Described are methods of analyzing cell free DNA based on combining analysis of cfDNA methylation with analysis of the cfDNA nucleosome footprint and/or with analysis of cfDNA copy number alteration. The diagnostic performance of these methods, in particular relating to early or earlier stage diseases or disorders, is increased compared to the diagnostic performance of the individual cfDNA analysis methods.

Described are methods of analyzing cell free DNA based on combining analysis of cfDNA methylation with analysis of the cfDNA nucleosome footprint and/or with analysis of cfDNA copy number alteration. The diagnostic performance of these methods, in particular relating to early or earlier stage diseases or disorders, is increased compared to the diagnostic performance of the individual cfDNA analysis methods.

Methods are provided for the epigenetic analysis of cell-free DNA using organic boranes to convert oxidized 5-methylcytosine residues in the cell-free DNA to dihydrouracil (DHU) residues. Cell-free DNA is contacted with an organic borane selected to successively bring about reduction, deamination, and decarboxylation of oxidized 5-methylcytosine residues such as 5-carboxylcytosine and 5-formylcytosine, resulting in DHU residues in place thereof. Following amplification, the treated cell-free DNA is sequenced, with the DHU residues read as thymine residues. Reaction mixtures, kits and additional methods are also provided, as are related methods for the epigenetic analysis of DNA, including cell-free DNA.

Methods are provided for the epigenetic analysis of cell-free DNA using organic boranes to convert oxidized 5-methylcytosine residues in the cell-free DNA to dihydrouracil (DHU) residues. Cell-free DNA is contacted with an organic borane selected to successively bring about reduction, deamination, and decarboxylation of oxidized 5-methylcytosine residues such as 5-carboxylcytosine and 5-formylcytosine, resulting in DHU residues in place thereof. Following amplification, the treated cell-free DNA is sequenced, with the DHU residues read as thymine residues. Reaction mixtures, kits and additional methods are also provided, as are related methods for the epigenetic analysis of DNA, including cell-free DNA.

The present disclosure provides methods, systems, and kits for processing nucleic acid molecules. A method may comprise providing a template nucleic acid fragment (e.g., within a cell, cell bead, or cell nucleus) within a partition (e.g., a droplet or well) and subjecting the template nucleic acid fragment to one or more processes including a barcoding process and a single primer extension or amplification process. The processed template nucleic acid fragment may then be recovered from the partition and subjected to further amplification to provide material for subsequent sequencing analysis. The methods provided herein may permit simultaneous processing and analysis of both DNA and RNA molecules originating from the same cell, cell bead, or cell nucleus.

The present disclosure provides methods of processing or analyzing a sample. A method for processing a sample may comprise hybridizing a probe molecule to a target region of a nucleic acid molecule (e.g., a ribonucleic acid (RNA) molecule), barcoding the probe-nucleic acid molecule complex, and performing extension, denaturation, and amplification processes. A method for processing a sample may comprise hybridizing first and second probes to adjacent or non-adjacent target regions of a nucleic acid molecule (e.g., an RNA molecule), linking the first and second probes to provide a probe-linked nucleic acid molecule, and barcoding the probe-linked nucleic acid molecule. One or more processes of the methods described herein may be performed within a partition, such as a droplet or well. One or more processes of the methods described herein may be performed on a cell, such as a permeabilized cell.

Provided herein is a method for analyzing polynucleotides such as genomic DNA. In certain embodiments, the method comprises: (a) treating chromatin isolated from a population of cells with an insertional enzyme complex to produce tagged fragments of genomic DNA; (b) sequencing a portion of the tagged fragments to produce a plurality of sequence reads; and (c) making an epigenetic map of a region of the genome of the cells by mapping information obtained from the sequence reads to the region. A kit for performing the method is also provided.

Provided herein is a method for analyzing polynucleotides such as genomic DNA. In certain embodiments, the method comprises: (a) treating chromatin isolated from a population of cells with an insertional enzyme complex to produce tagged fragments of genomic DNA; (b) sequencing a portion of the tagged fragments to produce a plurality of sequence reads; and (c) making an epigenetic map of a region of the genome of the cells by mapping information obtained from the sequence reads to the region. A kit for performing the method is also provided.

There is described herein, a method of capturing and analyzing cell-free methylated DNA in a sample. The method involves subjecting the sample to library preparation to permit subsequent sequencing of the cell-free methylated DNA. A predetermined amount of control synthetic DNA fragments are added to the sample. The control synthetic DNA fragments each have a known nucleic acid sequence that does not align to a target genome sequence, and at least some of the control synthetic DNA fragments are methylated. The sample is denatured, and cell-free methylated DNA and the control synthetic DNA fragments are captured using a binder selective for methylated polynucleotides. The captured DNA is amplified and sequenced.

There is described herein, a method of capturing and analyzing cell-free methylated DNA in a sample. The method involves subjecting the sample to library preparation to permit subsequent sequencing of the cell-free methylated DNA. A predetermined amount of control synthetic DNA fragments are added to the sample. The control synthetic DNA fragments each have a known nucleic acid sequence that does not align to a target genome sequence, and at least some of the control synthetic DNA fragments are methylated. The sample is denatured, and cell-free methylated DNA and the control synthetic DNA fragments are captured using a binder selective for methylated polynucleotides. The captured DNA is amplified and sequenced.