| CPC C12Q 1/6874 (2013.01) | 17 Claims |
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1. A method of sequencing a nucleic acid sequence, comprising the steps of:
providing a sample from a single cell, comprising a native single-stranded nucleic acid sequence or native double-stranded nucleic acid sequence in cell-free form,
adding at least 5 consecutive nucleotides to the 3′-terminus of the native single-stranded sequence, or a fragment thereof, in cell-free form, or to a strand from the native double-stranded nucleic acid sequence, or a fragment thereof, in cell-free form,
hybridizing a priming oligonucleotide sequence complementary to the added nucleotide sequence and synthesizing a cDNA sequence with a template dependent DNA polymerase to obtain a double-stranded nucleic acid sequence,
hybridizing to a strand of the generated double-stranded nucleic acid sequence, a template switching oligonucleotide (TSO) comprising a template switching motif sequence, and a random sequence made of at least 6 bases varying among molecules of the TSO, wherein the random sequence is linked to the template switching motif sequence, and wherein the template switching motif sequence hybridizes to overhanging and untemplated C nucleotides added by the template dependent DNA polymerase,
extending the 3′ end of the cDNA sequence to synthesize an extended double-stranded nucleic acid sequence, wherein one strand of the extended double-stranded nucleic acid sequence comprises the priming oligonucleotide and a cDNA sequence that is complementary to the native single-stranded nucleic acid sequence or the fragment thereof, or the strand from the native double-stranded nucleic acid sequence or the fragment thereof, and to the template switching oligonucleotide sequence,
performing a genetic amplification, and
performing a base sequencing.
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