| CPC G16B 20/10 (2019.02) [G16B 30/00 (2019.02); G16B 30/20 (2019.02); G16B 40/20 (2019.02); G16B 40/30 (2019.02)] | 19 Claims |
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1. A method, implemented using a nucleic acid sequencer and a computer system comprising one or more processors and memory, for processing a test sample comprising cell-free nucleic acid fragments originating from a mother and a fetus, the method comprising:
(A) sequencing, at a first depth of sequencing, the test sample comprising the cell-free nucleic acid fragments originating from a mother and a fetus;
(B) computationally analyzing sequence information from the test sample, the analyzing comprising:
(a) determining an observed value of a fetal fraction of the test sample, wherein the fetal fraction of the test sample indicates a relative amount of fetal origin cell-free nucleic acid fragments in the test sample;
(b) receiving, by the computer system, sequence reads obtained by sequencing the cell-free nucleic acid fragments in the test sample;
(c) aligning, by the computer system, the sequence reads of the cell-free nucleic acid fragments to a reference genome comprising a sequence of interest, thereby providing sequence tags;
(d) determining, by the computer system, a coverage of the sequence tags for at least a portion of the reference genome; and
(e) determining that the test sample is within an exclusion region based on the coverage of sequences tags determined in (d) and the observed value of the fetal fraction determined in (a), wherein the exclusion region is defined by at least a fetal fraction limit of detection (LOD) curve, wherein the fetal fraction LOD curve varies with coverage values and indicates, for a given coverage value of different coverage values, a minimum observed value of the fetal fraction to achieve a given level of confidence that a corresponding ground truth fetal fraction is greater than a minimum value of fetal fractions needed to achieve a detection criterion; and
(C) re-sequencing, at a second depth of sequencing that is greater than the first depth of sequencing, the test sample to obtain re-sequenced sequence reads;
(D) repeating (B) (a)-(d) for the re-sequenced sequence reads; and
(E) determining that the re-sequenced test sample is outside the exclusion region;
further comprising: obtaining the LOD curve by:
(i) selecting from a first plurality of training samples, for each observed fetal fraction of a plurality of observed fetal fractions and for each coverage level of a plurality of coverage levels, one or more selected training samples whose true fetal fractions have the given level of confidence, wherein each training sample comprises cell-free nucleic acid fragments originating from a mother and a fetus,
(ii) obtaining, using the one or more selected training samples for each observed fetal fraction and each coverage level, true fetal fraction versus observed fetal fraction curves of the plurality of coverage levels,
(iii) obtaining, using a second plurality of training samples, true fetal fractions having at least the minimum value of fetal fraction needed to achieve the detection criterion for the plurality of coverage levels,
(iv) obtaining, using the true fetal fraction versus observed fetal fraction curves and the true fetal fractions having at least the minimum value of fetal fraction needed to achieve the detection criterion, a plurality of minimal observed fetal fractions for the plurality of coverage levels required to have the given level of confidence that true fetal fractions have at least the minimum value of fetal fraction needed to achieve the detection criterion, and
(v) obtaining the fetal fraction LOD curve using the plurality of minimal observed fetal fractions for the plurality of coverage levels.
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