Methylation markers and targeted methylation probe panel

Inventors: Samuel S. Gross (Menlo Park, CA); Hamed Amini (Menlo Park, CA); Arash Jamshidi (Menlo Park, CA); Seyedmehdi Shojaee (Menlo Park, CA); Srinka Ghosh (Menlo Park, CA); Rongsu Qi (Menlo Park, CA); M. Cyrus Maher (Menlo Park, CA); Alexander P. Fields (Menlo Park, CA); Oliver Claude Venn (Menlo Park, CA)

Assignee: GRAIL, Inc.

C12Q1/6886 C12Q1/70 C40B40/06 C12Q2600/154

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Quick Facts

Patent No.: US 12,435,375
App. No.: 17/061,013
Granted: Oct 7, 2025
Kind: B2

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Abstract

The present description provides a cancer assay panel for targeted detection of cancer-specific methylation patterns. Further provided herein includes methods of designing, making, and using the cancer assay panel for diagnosis of cancer.

Claims (2408)

1. An assay panel for enriching nucleic acids, wherein the assay panel comprises at least 1,000 different polynucleotide probes;

wherein each of the at least 1,000 different polynucleotide probes comprises a nucleic acid sequence that comprises:

(1) a first sequence that is identical to the sequence of at least 45 contiguous nucleotides within a target genomic region,

(2) a second sequence that varies with respect to the sequence of at least 45 contiguous nucleotides within the target genomic region by one or more transitions comprising one or more transitions that occur at a nucleotide corresponding to a CpG site in the target genomic region, or

(3) a third sequence that varies with respect to the sequence of at least 45 contiguous nucleotides within the target genomic region by comprising an adenine in place of a guanine;

wherein the at least 1,000 different polynucleotide probes comprise at least 500 different pairs of polynucleotide probes;

wherein each pair of the at least 500 different pairs of polynucleotide probes comprises two different polynucleotide probes tiled with respect to the same target genomic region;

wherein the two different polynucleotide probes of each pair of the at least 500 pairs of polynucleotide probes comprise (i) an identical overlapping sequence comprising 30 contiguous nucleotides of the first, second or third sequence, and (ii) a different non-overlapping sequence of 15 contiguous nucleotides of the first, second or third sequence; and

wherein each of the at least 1,000 different polynucleotide probes is conjugated to an affinity moiety.

2. The assay panel of claim 1 , wherein each of the at least 1,000 different polynucleotide probes comprises an adenine at each position corresponding to an unmethylated cytosine in a DNA molecule from the target genomic region.

3. The assay panel of claim 2 , wherein each of the at least 1,000 different polynucleotide probes comprises:

a) an adenine at each position corresponding to a cytosine of a CpG site of the DNA molecule from the target genomic region; or

b) a guanine at each position corresponding to a cytosine of a CpG site of the DNA molecule from the target genomic region.

4. The assay panel of claim 1 , wherein the target genomic region comprises at least five CpG dinucleotides and wherein at least 80% of the at least five CpG dinucleotides are either methylated or unmethylated in an individual with cancer.

5. The assay panel of claim 1 , wherein at least 10% of the at least 1,000 different polynucleotide probes lack guanine (G).

6. The assay panel of claim 1 , wherein the at least 1,000 different polynucleotide probes together comprise at least 200,000 nucleotides.

7. The assay panel of claim 1 , wherein (a) the target genomic region is a region of a human genome; and (b) none of the at least 1,000 different polynucleotide probes comprises a contiguous 45 bases having at least 90% sequence complementarity to at least 20 off-target regions in a GRCh37/hg19 genome.

8. The assay panel of claim 1 , wherein each of the at least 1,000 different polynucleotide probes comprises at least 75 nucleotides.

9. The assay panel of claim 1 , further comprising a plurality of virus-specific probes,

wherein each of the plurality of virus-specific probes comprises a sequence that (i) is complementary to a sequence of a viral genome or (ii) varies from a sequence complementary to a sequence of a viral genome by comprising an adenine at each position corresponding to an unmethylated cytosine in a nucleic acid fragment from the viral genome.

10. The assay panel of claim 9 , wherein the viral genome is a MCV, EBV, HBV, HCMV, HCV, HHV5, HPV16, or HPV18 genome.

11. The assay panel of claim 1 , wherein the target genomic region comprises at least 500 different target genomic regions.

12. The assay panel of claim 11 , wherein the at least 500 different target genomic regions comprise at least 0.2 Mb.

13. The assay panel of claim 11 , wherein the at least 500 different target genomic regions of a GRCh37/hg19 genome are selected from Table 11, Table 12, Table 13,

Lengthy table referenced here

US17061013-20250804-T00025