ANALYSIS OF NUCLEIC ACIDS ASSOCIATED WITH SINGLE CELLS USING NUCLEIC ACID BARCODES
Provided herein are methods and compositions for analyzing nucleic acids associated with single cells using nucleic acid barcodes. According to some embodiments, a method for producing one or more polynucleotides of interest comprises: obtaining a plurality of RNAs associated with one or more samples, wherein the samples are obtained from one or more subjects, each RNA is associated with a single sample, and the RNAs associated with each sample are present in a separate reaction volume; adding an adapter molecule to the RNAs associated with each sample, wherein the adapter molecule is generated using an enzymatic reaction and comprises a universal priming sequence, a barcode sequence, and a binding site; and incorporating the barcode sequence into one or more polynucleotides associated with each sample, thereby producing the one or more polynucleotides of interest.
1 . A method for producing one or more polynucleotides of interest, the method comprising:
obtaining a plurality of RNAs associated with one or more samples, wherein
the samples are obtained from one or more subjects, and
the RNAs associated with a sample are present in a separate reaction volume;
adding an adapter molecule to the RNAs associated with the sample, wherein the adapter molecule is generated using an enzymatic reaction and comprises a universal priming sequence, a barcode sequence, and a binding site; and
incorporating the barcode sequence into one or more polynucleotides associated with the sample,
thereby producing the one or more polynucleotides of interest.
2 . The method of claim 1 , further comprising generating the adapter molecule using the enzymatic reaction.
3 . The method of claim 1 , wherein the adapter molecule is generated by contacting a template molecule with one or more enzymes.
4 . (canceled)
5 . (canceled)
6 . The method of claim 3 , wherein the template molecule is a DNA molecule comprising a nicking endonuclease restriction site, and the one or more enzymes include a nicking endonuclease and a strand-displacing DNA polymerase.
7 . The method of claim 6 , wherein the nicking endonuclease restriction site is selected from the group consisting of Nt.BbvCI, Nt.BspQI, Nt.BsmAI, Nt.BstNBI, Nt.AlwI, and Nt.BsmAI.
8 . The method of claim 6 , wherein the strand-displacing DNA polymerase is selected from the group consisting of Klenow exo-, Bst Large Fragment and engineered variants of Bst Large Fragment.
9 . The method of claim 3 , wherein:
the template molecule is bound to a solid support,
the solid support is contacted with an aqueous solution, and
the adapter molecule is released into the aqueous solution as it is generated.
10 . The method of claim 9 , wherein adding the adapter molecule to the RNAs associated with one sample comprises combining the aqueous solution with the reaction volume in which the RNAs are present.
11 . The method of claim 9 , wherein the aqueous solution is present in the same reaction volume as the RNAs associated with one sample.
12 . The method of claim 9 , wherein:
the template molecule comprises an endonuclease restriction site,
the one or more enzymes comprise a restriction endonuclease, and
the adapter molecule comprises a portion of the template molecule, said portion being generated and released into the aqueous solution upon contacting the template molecule with the restriction endonuclease.
13 . The method of claim 9 , wherein the solid support is a bead or a surface.
14 . The method of claim 1 , wherein the adapter molecule is free in solution prior to adding the adapter molecule to the RNAs associated with one sample.
15 . The method of claim 1 , wherein the adapter molecule is generated in a compartment, and adding the adapter molecule to the RNAs associated with one sample comprises combining the compartment with the reaction volume in which the RNAs are present.
16 . The method of claim 1 , wherein the adapter molecule is generated in the reaction volume in which the RNAs to which the adapter molecule is added are present.
17 . The method of claim 1 , wherein the adapter molecule is not generated in the reaction volume in which the RNAs to which the adapter molecule is added are present.
18 . The method of claim 1 , wherein the enzymatic reaction is an isothermal reaction.
19 . The method of claim 1 , wherein the adapter molecule further comprises a unique molecular identifier (UMI) sequence.
20 . (canceled)
21 . (canceled)
22 . The method of claim 1 , wherein the adapter molecule is a DNA molecule, and producing the one or more polynucleotides of interest comprises reverse-transcribing the RNAs associated with each sample, thereby synthesizing a plurality of first-strand cDNAs,
at least some of the RNAs associated with the sample comprise a sequence region complementary to the binding site of the adapter molecule, and
the adapter molecule is used as a primer for reverse transcription, such that the barcode sequence is incorporated into first-strand cDNAs associated with the sample.
23 . The method of claim 22 , wherein the adapter molecule is generated using DNA polymerase (DNAP).
24 .- 28 . (canceled)
29 . The method of claim 22 , wherein reverse-transcribing the RNAs associated with the sample occurs in the same reaction volume where the adapter molecule added to the RNAs is generated.
30 . The method of claim 1 , further comprising reverse-transcribing the RNAs associated with the sample to obtain a plurality of cDNAs, wherein reverse-transcribing an RNA comprises synthesizing a first strand of cDNA using a reverse-transcriptase and a first-strand primer.
31 .- 36 . (canceled)
37 . The method of claim 30 , wherein:
the reverse transcriptase has template switching activity,
at least some first strands of cDNA associated with the sample comprise a 3′ overhang,
the binding site of the adapter molecule comprises a 3′ portion complementary to the 3′ overhang, and
the adapter molecule serves as a template for the reverse transcriptase, such that the barcode sequence is incorporated into first strands of cDNAs associated with the sample.
38 . The method of claim 37 , wherein the 3′ overhang comprises one or more C nucleotides and the 3′ portion of the binding site comprises one or more G nucleotides.
39 .- 40 . (canceled)
41 . The method of claim 30 , wherein producing polynucleotides of interest comprises amplifying the first strands of cDNA for the sample using a first primer and a second primer, the second primer having the same sequence as at least a portion of the first-strand primer, wherein the first primer or the second primer is the adapter molecule.
42 .- 45 . (canceled)
46 . The method of claim 1 , wherein the sample comprises a cell.
47 . The method of claim 46 , wherein the cell is a blood cell, an immune cell, a tissue cell, or a tumor cell.
48 . The method of claim 47 , wherein the cell is a B cell or a T cell.
49 .- 68 . (canceled)
69 . An adapter template comprising a nicking endonuclease restriction site, a universal priming sequence, a barcode sequence, and a binding site.
70 . The adapter template of claim 69 , wherein the nicking endonuclease restriction site is selected from the group consisting of Nt.BbvCI, Nt.BspQI, Nt.BsmAI, Nt.BstNBI, Nt.AlwI, and Nt.BsmAI.
71 . The adapter template of claim 69 , further comprising a unique molecular identifier (UMI) sequence.
72 . A solid support comprising the adapter template of claim 69 .
73 .- 132 . (canceled)