US 11,705,218 B2
Nucleic acid analysis method, nucleic acid analysis program, and device for library preparation
Yusuke Osaki, Kanagawa (JP); Hirotaka Unno, Kanagawa (JP); Yudai Kawashima, Kanagawa (JP); Michie Hashimoto, Tokyo (JP); Masayuki Yumoto, Kanagawa (JP); Satoshi Nakazawa, Kanagawa (JP); Yuki Yonekawa, Kanagawa (JP); Takahiro Matsudaira, Kanagawa (JP); and Eri Nishiyama, Kanagawa (JP)
Assigned to Ricoh Company, Ltd., Tokyo (JP); and FASMAC CO., LTD., Kanagawa (JP)
Filed by RICOH COMPANY, LTD., Tokyo (JP); and FASMAC CO., LTD, Kanagawa (JP)
Filed on Dec. 18, 2019, as Appl. No. 16/718,390.
Claims priority of application No. 2018-236746 (JP), filed on Dec. 18, 2018; application No. 2019-015126 (JP), filed on Jan. 31, 2019; application No. 2019-046689 (JP), filed on Mar. 14, 2019; and application No. 2019-047881 (JP), filed on Mar. 15, 2019.
Prior Publication US 2020/0194100 A1, Jun. 18, 2020
Int. Cl. G16B 35/10 (2019.01); G16B 30/00 (2019.01); C12Q 1/6874 (2018.01)
CPC G16B 35/10 (2019.02) [C12Q 1/6874 (2013.01); G16B 30/00 (2019.02)] 11 Claims
 
1. A method for analyzing data of high-throughput sequencing reaction using at least one standard sample comprising a nucleic acid of specific copy number(s), the method comprising:
a) preparing a library for the at least one standard sample and at least one sequence sample under a same condition, wherein the library is prepared with a base material that comprises a plurality of filled sites and is formed by
discharging one or more cell suspension liquid droplets into at least one of a first set of filled sites, a cell of the cell suspension having the nucleic acid of the at least one standard sample, wherein each of the at least one of the first set of filled sites having the one or more cell suspension liquid droplets discharged therein has a specific copy number of the nucleic acid of the at least one standard sample;
counting a number of cells in the one or more cell suspension liquid droplets after discharging of the liquid droplets into the at least one of the first set of filled sites;
filling a sample comprising a nucleic acid of the at least one sequence sample into at least one of a second set of filled sites;
extracting the nucleic acid from the cell of the cell suspension in each of the at least one of the first set of filled sites and, optionally, extracting the at least one sequence sample, wherein a coefficient of variation (CV), which is the standard deviation of the specific copy number divided by an average copy number x, satisfies CV<1/√x for the average copy number;
b) subjecting the library prepared in the step a) to a sequencing reaction to obtain output data comprising reads derived from the at least one standard sample and the at least one sequence sample; and
c) separating the reads in the output data into a first group of reads having at least one read less than at least one threshold and a second group having at least one read more than the at least one threshold, wherein if the reads in the output data are equal to the at least one threshold, they are optionally included in the first group or the second group, wherein the at least one threshold is selected from the group consisting of a read number, the read number multiplied by a predetermined coefficient, the mean or median of read numbers, and a normalized value thereof.