Using nucleic acid size range for noninvasive cancer detection
Size-band analysis is used to determine whether a chromosomal region exhibits a copy number aberration or an epigenetic alteration. Multiple size ranges may be analyzed instead of focusing on specific sizes. By using multiple size ranges instead of specific sizes, methods may analyze more sequence reads and may be able to determine whether a chromosomal region exhibits a copy number aberration even when clinically-relevant DNA may be a low fraction of the biological sample. Using multiple ranges may allow for the use of all sequence reads from a genomic region, rather than a selected subset of reads in the genomic region. The accuracy of analysis may be increased with higher sensitivity at similar or higher specificity. Analysis may include fewer sequencing reads to achieve the same accuracy, resulting in a more efficient process.
1. A method of determining a cancer classification in a biological sample from a subject, wherein the biological sample includes a mixture of cell-free DNA molecules including tumor DNA molecules and non-tumor DNA molecules, wherein the biological sample comprises plasma or serum, the method comprising:
performing a physical size separation of the biological sample for a range of sizes of the cell-free DNA molecules to form an enriched biological sample;
performing methylation-aware sequencing to identify methylated cell-free DNA molecules in the enriched biological sample;
for each size range of a plurality of size ranges:
measuring a first amount of the methylated cell-free DNA molecules from the enriched biological sample corresponding to the size range, and
determining, by a computer system, a methylation level using the first amount of the methylated cell-free DNA molecules corresponding to the size range and a second amount of DNA molecules in a second size range that includes sizes not in the size range;
obtaining, by the computer system, a reference size pattern including a reference methylation level for each size range of the plurality of size ranges, wherein the reference size pattern is determined from a plurality of reference samples from subjects with cancer or from subjects without cancer;
comparing, by the computer system, a plurality of the methylation levels to the reference size pattern; and
determining a level of cancer based on the comparison.
2. The method of claim 1 , wherein the second amount is of methylated cell-free DNA molecules.
3. The method of claim 1 , wherein the methylated cell-free DNA molecules are from a chromosomal arm.
4. The method of claim 1 , wherein:
comparing the plurality of the methylation levels to the reference size pattern comprises:
comparing each methylation level of the plurality of size ranges to the reference methylation level at the corresponding size range,
determining that each methylation level is statistically similar to the reference methylation level at the corresponding size range.
5. The method of claim 1 , wherein:
comparing the plurality of the methylation levels to the reference size pattern comprises:
determining a size pattern including the plurality of the methylation levels for the plurality of size ranges;
comparing a slope or an inflection point of the size pattern with a threshold value for a reference slope or a reference inflection point of the reference size pattern, wherein the threshold value indicates a statistically significant difference between the slope and the reference slope or the inflection point with the reference inflection point,
based on the comparing, determining the size pattern has a similar shape as the reference size pattern when the slope or the inflection point is not statistically different than the reference slope or the reference inflection point of the reference size pattern.
6. The method of claim 4 , wherein:
the reference size pattern is determined from the plurality of reference samples from subjects with cancer,
the method further comprising:
determining that the subject has cancer.
7. The method of claim 1 , wherein the first amount of the methylated cell-free DNA molecules is from a genomic region.
8. The method of claim 7 , wherein the genomic region is a chromosomal arm, the chromosomal arm selected from the group consisting of 1p, 1q, 8p, 8q, 13q, and 14q.
9. The method of claim 1 , wherein comparing the plurality of the methylation levels to the reference size pattern comprises comparing the plurality of the methylation levels to a plurality of threshold values that are determined from the plurality of reference samples.
10. The method of claim 1 , wherein:
the plurality of size ranges comprises M size ranges,
measuring the first amount of the methylated cell-free DNA molecules comprises measuring the first amount of the methylated cell-free DNA molecules corresponding to the size range and corresponding to each genomic region for N genomic regions,
calculating the methylation level using the first amount of the methylated cell-free DNA molecules corresponding to the size range and corresponding to the genomic region and the second amount generates a measurement vector of N×M methylation levels, wherein N is an integer greater than or equal to 1, and M is an integer greater than 1,
the reference size pattern includes a reference vector of reference methylation levels for the N genomic regions and the M size ranges, and
comparing the plurality of the methylation levels to the reference size pattern comprises comparing the measurement vector to the reference vector.
11. The method of claim 1 , further comprising:
measuring a size of each methylated cell-free DNA molecule of the methylated cell-free DNA molecules by:
obtaining sequence reads from both ends of the methylated cell-free DNA molecule,
aligning the sequence reads to a reference genome to obtain genomic coordinates of the ends of the methylated cell-free DNA molecule, and
subtracting the genomic coordinate of one end from the genomic coordinate of the other end.
12. The method of claim 1 , further comprising:
measuring a size of each methylated cell-free DNA molecule of the methylated cell-free DNA molecules using sequence reads, wherein the sequence reads are obtained by massively parallel sequencing.
13. The method of claim 1 , wherein the methylated cell-free DNA molecules comprise 1,000 molecules.
14. The method of claim 1 , wherein the methylation-aware sequencing includes bisulfite sequencing, sequencing preceded by methylation-sensitive restriction enzyme digestion, immunoprecipitation using anti-methylcytosine antibody or methylation binding protein, or single molecule sequencing.
15. The method of claim 1 , wherein determining the methylation level comprises:
calculating a ratio of the first amount to the second amount,
determining a difference between the ratio and an average value for a control group, and
dividing the difference by a standard deviation for the control group.
16. The method of claim 1 , wherein the plurality of size ranges comprises at least 71 size ranges.
17. The method of claim 1 , wherein the comparing, by the computer system, the plurality of the methylation levels to the reference size pattern comprises using a machine learning model trained using datasets including samples from subjects with cancer and subjects without cancer.
18. The method of claim 10 , wherein N is at least 4 and M is at least 71.
19. The method of claim 1 , wherein the range of sizes comprises sizes including 100 bp to 140 bp.
20. The method of claim 1 , wherein the range of sizes comprises sizes including 50 bp to 400 bp.
21. The method of claim 1 , wherein the range of sizes comprises sizes including 385 bp to 425 bp.
22. The method of claim 1 , further comprising training the machine learning model, wherein training the machine learning model comprises:
collecting the datasets including the samples from the subjects with cancer and the subjects without cancer, and
training the machine learning model using the datasets.
23. The method of claim 1 , wherein the physical size separation includes gel electrophoresis, filtration, selective-size precipitation, or hybridization.