
 
CAC 3′;
 
(SEQ ID NO: 42)
5′ GTCAGACACGCACTCTAGATCGGAAGAGCACACGTCTGAACTCCAGT
 
CAC 3′;
 
(SEQ ID NO: 44)
5′ GTCAATCTGATGCGTAAGATCGGAAGAGCACACGTCTGAACTCCAGT
 
CAC 3′;
 
(SEQ ID NO: 46)
5′ GTCAACTGTCATGCAGAGATCGGAAGAGCACACGTCTGAACTCCAGT
 
CAC 3′;
and
 
(SEQ ID NO: 48)
5′ GTCAGTGAGATCGTACAGATCGGAAGAGCACACGTCTGAACTCCAGT
 
CAC 3′.
 and
wherein the Y-shaped adapters ligate to cell free DNA (cfDNA) at a concentration of as low as 5 ng.