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1. WO2020118200 - PROCÉDÉS DE PRÉPARATION D'ÉCHANTILLONS D'ADNC POUR SÉQUENÇAGE D'ARN, ET ÉCHANTILLONS D'ADNC ET LEURS UTILISATIONS

Note: Texte fondé sur des processus automatiques de reconnaissance optique de caractères. Seule la version PDF a une valeur juridique

[ EN ]

WHAT IS CLAIMED IS:

1. A method of generating a cDNA sample, comprising

adding to an RNA sample, a plurality of random priming oligonucleotides, each random priming oligonucleotide comprising a cell barcode (cID), a unique molecular index (UMI), and a random sequence region of 5 to 8 nucleotides, and

performing a reverse transcription reaction (RT).

2. The method of claim 1, wherein the random priming oligonucleotide further comprises a universal amplification sequence.

3. The method of claim 1 or 2, wherein two or more of the random priming oligonucleotides bind to a single RNA molecule.

4. The method of any one of claims 1-3, further comprising performing an amplification reaction to generate a cDNA sample.

5. The method of any one of claims 1-4, further comprising adding a template switching oligonucleotide (TSO) and performing a template switching reaction (TS) to generate a cDNA sample comprising universal PCR handles on the 5’ and 3’ ends.

6. The method of claim 5, wherein the TS is decoupled from the RT.

7. The method of claim 6, wherein the TS is cap dependent.

8. The method of claim 6, wherein the TS is cap independent.

9. The method of any one of claims 1-4, further comprising adding a homopolymer oligonucleotide tail to a first strand cDNA by adding a terminal transferase (TdT) and performing a primer extension reaction to generate a second strand cDNA comprising universal PCR handles on the 5’ and 3’ ends.

10. The method of claim 9, wherein the homopolymer oligonucleotide tail comprise adenines (A), cytosines (C), guanines (G), or thymines (T).

11. The method of any one of claims 1-10, wherein the random sequence region comprises random nucleotides selected from A, C, G, and T bases (N5-8).

12. The method of any one of claims 1-10, wherein the random sequence region comprises random nucleotides selected from A, C, and T bases (H5-8).

13. The method of claim 11, wherein the random sequence region comprises 5 random nucleotides selected from A, C, G, and T bases and 3 G bases (N5-8G3).

14. The method of claim 13, wherein the random sequence region comprises 5 random nucleotides selected from A, C, G, and T bases and 3 G bases (N5G3) in a 5’ to 3’ direction.

15. The method of any one of claims 1-13, wherein the random sequence region comprises 5 to 8 random nucleotides selected from A, C, G, and T bases and 3 T bases (N5-8T3).

16. The method of claim 15, wherein the random sequence region comprises 5 random nucleotides selected from A, C, G, and T bases and 3 T bases (N5T3) in a 5’ to 3’ direction.

17. The method of any one of claims 4- 16, further comprising sequencing the cDNA sample.

18. A random priming oligonucleotide comprising a random sequence region of 5 to 8 nucleotides, a unique molecular index (UMI), and a cell barcode (cID).

19. The oligonucleotide of claim 18, further comprises a universal amplification sequence.

20. The oligonucleotide of claim 18 or 19, wherein the random sequence region comprises random nucleotides selected from A, C, G, and T bases (N5-8).

21. The oligonucleotide of claim 18 or 19, wherein the random sequence region comprises random nucleotides selected from A, C, and T bases (H5-8).

22. The oligonucleotide of claim 19, wherein the random sequence region comprises 5 to 8 random nucleotides selected from A, C, G, and T and 3 G bases (N5-8G3).

23. The oligonucleotide of claim 22, wherein the random sequence region comprises 5 random nucleotides selected from A, C, G, and T and 3 G bases (N5G3) in a 5’ to 3’ direction.

24. The oligonucleotide of claim 19, wherein the random oligonucleotide region comprises 5 to 8 random nucleotides selected from A, C, G, and T bases and 3 T bases (N5-8T3).

25. The oligonucleotide of claim 24, wherein the random sequence region comprises 5 random nucleotides selected from A, C, G, and T and 3 T bases (N5T3) in a 5’ to 3’ direction.

26. A mixture of at least 2 oligonucleotides of claims 18-25.