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JP-7856650-B2 - Whole-cell transcriptome analysis in single cells

JP7856650B2JP 7856650 B2JP7856650 B2JP 7856650B2JP-7856650-B2

Inventors

  • オズトゥルク，セディーデ
  • ラニク，マーティン
  • ルベルト，フロリアン

Assignees

  • エフ． ホフマン－ラ ロシュ アーゲー

Dates

Publication Date
20260511
Application Date
20211201
Priority Date
20201203

Claims (14)

  1. A method for detecting multiple target nucleic acids in multiple cells, wherein the method is a. Contacting multiple target nucleic acids in each of the multiple cells in the sample with oligonucleotide primers in the presence of nucleic acid polymerase having terminal transferase activity and template switch activity . b. For each of the multiple target nucleic acids in the multiple cells, the oligonucleotide primer is extended with the nucleic acid polymerase to form a copy chain having one or more non-template nucleotides at the 3' end , where the one or more non-template nucleotides at the 3' end of the copy chain can function as anchoring sites for barcode subunits. c . Forming a cell characteristic compound barcode containing an attached barcode subunit for each of the multiple target nucleic acids in each of the multiple cells, wherein the multiple target nucleic acids in different cells of the multiple cells contain different cell characteristic compound barcodes, and the multiple target nucleic acids in any one individual cell of the multiple cells contain the same cell characteristic compound barcode, and the formation of the cell characteristic compound barcode in each of the multiple cells in the sample comprises repeatedly performing split pool synthesis rounds until each of the multiple target nucleic acids in each of the different cells of the multiple cells contains the different cell characteristic compound barcodes, and each split pool synthesis round is as follows: i. Randomly distributing the plurality of cells in the sample into a plurality of reaction volume sets , wherein each of the plurality of reaction volumes comprises a unique barcode subunit and the nucleic acid polymerase, and each of the unique barcode subunits comprises a barcode and a nucleic acid sequence complementary to the one or more non-template nucleic acids at the 3' end of the copy strand . ii. The 3' end of the copy strand is extended with the nucleic acid polymerase, wherein the extension of the 3' end of the copy strand copies the unique barcode subunit and adds a non-template nucleic acid strand to the 3' end of the copy strand. i . Combining the above multiple reaction volumes into a pool , Includes, The sequence of the extended copy strands obtained above of the plurality of target nucleic acids in each of the plurality of cells containing the formed cell characteristic compound barcode is determined, and thereby the plurality of target nucleic acids in the plurality of cells is detected . Methods that include...
  2. The method according to claim 1, further comprising the step of amplifying the extended copy strand containing the cell-characterizing compound barcode before sequencing.
  3. The method according to claim 1, wherein the oligonucleotide primer includes a barcode.
  4. The method according to claim 2, wherein the oligonucleotide primer includes a universal amplification primer binding site.
  5. The method according to claim 2, wherein the last barcode subunit to be copied includes a universal amplification primer binding site.
  6. The method according to claim 1, wherein the barcode subunit comprises one or more modified nucleotides that reduce the stability of subunit hybridization.
  7. The method according to claim 6, wherein the modified nucleotide is an isonucleotide located at the 5' end of the barcode subunit.
  8. The method according to claim 1, wherein the target nucleic acid is RNA.
  9. The method according to claim 1, wherein the oligonucleotide primer comprises a target-specific sequence.
  10. The method according to claim 1, wherein the oligonucleotide primer includes a barcode.
  11. The method according to claim 1, wherein the target nucleic acid is DNA.
  12. The method according to claim 1, wherein the nucleic acid polymerase is a reverse transcriptase.
  13. The method according to claim 1, wherein the one or more non-template nucleotides are deoxycytosine.
  14. The method according to claim 1, wherein the barcode subunit includes a portion complementary to one or more non-template nucleotides.

Description

This invention generally relates to single-cell analysis. More specifically, it relates to detecting multiple nucleic acid targets within individual cells without the need to isolate or separate individual cells. Single-cell analysis is becoming increasingly important in understanding biology and disease. Gene expression studies, including whole-transcriptome analysis, reveal variations in gene expression between cells. Previous methods required the physical isolation of each cell under a microscope (see Tang et al. (2009) mRNA-Seq, whole-transcriptome analysis of a single cell, Nature Methods 6:377). U.S. Patent No. 10,392,662 describes a technical solution involving encapsulating individual cells in droplets and treating a water-oil emulsion so that multiple individual reactions can occur simultaneously. An alternative, simpler, and more sophisticated method is described in U.S. Patent No. 10,144,950. This novel approach, called "Quantum Barcoding" or "QBC," does not require the separation of individual cells in droplets or by any other means. Instead, QBC involves passing multiple cells through a series of split-pool rounds, as a result of assembling a unique compound barcode on each cell. In each round, the solution containing multiple cells is divided into several reaction volumes, each containing a barcode sequence. After the barcodes are bound to the targets of each cell, the reaction volumes are pooled and divided again, resulting in each cell receiving a barcode in the next round. Each cell follows a unique path through a series of barcode-containing wells to acquire a unique cell-associated compound barcode. The QBC method allows these unique cell-associated compound barcodes to attach to any target of interest within the cell, i.e., DNA, RNA, proteins, or other cellular targets. The assembly of compound barcodes on nucleic acids typically involves a ligation step; see Rosenberg, et al. (2018) Single cell profiling of the developing mouse brain and spinal code by split-pool barcoding, Science, 360:176. Ligation requires additional reagents and reaction conditions and is less efficient than primer extension. Low efficiency is unacceptable in applications where nucleic acid targets are rare, such as whole transcriptome analysis. There is an unmet need for a more robust method for barcoding and detecting nucleic acid targets in individual cells. This invention includes a method for assembling compound barcodes onto nucleic acid targets in individual cells. Each of multiple targets in an individual cell is to be labeled with the same cell-specific barcode. The barcodes are assembled from barcode subunits via a split-pooling process. The barcode subunits are copied and the compound barcodes are assembled using the unique properties of reverse transcriptase. In one embodiment, the present invention is a method for detecting multiple target nucleic acids in multiple cells, comprising: contacting multiple cells in a sample with oligonucleotide primers for each target nucleic acid in the presence of nucleic acid polymerase having terminal transferase activity; extending the oligonucleotide primers to form copy strands having one or more non-template nucleotides at the 3' end of the copy strand; and distributing the sample into a first reaction volume set, where each volume contains the first barcode subunit and the 3' end of the copy strand to copy the first barcode subunit. The method comprises forming a cell-characterizing compound barcode on a copy strand by sequentially combining a first reaction volume set into a pool and distributing a nucleic acid polymerase for extending the end, each volume comprising a second barcode subunit and a nucleic acid polymerase for further extending the 3' end of the copy strand to copy the second barcode subunit, through a split-pool process comprising one or more rounds; determining the sequence of the extended copy strand containing the cell-characterizing compound barcode, thereby detecting multiple target nucleic acids in multiple cells. In some embodiments, the method further includes a step of amplifying the extended copy strand containing the cell-characterizing compound barcode before sequencing. In some embodiments, the oligonucleotide primer includes a barcode and/or a universal amplification primer binding site. In some embodiments, the copied barcode subunit includes a universal amplification primer binding site. In some embodiments, the target nucleic acid is DNA. In some embodiments, the target nucleic acid is RNA, such as messenger RNA, and the oligonucleotide primer includes a poly-dT sequence or a target-specific sequence. In some embodiments, the oligonucleotide primer includes a barcode. In some embodiments, the nucleic acid polymerase is a reverse transcriptase. RT may have reduced RNaseH activity. In some embodiments, the non-template nucleotide is deoxycytosine. In some embodiments, the barcode subunit includes a moiety complementary to one or mor