US 12,460,202 B2
Compositions and methods for preparing nucleic acid libraries
Zhihong Zhang, Guangzhou (CN); Tao Zheng, Guangzhou (CN); Bingsi Li, Guangzhou (CN); Wanglong Deng, Guangzhou (CN); and Yusheng Han, Guangzhou (CN)
Assigned to GUANGZHOU BURNING ROCK DX CO., LTD., Guangzhou (CN)
Filed by GUANGZHOU BURNING ROCK DX CO., LTD., Guangdong (CN)
Filed on Apr. 7, 2021, as Appl. No. 17/225,082.
Application 17/225,082 is a continuation of application No. 17/044,723, previously published as PCT/CN2019/081059, filed on Apr. 2, 2019.
Application 17/044,723 is a continuation of application No. PCT/CN2018/081748, filed on Apr. 3, 2018.
Prior Publication US 2021/0254051 A1, Aug. 19, 2021
Int. Cl. C12N 15/10 (2006.01)
CPC C12N 15/1093 (2013.01) 14 Claims
 
1. A method for preparing a polynucleotide library, the method comprising, in the following order:
(a) in a first tailing reaction, adding a first tail to each of a plurality of target polynucleotides by template-independent polymerization of a pool of nucleotides consisting of dCTP and dTTP, wherein the dCTP and the dTTP are in a ratio selected from the group consisting of 9:1 and 3:1, such that the first tail has a ratio of dCTP to dTTP selected from the group consisting of 9:1 and 3:1, and the first tail is a single-stranded polynucleotide,
wherein each of the plurality of target polynucleotides comprises a single-stranded polynucleotide,
wherein the first tailing reaction further comprises hybridizing a first adapter to the first tail,
wherein the first adapter comprises a first strand and a second strand forming a double-stranded region and an overhang within the first strand of the first adapter, and
wherein the overhang of the first adapter is hybridized to the first tail;
(b) in a first ligation reaction, ligating one end of the second strand of the first adapter that is on the same side of the overhang in the first adaptor to the end of the first tail;
(c) in a linear amplification reaction, hybridizing a first primer to the second strand of the first adaptor that is ligated to the first tail in (b) and extending the first primer, and repeating the hybridizing and extending for a total of 10 to 15 cycles to linearly amplify each of the plurality of target polynucleotides, thereby producing a plurality of complement target polynucleotides; and
(d) in a second ligation reaction, ligating a strand of a second adapter to the plurality of complement target polynucleotides at the end opposite to the first primer.