Combinatorial sets of nucleic acid barcodes for analysis of nucleic acids associated with single cells

Abstract

Provided herein are methods of identifying the origin of a nucleic acid sample. The methods include forming a reaction mixture comprising a nucleic acid sample comprising nucleic acid molecules from a single cell and a set of barcodes, incorporating the set of barcodes into the nucleic acid molecules of the sample, and identifying the set of barcodes incorporated into the nucleic acid molecules of the single cell thereby identifying the origin of the nucleic acid sample.

1 .- 44 . (canceled) 
     
     
         45 . A method of identifying the origin of a nucleic acid sample comprising the steps of:
 (a) providing a set of 3′ nucleic acid constructs each construct comprising a unique 3′ barcode sequence, a binding sequence, and a barcode association sequence;   (b) providing a nucleic acid sample comprising nucleic acid molecules from a single cell;   (c) contacting the nucleic acid sample with the set of 3′ nucleic acid constructs;   (d) separating the 3′ barcode sequence and binding sequence from the barcode association sequence;   (e) incorporating into the nucleic acid molecules the 3′ barcode sequences from the 3′ nucleic acid constructs; and   (f) identifying the set of 3′ barcode sequences using the barcode association sequences thereby identifying the origin of the nucleic acid sample from the single cell.   
     
     
         46 . The method of  claim 45 , wherein the set of 3′ nucleic acid constructs comprises 2, 3, 4, 5, 6, 7, or 8 3′ nucleic acid constructs. 
     
     
         47 . The method of  claim 45  or  46 , wherein the 3′ nucleic acid constructs further comprise unique molecular identifier (UMI) sequences. 
     
     
         48 . The method of any one of  claims 45 - 47 , wherein the 3′ nucleic acid construct is capable of forming a hairpin. 
     
     
         49 . The method of any one of  claims 45 - 48 , wherein the 3′ barcode sequence and binding sequence are separated from the barcode association segment by a restriction enzyme sequence or photocleavable linker. 
     
     
         50 . The method of any one of  claims 45 - 49 , wherein multiple copies of the 3′ nucleic acid constructs are attached to a solid surface. 
     
     
         51 . The method of  claim 50 , wherein the solid surface is a bead, microbead, microparticle, microsphere, nanoparticle, nanobead or hydrogel. 
     
     
         52 . The method of  claim 51 , a restriction enzyme releases the 3′ barcode sequence from the solid surface and separates the binding sequence from the barcode association segment. 
     
     
         53 . The method of  claim 51 , wherein UV light releases the 3′ barcode sequence from the solid surface and separates the binding sequence from the barcode association segment. 
     
     
         54 . The method of any one of  claims 45 - 53 , wherein the binding sequence on the 3′ nucleic acid constructs comprises a poly-T sequence. 
     
     
         55 . The method of  claim 54 , wherein the binding sequence of the 3′ nucleic acid constructs binds to the poly-A sequences on the nucleic acid molecules of the sample. 
     
     
         56 . The method of any one of  claims 45 - 55 , wherein the barcode sequences on the 3′ nucleic acid constructs are incorporated by reverse transcription. 
     
     
         57 . The method of  claim 56 , wherein the reverse transcription produces nucleic acid molecules comprising the 3′ barcode sequence. 
     
     
         58 . The method of any one of  claims 45 - 57 , wherein the barcode association segment comprises a priming sequence. 
     
     
         59 . The method of  claim 58 , wherein the priming sequence is a palindromic sequence. 
     
     
         60 . The method of  claim 58 , wherein the priming sequence of one barcode association segment is capable of binding the priming sequence of any other barcode association segment in the nucleic acid sample. 
     
     
         61 . The method of  claim 59 , wherein the barcode association segments that have contacted each other are extended by a DNA polymerase to incorporate both barcode association segments into a single nucleic acid. 
     
     
         62 . The method of any one of  claims 45 - 61 , wherein the method further comprises identifying the barcode sequences incorporated into the nucleic acid molecules. 
     
     
         63 . The method of  claim 62 , wherein the barcode sequences in the nucleic acid sample are identified by sequencing. 
     
     
         64 . The method of  claim 62 , wherein the barcode association segments are identified by sequencing.