US 12,264,364 B2
Methods of preparing dual-indexed DNA libraries for bisulfite conversion sequencing
Anton Valouev, Palo Alto, CA (US); and Arash Jamshidi, Redwood City, CA (US)
Assigned to GRAIL, Inc., Menlo Park, CA (US)
Filed by GRAIL, LLC, Menlo Park, CA (US)
Filed on Dec. 20, 2022, as Appl. No. 18/084,955.
Application 18/084,955 is a continuation of application No. 15/674,003, filed on Aug. 10, 2017, granted, now 11,566,284.
Claims priority of provisional application 62/397,650, filed on Sep. 21, 2016.
Claims priority of provisional application 62/373,261, filed on Aug. 10, 2016.
Prior Publication US 2023/0119938 A1, Apr. 20, 2023
This patent is subject to a terminal disclaimer.
Int. Cl. C12Q 1/6874 (2018.01); C12Q 1/6869 (2018.01)
CPC C12Q 1/6874 (2013.01) [C12Q 1/6869 (2013.01)] 20 Claims
 
1. A method of determining a methylation profile of an individual, the method comprising:
a) obtaining a biological sample from the individual;
b) converting an unmethylated cytosine to an uracil in a nucleic acid molecule of the biological sample, thereby producing a converted nucleic acid molecule, wherein the converted nucleic acid molecule is single stranded and has a converted nucleic acid sequence;
c) attaching a single stranded nucleic acid adapter to the converted nucleic acid molecule, thereby producing a single stranded adapter ligated product comprising the converted nucleic acid molecule;
d) annealing a primer to the single stranded nucleic acid adapter in the single stranded adapter ligated product and extending the primer to form a double stranded complex comprising the single stranded adapter ligated product and an extension product;
e) attaching an at least partially double-stranded nucleic acid adapter comprising a first strand having a 5′-phosphate and a second strand comprising a blocked 3′ end to a 3′ end of the extension product of the double stranded complex via the 5′-phosphate of the first strand, wherein the blocked 3′ end blocks ligation of the second strand of the at least partially double-stranded nucleic acid adapter to a 5′ end of the single stranded adapter ligated product of the double stranded complex;
f) amplifying the double stranded complex comprising the single stranded adapter ligated product, the extension product, and the at least partially double stranded nucleic acid adapter that is ligated to the 3′ end of the extension product, wherein the second strand comprising the blocked 3′ end and the single stranded adapter ligated product is not amplified during the amplifying step;
g) determining the converted nucleic acid sequence of the converted nucleic acid molecule of the single stranded adapter ligated product or amplification products thereof; and
h) comparing the converted nucleic acid sequence to a reference nucleic acid sequence to determine the methylation profile of the individual.