CPC C12Q 1/6806 (2013.01) [G06F 17/18 (2013.01); G16B 50/00 (2019.02); C12Q 2600/158 (2013.01); C12Q 2600/16 (2013.01)] | 24 Claims |
1. A method for the analysis of minimal residual disease, the method comprising:
(a) obtaining multiple pairs of primers designed to amplify sequences that contain a plurality of sequence variations that have been previously identified in a patient's tumor through sequencing nucleic acids from tumor material, wherein the primer pairs are compatible in a multiplex amplification reaction;
(b) performing a targeted multiplex amplification using the multiple pairs of primers to amplify the sequences containing the plurality of sequence variations from cell-free DNA (cfDNA) isolated from a plasma sample of the patient to obtain amplicons;
(c) sequencing the amplicons of step (b), or an amplification product thereof, to produce sequence reads;
(d) detecting two or more of the sequence variations in the sequence reads of step (c), wherein the detecting comprises comparing a quantity of sequence reads containing a sequence variation against a threshold value, wherein the threshold value for the two or more sequence variations varies; and
(e) calculating a score for the patient sample based on the combined allele frequencies of the detected two or more sequence variations of step (d), wherein the score indicates the presence of minimal residual disease.
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