	US 12,252,743 B2
	Safe sequencing system
Bert Vogelstein, Baltimore, MD (US); Kenneth W. Kinzler, Frankford, DE (US); Nickolas Papadopoulos, Towson, MD (US); and Isaac A. Kinde, Beaumont, CA (US)
Assigned to The Johns Hopkins University, Baltimore, MD (US)
Filed by The Johns Hopkins University, Baltimore, MD (US)
Filed on Nov. 13, 2023, as Appl. No. 18/507,776.
Application 15/240,034 is a division of application No. 15/090,773, filed on Apr. 5, 2016, abandoned.
Application 15/090,773 is a division of application No. 14/814,030, filed on Jul. 30, 2015, granted, now 9,487,829, issued on Nov. 8, 2016.
Application 14/814,030 is a division of application No. 14/111,715, granted, now 9,476,095, issued on Oct. 25, 2016, previously published as PCT/US2012/033207, filed on Apr. 12, 2012.
Application 18/507,776 is a continuation of application No. 17/837,591, filed on Jun. 10, 2022.
Application 17/837,591 is a continuation of application No. 17/324,380, filed on May 19, 2021, granted, now 11,453,913, issued on Sep. 27, 2022.
Application 17/324,380 is a continuation of application No. 15/240,034, filed on Aug. 18, 2016, granted, now 11,180,803, issued on Nov. 23, 2021.
Claims priority of provisional application 61/484,482, filed on May 10, 2011.
Claims priority of provisional application 61/476,150, filed on Apr. 15, 2011.
Prior Publication US 2024/0084381 A1, Mar. 14, 2024
This patent is subject to a terminal disclaimer.
Int. Cl. C12Q 1/6869 (2018.01); C12Q 1/6806 (2018.01); C12Q 1/6874 (2018.01); C12Q 1/6876 (2018.01)

CPC C12Q 1/6874 (2013.01) [C12Q 1/6806 (2013.01); C12Q 1/6869 (2013.01); C12Q 1/6876 (2013.01); C12Q 2525/179 (2013.01); C12Q 2525/191 (2013.01); C12Q 2563/179 (2013.01); C12Q 2600/158 (2013.01)]

8 Claims

1. A method of determining that both strands of an initial UID strand pair double-stranded template molecule are mutated comprising:

a) providing an initial UID strand pair double-stranded template molecule comprising a first nucleic acid strand and a second nucleic acid strand, and

wherein said initial UID strand pair double-stranded template molecule comprises a first UID at one end, and a second UID at the other end,

b) subjecting said initial UID strand pair double-stranded template molecule or amplicons thereof to paired-end run sequencing on a massively parallel sequencer to generate first paired-end reads and second paired-end reads;

c) differentiating which strand of said initial UID strand pair double-stranded template molecule is represented by said first and second paired-end reads based on the observed orientation and order of said first and second UIDs in said first and second paired-end reads when sequence information is obtained from said paired-end run sequencing;

d) grouping said first and second paired-end reads to yield a first UID family and a second UID family, wherein said first UID family comprises said first paired-end reads and said second UID family comprises said second paired-end reads;

e) comparing nucleotide sequences between members of said first UID family, and comparing nucleotide sequences between members of said second UID family, and identifying a first nucleotide sequence as accurately representing said first nucleic acid strand, and identifying a second nucleotide sequence as accurately representing said second nucleic acid strand of said initial UID strand pair double-stranded template molecule when at least 50% of members of said first UID family, and said second UID family, contain said first nucleotide sequence and said second nucleotide sequence respectively; and

f) determining that both strands of said initial UID strand pair double-stranded template molecule are mutated by detecting a difference in said first and second nucleotide sequences at the same position with respect to a reference.