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US 12,378,596 B2Granted
Whole transcriptome analysis in single cells
AssigneeRoche Sequencing Solutions, Inc.
InventorsMartin Ranik, Florian Rubelt, Sedide Ozturk
Life of this patent
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Abstract
The invention is a method of single cell transcriptome analysis. The method comprises detecting multiple transcripts in each individual cell of the plurality of cells by barcoding the transcripts with a cell-specific compound barcode formed using a DNA polymerase and a terminal transferase, optionally in a single enzyme such as a reverse transcriptase.
Claims20
A method of detecting a plurality of target nucleic acids in a plurality of cells, the method comprising
- (a) contacting the plurality of cells in a sample with an oligonucleotide primer for each target nucleic acid in the presence of a nucleic acid polymerase having a terminal transferase activity and a template switch activity, and extending the oligonucleotide primer to form a copy strand having one or more non-templated nucleotides at the 3′-end of the copy strand capable of functioning as an anchoring site for a barcode subunit;
- (b) for each target nucleic acid in each cell of the plurality of cells, forming an extended copy strand comprising a cell-characteristic compound barcode on the copy strand by sequentially attaching a series of barcode subunits to the 3′-end of a copy strand, wherein each barcode subunit comprises a sequence complementary to the one or more non-templated nucleotides at the 3′ end of the copy strand and a barcode, wherein each barcode subunit includes (i) one or more 5′-nucleotide isomers, and (ii) one or more Tm-modified nucleotides, and wherein the attaching of the barcode subunits is performed by a split-pool process comprising one or more rounds of:
- (i) distributing the sample into a first set of reaction volumes, each volume containing a first barcode subunit and a nucleic acid polymerase to extend the 3′-end of the copy strand to copy the first barcode subunit;
- (ii) combining the first set of reaction volumes into a pool;
- and
- (iii) distributing the pool into a second set of reaction volumes, each volume containing a second barcode subunit and a nucleic acid polymerase to further extend the 3′-end of the copy strand to copy the second barcode subunit;
- and
- (c) determining the sequence of the extended copy strands including the cell-characteristic compound barcodes, thereby detecting the plurality of target nucleic acids in the plurality of cells;
- wherein the method does not comprise a separation or encapsulation of the plurality of cells prior to or during the split-pool process.
6 more elements
A method of detecting a plurality of target nucleic acids in a plurality of cells, the method comprising
- (a) contacting the plurality of cells in a sample with an oligonucleotide primer for each target nucleic acid in the presence of a nucleic acid polymerase having a terminal transferase activity and a template switch activity, wherein the oligonucleotide primer includes a motif-specific sequence or a random sequence, and extending the oligonucleotide primer to form a copy strand having one or more non-templated nucleotides at the 3′-end of the copy strand capable of functioning as an anchoring site for a barcode subunit;
- (b) for each target nucleic acid in each cell of the plurality of cells, forming an extended copy strand comprising a cell-characteristic compound barcode on the copy strand by sequentially attaching a series of barcode subunits to the 3′-end of a copy strand, wherein each barcode subunit comprises a sequence complementary to the one or more non-templated nucleotides at the 3′ end of the copy strand and a barcode, wherein each barcode subunit includes one or more 5′-nucleotide isomers, and the attaching step is performed by a split-pool process comprising one or more rounds of:
- (i) distributing the sample into a first set of reaction volumes, each volume containing a first barcode subunit and a nucleic acid polymerase to extend the 3′-end of the copy strand to copy the first barcode subunit;
- (ii) combining the first set of reaction volumes into a pool;
- and
- (iii) distributing the pool into a second set of reaction volumes, each volume containing a second barcode subunit and a nucleic acid polymerase to further extend the 3′-end of the copy strand to copy the second barcode subunit;
- and
- (c) determining the sequence of the extended copy strands including the cell-characteristic compound barcodes, thereby detecting the plurality of target nucleic acids in the plurality of cells;
- wherein the method does not comprise a separation or encapsulation of the plurality of cells prior to or during the split-pool process.
6 more elements
A method of detecting a plurality of target nucleic acids in a plurality of cells, the method comprising
- (a) contacting the plurality of cells in a sample with an oligonucleotide primer for each target nucleic acid in the presence of a nucleic acid polymerase having a terminal transferase activity and a template switch activity, wherein the oligonucleotide primer includes a universal capture sequence, and extending the oligonucleotide primer to form a copy strand having one or more non-templated nucleotides at the 3′-end of the copy strand capable of functioning as an anchoring site for a barcode subunit;
- (b) for each target nucleic acid in each cell of the plurality of cells, forming an extended copy strand comprising a cell-characteristic compound barcode on the copy strand by sequentially attaching a series of barcode subunits to the 3′-end of a copy strand, wherein each barcode subunit comprises a sequence complementary to the one or more non-templated nucleotides at the 3′ end of the copy strand and a barcode, wherein each barcode subunit includes one or more 5′-nucleotide isomers, and the attaching step is performed by a split-pool process comprising one or more rounds of:
- (i) distributing the sample into a first set of reaction volumes, each volume containing a first barcode subunit and a nucleic acid polymerase to extend the 3′-end of the copy strand to copy the first barcode subunit;
- (ii) combining the first set of reaction volumes into a pool;
- and
- (iii) distributing the pool into a second set of reaction volumes, each volume containing a second barcode subunit and a nucleic acid polymerase to further extend the 3′-end of the copy strand to copy the second barcode subunit;
- and
- (c) determining the sequence of the extended copy strands including the cell-characteristic compound barcodes, thereby detecting the plurality of target nucleic acids in the plurality of cells;
- wherein the method does not comprise a separation or encapsulation of the plurality of cells prior to or during the split-pool process.
6 more elements
Dependencies are inferred from the claim text.