BACKGROUND

The discovery that tumor cells release tumor-derived DNA into the blood stream has sparked the development of non-invasive methods capable of determining the presence, location and/or type of tumor in a subject using cell-free samples (e.g., plasma). Many tumors can be treatable if detected early in their development. However, current methods can lack the sensitivity and/or specificity to detect a tumor at an early stage and can return a large number of false positive or false negative results. The sensitivity of a test can refer to the likelihood that a subject that is positive for a condition tests positive for the condition. The specificity of a test can refer to the likelihood that a subject that is negative for a condition tests negative for that condition. The problems of sensitivity and specificity can be exaggerated in assays for the early detection of tumors, e.g., because samples on which such tumor detection methods are performed can have relatively small amounts of tumor-derived DNA and because the condition itself can have a relatively low prevalence among individuals tested in the early stage. Accordingly, there is a clinical need for methods having higher sensitivity and/or specificity for the detection of tumors.

SUMMARY

Various embodiments are directed to applications (e.g., classification of biological samples) of the analysis of the count, the fragmentation patterns, and size of cell-free nucleic acids, e.g., plasma DNA and serum DNA, including nucleic acids from pathogens, such as viruses. Embodiments of one application can determine if a subject has a particular condition. For example, a method of present disclosure can determine if a subject has cancer or a tumor, or other pathology. Embodiments of another application can be used to assess the stage of a condition, or the progression of a condition over time. For example, a method of the present disclosure may be used to determine a stage of cancer in a subject, or the progression of cancer in a subject over time (e.g., using samples obtained from a subject at different times).

According to one embodiment, sequence reads obtained from a sequencing of the mixture of cell free nucleic acid molecules can be used to determine an amount of the sequence reads aligning to a reference genome corresponding to the virus. The amount of sequence reads aligning to the reference genome can be compared to a cutoff value to screen for the pathology.

According to another embodiment, sizes of viral nucleic acid molecules (e.g., those aligning to a reference genome corresponding to the virus) can be used. A statistical value of a size distribution of the nucleic acid molecules from the virus can be determined. A level of pathology in the subject can be determined by processing the statistical value against a cutoff value.

According to another embodiment, a first amount of cell-free nucleic acid molecules that end within one or more first windows of a reference genome corresponding to the virus is determined. Each first window comprising at least one of a first set of genomic positions at which ends of cell-free nucleic acid molecules are present at a rate above a first threshold in subjects with a cancer (or other pathology) associated with the virus. A relative abundance can be computed by normalizing the first amount using a second amount of cell-free nucleic acid molecules, which includes cell-free nucleic acid molecules ending at a second set of genomic positions outside of the one or more first windows including the first set of genomic positions. A level of cancer in the subject can be determined by processing the relative abundance against a cutoff value.

Embodiments can combine various techniques. For example, a first assay can be count-based, size-based, or fragmentation-based. A second assay can be one of the other techniques. As examples a majority voting can be used, or cutoff values can be determined for both techniques, thereby determining a set of data points from the two techniques that correspond to a particular level of pathology.

Other embodiments are directed to systems, portable consumer devices, and computer readable media associated with methods described herein.

Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

