LIQUID BIOPSY ASSAY FOR GENOMIC PROFILING OF CIRCULATING TUMOR DNA

1 - 25 . (canceled)

26 . A method of performing copy number noise reduction for a batch comprising a plurality of blood samples, wherein the method comprises:

obtaining sequencing data for the plurality of blood samples;

averaging raw coverages per gene per sample in the sequencing data, the raw coverages measured at a center of each of a plurality of probes;

normalizing the normalized coverages to a median gene-level coverage;

filtering the normalized coverages for well-behaved probes;

creating a model for each sample to predict sample-normalized coverage for guanine-cytosine (GC) content of probes;

normalizing the sample-normalized coverages to expected values from the models corresponding to the samples to determine per-sample GC normalized coverage;

removing, for all of the plurality of samples, probe values that deviate from the expected value by at least a threshold amount for at least one of the plurality of samples;

measuring a median per-sample GC normalized coverage for each probe across the batch;

normalizing the per-sample GC normalized coverage to generate expected GC normalized values; and

calculating probe copy numbers from the expected GC normalized values.

27 . A method for genomic profiling of plasma-derived circulating tumor DNA (ctDNA), the method comprising:

obtaining a mixture including plasma from a blood sample of a subject, the mixture including cell-free DNA (cfDNA) from the subject;

performing processing steps on the mixture to produce a sequencing library, wherein at least some of the processing steps including adding a corresponding set of Quantitative Counting Template (QCT) molecules to the mixture;

sequencing the sequencing library to produce sequence reads;

performing quality tracking based on a subset of the sequence reads that correspond to the QCT molecules; and

responsive to the quality tracking indicating success of the processing steps, constructing a tumor mutational profile for the subject based on the sequence reads.

28 . The method of claim 27 , further comprising estimating a tumor fraction of the ctDNA, wherein the tumor mutation profile is further based on the tumor fraction.

29 . A method of genomic profiling of circulating tumor DNA (ctDNA), the method comprising:

extracting buffy coat DNA from a blood sample;

performing multiplex PCR or ddPCR on the buffy coat DNA for a first set of mutations;

identifying, from sequencing data for the first set of mutations, one or more mutations present in the buffy coat DNA;

analyzing plasma extracted from the blood sample using a hybrid capture method that interrogates millions of base pairs to identify potential tumor-derived mutations; and

generating a tumor mutation profile that reports mutations detected by the hybrid capture method that were not present in the buffy coat DNA, wherein a potential tumor-derived mutation that also occurs in the buffy coat DNA is not a tumor-derived mutation.

30 . The method of claim 29 , wherein the first set of mutations comprises 1-10 mutations.

31 . The method of claim 26 , wherein each of the plurality of blood samples comprises cell-free DNA.

32 . The method of claim 26 , wherein each of the plurality of blood samples comprises circulating tumor DNA.

33 . The method of claim 26 , wherein obtaining the sequencing data comprises:

obtaining a mixture including plasma from a blood sample of the plurality of blood samples, the mixture including cell-free DNA (cfDNA) from the subject;

performing processing steps on the mixture to produce a sequencing library, wherein at least some of the processing steps including adding a corresponding set of Quantitative Counting Template (QCT) molecules to the mixture;

sequencing the sequencing library to produce sequence reads; and

generating the sequencing data using the sequence reads.

34 . The method of claim 33 , further comprising performing genomic profiling of the cfDNA by:

performing quality tracking based on a subset of the sequence reads that correspond to the QCT molecules; and

responsive to the quality tracking indicating success of the processing steps, constructing a tumor mutational profile for the subject based on the sequence reads.

35 . The method of claim 34 , further comprising estimating a tumor fraction of the ctDNA, wherein the tumor mutation profile is further based on the tumor fraction.

36 . The method of claim 35 , wherein the tumor fraction estimation is based on one or more signals derived from plasma of the blood sample, the one or more signals including at least one of: a maximum/average SNV signal, an aneuploidy signal, or a methylation signal.

37 . The method of claim 36 , wherein the aneuploidy signal is determined by:

obtaining copy number data for the plasma of the blood sample;

fitting a Gaussian Mixture Model (GMM) to the copy number data, the fitting of the GMM generating a plurality of peaks distributed around a central peak; and

inferring a tumor fraction from distances between the plurality of peaks from the central peak.

38 . The method of claim 34 , wherein the tumor mutational profile further includes an indication of presence or absence of at least one of single nucleotide variants (SNVs), insertions and deletions (indels), fusions, and microsatellite instability (MSI).

39 . The method of claim 38 , wherein the method uses a panel of multiple genes with a limit of detection of 0.13-0.16% allele fraction for SNVs and indels, and 2.100-2.160 for Copy Number amplification, 1.7-1.9 copies for Copy Number loss, 0.25-0.40% allele fraction for fusions, and 0.07-0.40% allele fraction for MSI.

40 . The method of claim 34 , wherein the tumor mutational profile further includes an indication of presence of a tumor mutation having a variant allele fraction (VAF) in a range from 0.05% to 0.2%.

41 . The method of claim 33 , wherein the sequencing has a median probe coverage of at least 40,000×.

42 . The method of claim 33 , wherein the cfDNA is circulating tumor DNA.

43 . The method of claim 29 , further comprising:

estimating tumor fraction from cell-free DNA (cfDNA); and

determining an in-tissue CNV for one or more variants identified in the tumor mutation profile.

44 . The method of claim 43 , wherein estimating the tumor fraction comprises:

applying a Gaussian Mixture Model (GMM) to quantify a liquid aneuploidy signal on measured genes; and

estimating the tumor fraction based on a periodicity pattern determined by the GMM.

45 . The method of claim 29 , further comprising a selective enrichment step that selectively amplifies target sequences in the buffy coat DNA and/or the plasma DNA, during or before which target enrichment QCTs are added, wherein a number of sequence reads corresponding to the target enrichment QCTs indicates success of or an issue with the selective enrichment step.