US 11,702,653 B2
Control compositions and methods for sequencing
Rachel R. Spurbeck, Columbus, OH (US); Richard Mon Che Chou, Columbus, OH (US); and Anthony D. Duong, Columbus, OH (US)
Assigned to BATTELLE MEMORIAL INSTITUTE, Columbus, OH (US)
Filed by BATTELLE MEMORIAL INSTITUTE, Columbus, OH (US)
Filed on May 21, 2019, as Appl. No. 16/418,521.
Claims priority of provisional application 62/801,520, filed on Feb. 5, 2019.
Claims priority of provisional application 62/703,266, filed on Jul. 25, 2018.
Claims priority of provisional application 62/674,533, filed on May 21, 2018.
Prior Publication US 2019/0382757 A1, Dec. 19, 2019
Int. Cl. C12N 15/10 (2006.01); C12N 15/113 (2010.01); C12Q 1/6869 (2018.01); C12Q 1/6876 (2018.01)
CPC C12N 15/1089 (2013.01) [C12N 15/113 (2013.01); C12Q 1/6869 (2013.01); C12Q 1/6876 (2013.01); C12Q 2563/185 (2013.01); C12Q 2600/166 (2013.01)] 16 Claims
 
1. A method for monitoring sample cross-contamination and/or sample swapping, and for quantitation of nucleic acids during sequencing, the method comprising,
a) spiking a first sample comprising prokaryotic or eukaryotic cells with a first control composition comprising a first nucleic acid construct wherein the first nucleic acid construct comprises at least one barcode sequence fragment, at least one universal sequence fragment, and at least one GC content fragment, and wherein the first nucleic acid construct is a deoxyribonucleic acid construct;
b) extracting total DNA from the first sample wherein total DNA comprises the DNA from the first sample and the DNA from the first nucleic acid construct;
c) purifying total DNA;
d) preparing a library from total DNA;
e) sequencing total DNA;
f) detecting and quantifying the first nucleic acid construct in total DNA;
g) spiking a second sample comprising the prokaryotic or eukaryotic cells with a second control composition, wherein the second control composition comprises a different nucleic acid construct than the first control composition with a different barcode sequence fragment linked to at least one universal sequence fragment and then performing steps b) to f) for the second sample wherein the second nucleic acid construct is detected and quantified in step f), and wherein detection of the different barcode sequence fragments in the first sample and the second sample is used to determine if cross-contamination between the first sample and the second sample or sample swapping between the first sample and the second sample occurred during sample preparation or processing and wherein the quantification of the GC content fragments in the first sample and the second sample is used to control for enzyme GC content bias; and
h) comparing the extraction efficiency of the first and second nucleic acid constructs with the extraction efficiency of the DNA in the first and second samples to control for extraction efficiency wherein the first nucleic acid construct and the second nucleic acid construct are separately encapsulated in a simulated cell membrane that mimics the cell membrane of the prokaryotic or eukaryotic cells in the first sample and the second sample to control for extraction efficiency.