Provided herein are methods that enable parallel evaluation of multiple functional nucleic acids in individual cells or subpopulations of cells, in the context of incubation with other types of single cells. The key insight is concurrent measurement of polynucleic acids derived from small populations of at least two different cell types, such that function in one cell type is linked to the clonal identity of another cell. These methods simultaneously process thousands, millions, or more single cells or small populations of cells. The method integrates molecular, algorithmic, and engineering approaches. This invention has broad and useful application in a number of biological and medical fields, including immunology and drug discovery.

This invention relates to methods, compositions and kits for extending a polynucleotide and for preparing sequencing library of polynucleotides involving generating modified target polynucleotide on an adaptor template oligonucleotide and tagging one or two strands of a target sequence. The sequencing library is suitable for massive parallel sequencing and comprises a plurality of double-stranded nucleic acid molecules.

This invention relates to methods, compositions and kits for extending a polynucleotide and for preparing sequencing library of polynucleotides involving generating modified target polynucleotide on an adaptor template oligonucleotide and tagging one or two strands of a target sequence. The sequencing library is suitable for massive parallel sequencing and comprises a plurality of double-stranded nucleic acid molecules.

Described herein is a method of sequencing, comprising: splitting an asymmetrically tagged library into a plurality of subsamples, tagging the adaptor-ligated DNA in the sub-samples with sequence tags that identify the subsamples, optionally pooling the sub-samples, sequencing polynucleotides from each of the tagged sub-samples, or copies of the same, to produce sequence reads each comprising: i. a sub-sample identifier sequence and ii. the sequence of at least part of a fragment in the sample, wherein some of the sequence reads are derived from the top strand of a fragment in the sample and some of the sequence reads of are derived from the bottom strand of the same fragment.

Described herein is a method of sequencing, comprising: splitting an asymmetrically tagged library into a plurality of subsamples, tagging the adaptor-ligated DNA in the sub-samples with sequence tags that identify the subsamples, optionally pooling the sub-samples, sequencing polynucleotides from each of the tagged sub-samples, or copies of the same, to produce sequence reads each comprising: i. a sub-sample identifier sequence and ii. the sequence of at least part of a fragment in the sample, wherein some of the sequence reads are derived from the top strand of a fragment in the sample and some of the sequence reads of are derived from the bottom strand of the same fragment.

The present invention relates to novel methods for generating a population of double-stranded polynucleotide molecules from a sample containing at least one polynucleotide.

The present invention relates to novel methods for generating a population of double-stranded polynucleotide molecules from a sample containing at least one polynucleotide.

One or more embodiments of the present invention are intended to dissolve the problem of lowering in reaction efficiency of the nucleic acid amplification reaction using a primer with a polynucleotide tag to prepare an amplified product of a target nucleic acid that can be detected on a solid-phase support. One or more other embodiments of the present invention relate to a set of primers comprising: the first primer comprising the first polynucleotide comprising, at the 3′ terminus, polynucleotide A capable of hybridizing to a complementary strand of partial polynucleotide A′ at the 5′ terminus of the target nucleic acid and the first polynucleotide tag, which is a polynucleotide independent of the nucleic acid amplification reaction; the second primer comprising the second polynucleotide comprising, at the 3′ terminus, polynucleotide B capable of hybridizing to partial polynucleotide B′ at the 3′ terminus of the target nucleic acid; and the third primer comprising the third polynucleotide hybridizing to the complementary strand of the target nucleic acid competitively with the polynucleotide A of the first primer but comprising no polynucleotide independent of the nucleic acid amplification reaction.

One or more embodiments of the present invention are intended to dissolve the problem of lowering in reaction efficiency of the nucleic acid amplification reaction using a primer with a polynucleotide tag to prepare an amplified product of a target nucleic acid that can be detected on a solid-phase support. One or more other embodiments of the present invention relate to a set of primers comprising: the first primer comprising the first polynucleotide comprising, at the 3′ terminus, polynucleotide A capable of hybridizing to a complementary strand of partial polynucleotide A′ at the 5′ terminus of the target nucleic acid and the first polynucleotide tag, which is a polynucleotide independent of the nucleic acid amplification reaction; the second primer comprising the second polynucleotide comprising, at the 3′ terminus, polynucleotide B capable of hybridizing to partial polynucleotide B′ at the 3′ terminus of the target nucleic acid; and the third primer comprising the third polynucleotide hybridizing to the complementary strand of the target nucleic acid competitively with the polynucleotide A of the first primer but comprising no polynucleotide independent of the nucleic acid amplification reaction.

A method for quantitating a plurality of nucleic acid molecules is provided. The method includes contacting a plurality of detectably-labeled probes and a plurality of extension primers with the plurality of nucleic acid molecules, each detectably-labeled probe including a first labeled nucleic acid domain having a first label; producing an extension product of each of the plurality of target nucleic acid fragments by extending a respective one of the plurality of extension primers with a polymerase; hydrolyzing each of the plurality of detectably-labeled probes hybridized to the respective one of the target nucleic acid fragments during extending the respective one of the plurality of extension primers with the polymerase; detecting a first signal produced as a result of hydrolyzing the plurality of detectably-labeled probes; and calculating a number of the plurality of target nucleic acid fragments based on signals detected upon a single cycle of extension reactions.