The present invention provides methods, compositions and kits for assembling an enzyme-deoxyribonucleic acid (DNA) complex for use in preparing a double stranded DNA molecule comprising one or more loci of interest for determining the methylation status of the one or more loci of interest therein.

Provided herein are methods, compositions and kits for the generation of bisulfite-converted next generation sequencing (NGS) libraries. The methods, compositions and kits provided herein can be useful, for example, for the production of libraries from genomic DNA that allow for determination of the methylation status across the genome, i.e. the methylome. The methods, compositions and kits provided herein can also be utilized to query methylation status at a particular genomic locus or loci. Moreover, the methods provided herein can be employed for high-throughput sequencing of bisulfite-converted DNA while maintaining the directional (strandedness) information of the original nucleic acid sample.

Provided herein are methods, compositions and kits for the generation of bisulfite-converted next generation sequencing (NGS) libraries. The methods, compositions and kits provided herein can be useful, for example, for the production of libraries from genomic DNA that allow for determination of the methylation status across the genome, i.e. the methylome. The methods, compositions and kits provided herein can also be utilized to query methylation status at a particular genomic locus or loci. Moreover, the methods provided herein can be employed for high-throughput sequencing of bisulfite-converted DNA while maintaining the directional (strandedness) information of the original nucleic acid sample.

DNA is sequenced by: (a) combining dsDNA fragments with Y-adapters and hairpin adapters comprising an affinity-label under conditions wherein the adapters ligate to fragments forming a mixture of fragment inserts flanked by two Y-adapters, a Y-adapter and a hairpin adapter, and two hairpin adapters; and (b) sequencing the selected fragment inserts with sequencing primers selecting for the Y-adapters.

DNA is sequenced by: (a) combining dsDNA fragments with Y-adapters and hairpin adapters comprising an affinity-label under conditions wherein the adapters ligate to fragments forming a mixture of fragment inserts flanked by two Y-adapters, a Y-adapter and a hairpin adapter, and two hairpin adapters; and (b) sequencing the selected fragment inserts with sequencing primers selecting for the Y-adapters.

Methods of repairing a partially double-stranded DNA fragment are provided. In some embodiments, the methods comprise (a) contacting the partially double-stranded DNA fragment with one or more primers of a primer population, wherein the partially double-stranded DNA fragment comprises a 3′ overhang and the primer population comprises a random target-hybridizing sequence; (b) extending one or more primers of the primer population along the DNA fragment using a DNA polymerase, thereby producing one or more extended primers annealed to the DNA fragment; and (c) ligating the 3′ end of one or more extended primers to the 5′ end of an extended primer or a strand of the partially double-stranded DNA fragment, thereby providing a repaired DNA fragment.

Methods of repairing a partially double-stranded DNA fragment are provided. In some embodiments, the methods comprise (a) contacting the partially double-stranded DNA fragment with one or more primers of a primer population, wherein the partially double-stranded DNA fragment comprises a 3′ overhang and the primer population comprises a random target-hybridizing sequence; (b) extending one or more primers of the primer population along the DNA fragment using a DNA polymerase, thereby producing one or more extended primers annealed to the DNA fragment; and (c) ligating the 3′ end of one or more extended primers to the 5′ end of an extended primer or a strand of the partially double-stranded DNA fragment, thereby providing a repaired DNA fragment.

The present disclosure provides methods, systems and compositions for detecting nucleic acid sequences in a biological sample having a three-dimensional matrix. The present disclosure also provides methods, systems and compositions for processing a biological sample for use in nucleic acid sequence detection

The present disclosure provides methods, systems and compositions for detecting nucleic acid sequences in a biological sample having a three-dimensional matrix. The present disclosure also provides methods, systems and compositions for processing a biological sample for use in nucleic acid sequence detection

A method for analyzing target nucleic acid from a cell, including: 1) providing a discrete partition: target nucleic acid derived from a single cell and added with an oligonucleotide adaptor sequence, and a solid support with at least one oligonucleotide tag attached, wherein each oligonucleotide tag includes a first and second strand, the first strand includes a barcode sequence and a hybridization sequence located at the 3′-end of the barcode sequence, the second strand includes a first portion, complementary to the hybridization sequence of the first strand, and a second portion, complementary to the oligonucleotide adaptor sequence attached to the target nucleic acid, and the first and second strand form a partial double-strand, or the second strand and target nucleic acid attached form a partial double-strand; and (2) in the discrete partition, the oligonucleotide tag is linked to the target nucleic acid attached, thereby producing barcoded target nucleic acid.