| CPC C12Q 1/6886 (2013.01) [C12Q 1/6869 (2013.01); G16B 5/20 (2019.02); C12Q 2600/118 (2013.01); C12Q 2600/154 (2013.01); C12Q 2600/156 (2013.01); G16B 20/20 (2019.02); G16B 40/00 (2019.02)] | 10 Claims |
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1. A tiered, multipart method for detecting circulating tumor DNA in a biological sample of a subject, the method comprising:
performing a first analysis of nucleic acid sequence information that was derived from a first assay performed on the biological sample to identify whether the biological sample is not at risk of containing circulating tumor DNA, wherein the first analysis comprises analyzing methylation statuses of fewer than 1000 CGIs of nucleic acids from the biological sample each selected from Tables 1-4, wherein the first analysis achieves at least 80% specificity that the biological sample indicates the subject is not at risk of a disease,
and then if the biological sample is not identified as not at risk:
performing an intra-individual analysis for the subject comprising:
obtaining target nucleic acids and reference nucleic acids from the biological sample or an additional biological sample obtained from the subject, wherein the reference nucleic acids comprise genomic DNA from peripheral blood mononuclear cells (PBMCs) or polymorphonuclear cells of the subject;
performing bisulfite conversion of the target nucleic acids and the reference nucleic acids;
selectively amplifying at least 1000 CGIs selected from Tables 1-4 of the bisulfite converted target nucleic acids and reference nucleic acids;
generating a dataset comprising methylation information of the at least 1000 CGIs selected from Tables 1-4 from the target nucleic acids and methylation information of the at least 1000 CGIs selected from Tables 1-4 from the reference nucleic acids;
using a computer processor, combining the methylation information of the at least 1000 CGIs selected from Tables 1-4 from the target nucleic acids and the methylation information of the at least 1000 CGIs selected from Tables 1-4 from the reference nucleic acids to generate background-corrected methylation information of the at least 1000 CGIs selected from Tables 1-4 for the target nucleic acids;
performing a second analysis comprising analyzing the background-corrected methylation information of the at least 1000 CGIs selected from Tables 1-4 to detect the presence of the circulating tumor DNA in the biological sample, wherein the second analysis achieves at least a 70% positive predictive value; and
performing a third analysis comprising determining a longitudinal change between the background-corrected methylation information of the second analysis and methylation information of a further additional sample obtained from the subject.
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