US 12,460,247 B2
Methods for standardized sequencing of nucleic acids and uses thereof
James C. Willey, Toledo, OH (US); Thomas Blomquist, Waterville, OH (US); and Erin Crawford, Rossford, OH (US)
Assigned to The University of Toledo, Toledo, OH (US)
Filed by The University of Toledo, Toledo, OH (US)
Filed on Apr. 25, 2022, as Appl. No. 17/728,463.
Application 17/728,463 is a continuation of application No. 15/909,525, filed on Mar. 1, 2018, granted, now 11,312,987.
Application 15/909,525 is a continuation of application No. 14/646,585, granted, now 9,944,973, issued on Apr. 17, 2018, previously published as PCT/US2013/071656, filed on Nov. 25, 2013.
Claims priority of provisional application 61/784,394, filed on Mar. 14, 2013.
Claims priority of provisional application 61/730,463, filed on Nov. 27, 2012.
Claims priority of provisional application 61/729,853, filed on Nov. 26, 2012.
Prior Publication US 2022/0282303 A1, Sep. 8, 2022
This patent is subject to a terminal disclaimer.
Int. Cl. C12Q 1/6806 (2018.01); C12Q 1/6851 (2018.01); C12Q 1/6869 (2018.01); C40B 20/04 (2006.01)
CPC C12Q 1/6806 (2013.01) [C12Q 1/6851 (2013.01); C12Q 1/6869 (2013.01); C40B 20/04 (2013.01)] 19 Claims
 
1. A method for controlling for non-systematic errors by reducing oversampling of overrepresented native nucleic acid targets and stochastic sampling error associated with deep sequencing, the method comprising:
i) preparing a mixture comprising a known number of internal amplification control (IAC) nucleic acid molecules corresponding to each native nucleic acid target; and
ii) mixing the IAC mixture of step i) with a native nucleic acid target-containing sample;
wherein each native nucleic acid target is similar to its respective IAC, with the exception of one or more changes to the nucleic acid sequence that are identifiable with sequencing; and,
wherein such changes can include one or more of deletions, additions, or alteration to the ordering or composition of nucleotides used;
iii) sequencing the mixture of step ii);
iv) assessing the ratio of sequencing events between the native nucleic acid target and its respective IAC, along with the known number of IAC nucleic acid molecules input into the sample; and
v) quantifiably determining the original amount of each native nucleic acid target in the original sample;
wherein the non-systematic error comprises one or more of the following errors:
a) library preparation steps;
b) preparation sequencing library replicates;
c) different time of replicates;
d) different technicians preparing the library; and
e) preparing the library in a different laboratory.