Disclosed herein include systems, methods, compositions, and kits for determining expressions of proteins and genes simultaneously, and for sample indexing. The method can comprise extending a cellular component-binding reagent specific oligonucleotide hybridized to an oligonucleotide barcode to generate an extended cellular component-binding reagent specific oligonucleotide. The extended cellular component-binding reagent specific oligonucleotide can be separated from the oligonucleotide barcodes. The separated extended cellular component-binding reagent specific oligonucleotide can be amplified separately from barcoded cDNA.

Disclosed herein include systems, methods, compositions, and kits for determining expressions of proteins and genes simultaneously, and for sample indexing. The method can comprise extending a cellular component-binding reagent specific oligonucleotide hybridized to an oligonucleotide barcode to generate an extended cellular component-binding reagent specific oligonucleotide. The extended cellular component-binding reagent specific oligonucleotide can be separated from the oligonucleotide barcodes. The separated extended cellular component-binding reagent specific oligonucleotide can be amplified separately from barcoded cDNA.

A method for preparing a homopolymer recalibration panel includes: extracting, from a set of amplicons used in sequencing-by-synthesis, a set of candidate amplicons satisfying a first set of criteria, wherein the first set of criteria includes amplicons known to belong to high-confidence regions of a reference genome with no variants; and selecting, from the set of candidate amplicons, a reduced set of amplicons satisfying a second set of criteria, wherein the second set of criteria includes amplicons that together comprise at least a minimal threshold number of homopolymers of each homopolymer length between a predetermined minimal homopolymer length and a predetermined maximal homopolymer length for one or more of homopolymer types A, T, C, and G.

A method for preparing a homopolymer recalibration panel includes: extracting, from a set of amplicons used in sequencing-by-synthesis, a set of candidate amplicons satisfying a first set of criteria, wherein the first set of criteria includes amplicons known to belong to high-confidence regions of a reference genome with no variants; and selecting, from the set of candidate amplicons, a reduced set of amplicons satisfying a second set of criteria, wherein the second set of criteria includes amplicons that together comprise at least a minimal threshold number of homopolymers of each homopolymer length between a predetermined minimal homopolymer length and a predetermined maximal homopolymer length for one or more of homopolymer types A, T, C, and G.

The present invention provides a method for amplifying a sequence adjacent to a specific sequence, comprising the steps of: annealing a first forward primer to the specific sequence to synthesize a complementary strand; sequentially polymerically adding a first deoxynucleotide and a second deoxynucleotide to a 3′-end of the complementary strand; annealing a first reverse primer to a binding site between the 3′-end of the complementary strand and a polydeoxynucleotide strand composed of the first deoxynucleotide to synthesize a double-stranded DNA; performing a PCR with the double-stranded DNA as a template by using a second forward primer complementary to the specific sequence and a first reverse primer; and further performing a PCR by using a third forward primer complementary to the specific sequence and a second reverse primer.

The present invention provides a method for amplifying a sequence adjacent to a specific sequence, comprising the steps of: annealing a first forward primer to the specific sequence to synthesize a complementary strand; sequentially polymerically adding a first deoxynucleotide and a second deoxynucleotide to a 3′-end of the complementary strand; annealing a first reverse primer to a binding site between the 3′-end of the complementary strand and a polydeoxynucleotide strand composed of the first deoxynucleotide to synthesize a double-stranded DNA; performing a PCR with the double-stranded DNA as a template by using a second forward primer complementary to the specific sequence and a first reverse primer; and further performing a PCR by using a third forward primer complementary to the specific sequence and a second reverse primer.

We provide a method to efficiently analyze coding RNA and non-coding RNA at single cell level in the present disclosure. A tag sequence is first added to the 3′ of RNA molecules in a single cell, and the tag sequence is subsequently used to capture said RNA and prime reverse transcription of the RNA to cDNA. The resulting cDNA can be amplified and analyzed. The tag sequence can be combined with a cell barcode sequence to decode the identity of single cells, so that a plurality of single cells can be analyzed in parallel.

We provide a method to efficiently analyze coding RNA and non-coding RNA at single cell level in the present disclosure. A tag sequence is first added to the 3′ of RNA molecules in a single cell, and the tag sequence is subsequently used to capture said RNA and prime reverse transcription of the RNA to cDNA. The resulting cDNA can be amplified and analyzed. The tag sequence can be combined with a cell barcode sequence to decode the identity of single cells, so that a plurality of single cells can be analyzed in parallel.

Provided include methods, compositions and kits for single cell target sequencing, including but not limited to, high-throughput detection of nucleic acid sequences of single cell T cell receptor, high-throughput detection of expressed viral sequences in host cells, detection of cancer druggable mutations (e.g., lung cancer druggable mutations) in single cells, and simultaneous detection of targeted regions and whole transcriptome in single cells.

Provided include methods, compositions and kits for single cell target sequencing, including but not limited to, high-throughput detection of nucleic acid sequences of single cell T cell receptor, high-throughput detection of expressed viral sequences in host cells, detection of cancer druggable mutations (e.g., lung cancer druggable mutations) in single cells, and simultaneous detection of targeted regions and whole transcriptome in single cells.