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.

Methods, compositions, and systems are provided for characterization of modified nucleic acids. In certain preferred embodiments, single molecule sequencing methods are provided for identification of modified nucleotides within nucleic acid sequences. Modifications detectable by the methods provided herein include chemically modified bases, enzymatically modified bases, abasic sites, non-natural bases, secondary structures, and agents bound to a template nucleic acid.

Methods, compositions, and systems are provided for characterization of modified nucleic acids. In certain preferred embodiments, single molecule sequencing methods are provided for identification of modified nucleotides within nucleic acid sequences. Modifications detectable by the methods provided herein include chemically modified bases, enzymatically modified bases, abasic sites, non-natural bases, secondary structures, and agents bound to a template nucleic acid.

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.

Systems and methods for using determination of base modification in analyzing nucleic acid molecules and acquiring data for analysis of nucleic acid molecules are described herein. Base modifications may include methylations. Methods to determine base modifications may include using features derived from sequencing. These features may include the pulse width of an optical signal from sequencing bases, the interpulse duration of bases, and the identity of the bases. Machine learning models can be trained to detect the base modifications using these features. The relative modification or methylation levels between haplotypes may indicate a disorder. Modification or methylation statuses may also be used to detect chimeric molecules.

Systems and methods for using determination of base modification in analyzing nucleic acid molecules and acquiring data for analysis of nucleic acid molecules are described herein. Base modifications may include methylations. Methods to determine base modifications may include using features derived from sequencing. These features may include the pulse width of an optical signal from sequencing bases, the interpulse duration of bases, and the identity of the bases. Machine learning models can be trained to detect the base modifications using these features. The relative modification or methylation levels between haplotypes may indicate a disorder. Modification or methylation statuses may also be used to detect chimeric molecules.

Disclosed herein are methods and compositions for detection of one or more specific sequences of polynucleotides in a solution using a nanopore. In some embodiments, methods and compositions for identifying a polynucleotide in a sample or for target sequence detection of a polynucleotide are disclosed herein.

Compositions and methods for carboxymethylation of cytosine containing DNA and applications thereof for direct sequencing of 5mC are disclosed.

Compositions and methods for carboxymethylation of cytosine containing DNA and applications thereof for direct sequencing of 5mC are disclosed.

The present invention relates to the field of pharmacogenomics and in particular to detecting the presence or absence of hypermethylated DNA. The detection of CpG methylation in marker DNA is useful for the diagnosis of cancers and the invention provides improved methods for this purpose. These improved methods allow in particular for a more sensitive detection of methylated marker DNA with high backgrounds of unmethylated marker DNA.