Granted Patent B2

US 12,534,759 · App. 18/738,910 · Granted Jan 27, 2026

Inventors: Bert Vogelstein (Baltimore, MD); Kenneth W. Kinzler (Frankford, DE); Nickolas Papadopoulos (Towson, MD); Isaac A Kinde (Beaumont, CA)

Assignee: The Johns Hopkins University

C12Q1/6874C12Q1/6806C12Q1/6869C12Q1/6876C12Q2525/179C12Q2525/191C12Q2563/179C12Q2600/158

Abstract

Claims (25)

1 . A method comprising:

a) attaching paired-end adapters to first and second ends of an initial double-stranded template nucleic acid molecule (IDSTNM) having top and bottom strands to form an adapted fragment, wherein a first endogenous UID is at said first end of said IDSTNM and a second endogenous UID is at said second end of said IDSTNM;

b) amplifying said adapted fragment such that a plurality of double-stranded amplicons are generated that each comprise a first paired-end adapter at one end and a second paired-end adapter at the other end that is different from said first paired-end adapter,

c) subjecting said plurality of double-stranded amplicons to paired-end sequencing on a massively parallel sequencer using a first primer to said first paired-end adapter and a second primer to said second paired-end adapter to generate the following reads:

i) first primer top strand reads and second primer top strand reads, and

ii) first primer bottom strand reads and second primer bottom strand;

d) differentiating said top and bottom strands of said IDSTNM based on the observed orientation and order of said first and second endogenous UIDs as they appear in said reads when sequence information is obtained from said paired-end sequencing;

e) grouping said reads by chromosome position of said first and second endogenous UIDs yielding:

i) a first family comprising said first primer top strand reads and said second primer top strand reads, and

ii) a second family comprising said first primer bottom strand reads and said second primer bottom strand reads;

f) comparing nucleotide sequences between members of said first family, and comparing nucleotide sequences between members of said second family; and

g) identifying a nucleotide sequence as accurately representing said top strand or bottom strand of said initial double-stranded template molecule when at least 50% of members of said first family, or said second family, contain the sequence.

2 . The method of claim 1 , wherein said first and second endogenous UIDs are 2 to 1000 bases in length.

3 . The method of claim 1 , wherein said first and second endogenous UIDs are 4 to 400 bases in length.

4 . The method of claim 1 , wherein said at least 50% is at least 70%.

5 . The method of claim 1 , wherein said attaching adapters is performed by ligation.

6 . The method of claim 1 , wherein said amplification employs primers complementary to at least a portion of said paired-end adapters.

7 . The method of claim 6 , wherein said primers comprise an indexing and/or grafting sequence.

8 . The method of claim 1 , wherein said initial double-stranded template nucleic acid molecule is from a human sample.

9 . The method of claim 8 , wherein said human sample comprises tissue.

10 . The method of claim 1 , wherein said plurality of double-stranded amplicons are subjected to an enrichment step where it is captured by a complementary oligonucleotide prior to said paired-end sequencing.

11 . The method of claim 1 , wherein said differentiating said top and bottom strands of said IDSTNM is further based on the observed orientation and order of at least part of said first and second paired-end adapters.

12 . The method of claim 1 , wherein said members of said first family comprise more than two members, and wherein said members of said second family comprise more than two members.

13 . The method of claim 1 , wherein said members of said first family comprise more than five members, and wherein said members of said second family comprise more than five members.

14 . The method of claim 1 , wherein said members of said first family comprise more than ten members, and wherein said members of said second family comprise more than ten members.

Assignments (1)

Continuity (10)

References Cited (400)