Disclosed herein include methods and compositions for nucleic acid synthesis using a terminal deoxynucleotidyl transferase with deoxyribonucleotide trisphosphates each comprising a modified base with a photocleavable carbon chain moiety that enables single incorporations when present.

The invention relates to methods for preparing cDNA samples for RNA sequencing using random priming oligonucleotides comprising a cell barcode (cID), a unique molecular index (UMI), and a random sequence region, and performing a reverse transcription reaction (RT). The invention also relates to cDNA samples prepared by the methods and uses thereof.

The invention relates to methods for preparing cDNA samples for RNA sequencing using random priming oligonucleotides comprising a cell barcode (cID), a unique molecular index (UMI), and a random sequence region, and performing a reverse transcription reaction (RT). The invention also relates to cDNA samples prepared by the methods and uses thereof.

Disclosed herein include methods of generating a single stranded deoxyribonucleic acid (ssDNA) scaffold comprising nucleotides with modified bases using a recombinant terminal deoxynucleotidyl transferase (TdT). The recombinant TdT can comprise an amino acid sequence that is at least 80% identical to a Bos taurus TdT, or a fragment thereof, and for example comprise one or more amino acid substitution mutations at one or more positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His420 in the Bos taurus TdT.

The present disclosure provides a method for analyzing a polyribonucleotide, wherein the polyribonucleotide is an mRNA molecule with a poly(A) tail, said method comprising: (a) obtaining a labeled polyribonucleotide by GI tailing; followed by (b) providing a second molecule comprising a first primer recognition sequence followed by a sequence of C nucleotide residues linked to a sequence of T nucleotide residues; followed by (c) obtaining a complex of said labeled polyribonucleotide and said second molecule; followed by (d) obtaining an extended second molecule by (d1) extending the 3′ end of the second molecule by synthesizing a sequence that is complementary to the labeled polyribonucleotide; followed by (d2) extending the 3′ end of the second molecule by adding at least 1 to 5 C nucleotide residues, followed by a second primer recognition sequence; said method providing information on entire polyribonucleotides, in particular mRNA isoforms and their respective poly(A) tails.

The present disclosure provides a method for analyzing a polyribonucleotide, wherein the polyribonucleotide is an mRNA molecule with a poly(A) tail, said method comprising: (a) obtaining a labeled polyribonucleotide by GI tailing; followed by (b) providing a second molecule comprising a first primer recognition sequence followed by a sequence of C nucleotide residues linked to a sequence of T nucleotide residues; followed by (c) obtaining a complex of said labeled polyribonucleotide and said second molecule; followed by (d) obtaining an extended second molecule by (d1) extending the 3′ end of the second molecule by synthesizing a sequence that is complementary to the labeled polyribonucleotide; followed by (d2) extending the 3′ end of the second molecule by adding at least 1 to 5 C nucleotide residues, followed by a second primer recognition sequence; said method providing information on entire polyribonucleotides, in particular mRNA isoforms and their respective poly(A) tails.

This disclosure describes a technique for performing random access in a pool of polynucleotides by using one unique primer and one homopolymer primer to selectively amplify some but not all of the polynucleotides in the pool. The polynucleotides are synthesized by a template independent polymerase such as terminal deoxynucleotide transferase (TdT) rather than by phosphoramidite synthesis. Enzymatic synthesis efficiently creates homopolymer sequences through unregulated synthesis. Use of one homopolymer primer instead of two unique primers decreases the complexity, time, and cost of synthesizing the polynucleotides. Use of a unique primer provides a sequence that can be varied to uniquely identify multiple different groups of polynucleotides. This enables random access by polymerase chain reaction (PCR) amplification while still benefiting from the efficiency of homopolymer synthesis. The polynucleotides may include payload regions that use a sequence of nucleotides to encode digital data.

This disclosure describes a technique for performing random access in a pool of polynucleotides by using one unique primer and one homopolymer primer to selectively amplify some but not all of the polynucleotides in the pool. The polynucleotides are synthesized by a template independent polymerase such as terminal deoxynucleotide transferase (TdT) rather than by phosphoramidite synthesis. Enzymatic synthesis efficiently creates homopolymer sequences through unregulated synthesis. Use of one homopolymer primer instead of two unique primers decreases the complexity, time, and cost of synthesizing the polynucleotides. Use of a unique primer provides a sequence that can be varied to uniquely identify multiple different groups of polynucleotides. This enables random access by polymerase chain reaction (PCR) amplification while still benefiting from the efficiency of homopolymer synthesis. The polynucleotides may include payload regions that use a sequence of nucleotides to encode digital data.

A universal template strand built with universal base analogs is used as a template for polynucleotide synthesis. The universal template strand can hybridize to any sequence of nucleotides. A new polynucleotide is synthesized by using a polymerase to extend a primer hybridized to the universal template strand. Unlike primer extension in polymerase chain reactions, base pairing with nucleotides in the template strand does not specify the sequence of the new polynucleotide. Instead, the sequence of the new polynucleotide is specified by the order of addition of protected nucleotides. After addition of a single species of protected nucleotide, the blocking group is removed and another protected nucleotide is added. The order of nucleotide addition can be varied to create any sequence. After synthesis, the polynucleotide can be dehybridized from the universal template strand. The universal template strand may then be reused to synthesize a different polynucleotide.

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.