An example of a kit includes a flow cell, a primer fluid, and a cleaving fluid. The flow cell includes at least one surface functionalized with a polymeric hydrogel including azide functional groups or amine functional groups. The primer fluid includes a plurality of alkyne-containing primers, each alkyne-containing primer having an amino cleavable group attaching a primer sequence of the alkyne-containing primer to an alkyne-containing moiety of the alkyne-containing primer. The cleaving fluid includes a substance that is reactive with the amino cleavable group.

An example of a kit includes a flow cell, a primer fluid, and a cleaving fluid. The flow cell includes at least one surface functionalized with a polymeric hydrogel including azide functional groups or amine functional groups. The primer fluid includes a plurality of alkyne-containing primers, each alkyne-containing primer having an amino cleavable group attaching a primer sequence of the alkyne-containing primer to an alkyne-containing moiety of the alkyne-containing primer. The cleaving fluid includes a substance that is reactive with the amino cleavable group.

The present invention provides methods and compositions for carrying out nucleic acid sequencing, particularly paired-end sequencing. The methods use concatemeric sequencing templates that can be produced by rolling circle amplification of asymmetric circular nucleic acids having a central double-stranded region comprising a target nucleic acid sequence that is connected at each end to form a circular construct.

The present invention provides methods and compositions for carrying out nucleic acid sequencing, particularly paired-end sequencing. The methods use concatemeric sequencing templates that can be produced by rolling circle amplification of asymmetric circular nucleic acids having a central double-stranded region comprising a target nucleic acid sequence that is connected at each end to form a circular construct.

The disclosed invention is related to a universal strand-specific protocol for the sequencing preparation of all classes of RNA. The protocol allows for sequencing for dozens to more than thousands of samples simultaneously. Specifically, the disclosed invention is a method for parallel sequencing target RNA from samples from multiple sources while maintaining source identification. The method includes providing samples of RNA comprising target RNA from two or more sources; labeling, at the 3′ end, the RNA from the two or more sources with a first nucleic acid adaptor that comprises a nucleic acid sequence that differentiates between the RNA from the two or more sources; reverse transcribing the two or more sources to create a single stranded DNA comprising the nucleic acid sequence that differentiates between the RNA from the two or more sources; amplifying the single stranded DNA to create DNA amplification products that comprise the nucleic acid sequence that differentiates between the RNA from the two or more sources; sequencing the DNA amplification products thereby parallel sequencing target RNA from samples from multiple sources while maintaining source identification.

This disclosure provides methods and compositions for sample processing, particularly for sequencing applications. Included within this disclosure are bead compositions, such as diverse libraries of beads attached to large numbers of oligonucleotides containing barcodes. Often, the beads provides herein are degradable. For example, they may contain disulfide bonds that are susceptible to reducing agents. The methods provided herein include methods of making libraries of barcoded beads as well as methods of combining the beads with a sample, such as by using a microfluidic device.

This disclosure provides methods and compositions for sample processing, particularly for sequencing applications. Included within this disclosure are bead compositions, such as diverse libraries of beads attached to large numbers of oligonucleotides containing barcodes. Often, the beads provides herein are degradable. For example, they may contain disulfide bonds that are susceptible to reducing agents. The methods provided herein include methods of making libraries of barcoded beads as well as methods of combining the beads with a sample, such as by using a microfluidic device.

The present disclosure provides methods and kits for performing high multiplex PCR using molecular barcodes. The methods disclosed herein separately extend a set of primers (BC primers) that each comprise a target-specific sequence, a molecular barcode and a universal sequence, and amplify the resulting extension products using another set of primers (LA primers) that each comprise another target-specific sequence and a universal sequence. The methods may further comprise amplification using universal primers (preferably comprising an adapter).

The present disclosure provides methods and kits for performing high multiplex PCR using molecular barcodes. The methods disclosed herein separately extend a set of primers (BC primers) that each comprise a target-specific sequence, a molecular barcode and a universal sequence, and amplify the resulting extension products using another set of primers (LA primers) that each comprise another target-specific sequence and a universal sequence. The methods may further comprise amplification using universal primers (preferably comprising an adapter).

In various aspects, the present disclosure provides methods, compositions, reactions mixtures, kits, and systems for sequencing both RNA and DNA from a single source sample. In some embodiments, RNA is treated so as to differentiate RNA sequences from DNA sequences derived from the same sample. In some embodiments, the RNA and DNA are cell-free polynucleotides.