Embodiments of the disclosure encompass highly sensitive and quantitative methods for single-cell sequencing of total RNA. In particular cases, methods utilize annealing of multiple primers to RNA, polytailing of single stranded DNA reverse transcribed therefrom, and utilization of bar codes in primers for amplification of amplicons produced from second strand synthesis.

Provided herein are compositions and methods for generating libraries for high throughput sequencing without using ligation, and methods of using same.

Provided herein are compositions and methods for generating libraries for high throughput sequencing without using ligation, and methods of using same.

Disclosed herein include systems, methods, compositions, and kits for labeling nucleic acid targets in a sample. In some embodiments, nucleic acid targets (e.g., mRNAs) are initially barcoded on the 3′ end and are subsequently barcoded on the 5′ end following a template switching reaction and intermolecular and/or intramolecular hybridization and extension. 5′- and/or 3′-barcoded nucleic acid targets can serve as templates for amplification reactions and/or random priming and extension reactions to generate a sequencing library. The method can comprise generating a full-length sequence of the nucleic acid target by aligning a plurality of sequencing reads. Immune repertoire profiling methods are also provided in some embodiments.

Disclosed herein include systems, methods, compositions, and kits for labeling nucleic acid targets in a sample. In some embodiments, nucleic acid targets (e.g., mRNAs) are initially barcoded on the 3′ end and are subsequently barcoded on the 5′ end following a template switching reaction and intermolecular and/or intramolecular hybridization and extension. 5′- and/or 3′-barcoded nucleic acid targets can serve as templates for amplification reactions and/or random priming and extension reactions to generate a sequencing library. The method can comprise generating a full-length sequence of the nucleic acid target by aligning a plurality of sequencing reads. Immune repertoire profiling methods are also provided in some embodiments.

Provided are methods for determining a location of a target nucleic acid in a biological sample including: disposing the biological sample onto an array including a plurality of capture probes, where a first capture probe includes a first spatial barcode and a capture domain and a second capture probe includes a second spatial barcode and the capture domain. The second capture probe is not covered by the biological sample on the array and is contacted with a solution comprising TdT and one or more dideoxynucleotides, such that a dideoxynucleotide is incorporated into the second capture domain. Target nucleic acids are captured by the first capture probe, and the sequence of the first spatial barcode or a complement thereof and all or a portion of a sequence of the target nucleic acid, or a complement thereof, are used to determine the location of the target nucleic acid in the biological sample.

Provided are methods for determining a location of a target nucleic acid in a biological sample including: disposing the biological sample onto an array including a plurality of capture probes, where a first capture probe includes a first spatial barcode and a capture domain and a second capture probe includes a second spatial barcode and the capture domain. The second capture probe is not covered by the biological sample on the array and is contacted with a solution comprising TdT and one or more dideoxynucleotides, such that a dideoxynucleotide is incorporated into the second capture domain. Target nucleic acids are captured by the first capture probe, and the sequence of the first spatial barcode or a complement thereof and all or a portion of a sequence of the target nucleic acid, or a complement thereof, are used to determine the location of the target nucleic acid in the biological sample.

##STR00001##

The present invention relates to a compound according to Formula (1c) or (1d): wherein, R.sup.1; R.sup.2; R.sup.3 and R.sup.6 are defined herein, and their use in methods of nucleic acid synthesis.

##STR00001##

The present invention relates to a compound according to Formula (1c) or (1d): wherein, R.sup.1; R.sup.2; R.sup.3 and R.sup.6 are defined herein, and their use in methods of nucleic acid synthesis.

Array-based enzymatic oligonucleotide synthesis creates a large number of polynucleotides using an uncontrolled and template independent polymerase such as terminal deoxynucleotidyl transferase (TdT). Spatial control of reaction conditions on the surface of the array allows creation of polynucleotides with a variety of arbitrary sequences. Spatial control may be implemented by removing protecting groups attached to nucleotides only at a selected location on the array or by other techniques such as location-specific regulation of enzymatic activity. The ratio of polynucleotides with protecting groups to unprotected polynucleotides used during a cycle of synthesis is adjusted to control the length of homopolymers created by the polymerase. Digital information may be encoded in the enzymatically synthesized polynucleotides. An encoding scheme for representing digital information in a nucleotide sequence accounts for homopolymers in the polynucleotides by collapsing homopolymer strings in the sequence data to a single nucleotide or to a shorter homopolymer.