Hydroxymethylation analysis of cell-free nucleic acid samples for assigning tissue of origin, and related methods of use

Abstract

A method is provided for probabilistically assigning a tissue of origin to a nucleic acid in a sample, e.g., DNA in a cell-free fluid sample obtained from a human subject. A hydroxymethylation profile is generated for the sample DNA and then compared across a reference data set of hydroxymethylation profile vectors, where each hydroxymethylation profile vector identifies the hydroxymethylation profile at a specific reference locus, the tissue-specific gene associated with the reference locus, and the tissue with which the gene and reference locus are associated. A tissue of origin can be probabilistically assigned to the sample nucleic acid using the results of the comparison. Other methods of use are also provided.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims priority under 35 U.S.C. § 119(e)(1) to provisional U.S. Applications Ser. No. 62 / 688,975, filed Jun. 22, 2018, and Ser. No. 62 / 746,237, filed Oct. 16, 2018. The disclosures of the foregoing applications are incorporated by reference in their entireties.TECHNICAL FIELD[0002]The present invention relates generally to biotechnology, and more particularly relates to applications of the epigenetic analysis of cell-free DNA. The invention finds utility in the fields of genomics, medicine, diagnostics, and epigenetic research.BACKGROUND[0003]In many fields related to medicine, such as diagnostics, theranostics, and patient monitoring, there is an ongoing need for more precise methods of analyzing biological samples and extracting useful information from those analyses, with the often competing goals of accuracy and efficiency. Analytical methods should lend themselves to high speed processing and automation, yield the ne...

AI Technical Summary

Benefits of technology

[0047]In one aspect of the embodiment, the method further includes estimating the level of PBMC genomic contamination from the comparison in step (b). It will be appreciated that PBMC genomic contamination may be seen in cell-free samples as a result of post-collection PBMC lysis ex vivo, i.e., after sample collection. The method is thus useful as a measure of sample quality and an assessment as to whether a particular sample is suitable for use in cell-free DNA analyses.

[0048]In another aspect of the embodiment, the method further includes providing a correction vector for a cell-free DNA analysis based on a known hydroxymethylation vector for PBMC DNA, where the hydroxymethylation vector comprises a plurality of reference hydroxymethylation profiles corresponding to PBMC DNA. The correction vector can be used to offset, lower, or subtract the effects of poor sample quality due to PBMC DNA contamination, e.g., from post-collection PBMC lysis. The correction vector, providing that PBMC sub-type hydroxymethylation data is included in the vector, can also be used to specifically identify the proportions of PBMC sub-types in cell-free DNA contamination, including their activation or state.

Problems solved by technology

However, analysis of cell-free DNA also presents unique challenges, particularly since cell-free DNA samples are very small and typically contain only a few nanograms of DNA per mL of plasma.

The problem is compounded when cell-free DNA is used in conjunction with epigenetic analyses, in which relatively rare DNA modifications, such as 5-methylcytosine and the even rarer 5-hydroxymethylcytosine, require detection.

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