Methods and reagents for preparing nucleic acid samples for sequencing are provided. The samples include formalin-fixed paraffin-embedded (FFPE) samples. The methods comprise contacting a nucleic acid sample with a multimeric barcoding reagent comprising barcode regions linked together and appending barcode sequences to nucleic acid sequences of a target nucleic acid molecule. Methods are also provided that additionally use in-vitro transposition, coupling sequences and/or primer-extension to append barcode sequences to nucleic acid sequences of a target nucleic acid molecule.

Provided are high throughput methods for physically linking cDNA molecules derived from mRNA molecules expressed by the same cell, and libraries of linked cDNA molecules produced by the methods. The methods comprise reverse transcribing mRNA from a single cell in a first container to produce cDNA molecules, and linking the cDNA molecules in a second container. The methods unexpectedly produced libraries of cDNA molecules with an increase in the number of molecules that are correctly linked to other molecules derived from the same cell.

Provided are high throughput methods for physically linking cDNA molecules derived from mRNA molecules expressed by the same cell, and libraries of linked cDNA molecules produced by the methods. The methods comprise reverse transcribing mRNA from a single cell in a first container to produce cDNA molecules, and linking the cDNA molecules in a second container. The methods unexpectedly produced libraries of cDNA molecules with an increase in the number of molecules that are correctly linked to other molecules derived from the same cell.

Provided herein are methods of releasing an extended capture probe from a substrate and uses of the same.

Provided herein are methods of releasing an extended capture probe from a substrate and uses of the same.

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 invention relates to methods for pairwise sequencing of a polynucleotide template which result in the sequential determination of nucleotide sequence in two distinct and separate regions of the polynucleotide template.

The invention relates to methods for pairwise sequencing of a polynucleotide template which result in the sequential determination of nucleotide sequence in two distinct and separate regions of the polynucleotide template.

The present disclosure provides methods and compositions for molecular tagging of complex populations of nucleic acid molecules. The disclosure provides methods and compositions to obtain phase information of tagged nucleic acid molecules from high-throughput nucleic acid sequencing data.