Methods and compositions for nucleic acid sequencing

Abstract

Embodiments of the present invention relate to sequencing nucleic acids. In particular, embodiments of the methods and compositions provided herein relate to preparing nucleic acid templates and obtaining sequence data therefrom.

What is claimed is: 
     
         1 . A method for obtaining sequence information for a single cell, comprising:
 generating indexed template nucleic acid fragments derived from template nucleic acid of single cells, wherein the indexed template nucleic acid fragments comprise a first index and wherein the first index is associated with the template nucleic acid derived from the single cell, combining the cells and distributing the cells containing the indexed template nucleic acid fragments into a plurality of vessels;   providing a second index to at least a portion of the indexed template nucleic acid fragments in a vessel of the plurality of vessels to generate second indexed template nucleic acid fragments comprising the first index and the second index in the vessel;   obtaining sequence data from the indexed template nucleic acid fragments; and   assembling a sequence representation of the template nucleic acid from the sequence data.   
     
     
         2 . The method of  claim 1 , wherein the indexed template nucleic acid is prepared by undergoing at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more rounds of compartmentalizing and indexing. 
     
     
         3 . The method of  claim 1 , wherein generating the indexed template nucleic acid fragments comprises using transposomes comprising the first index to tagment the template nucleic acid. 
     
     
         4 . The method of  claim 1 , wherein generating the indexed template nucleic acid fragments comprises transposition, and where subsequently the index is introduced using ligation 
     
     
         5 . The method of  claim 1 , wherein the indexed nucleic acid fragments of the cells are amplified with PCR. 
     
     
         6 . The method of  claim 1 , wherein the vessel of the plurality of vessels comprises at least 2 or more cells. 
     
     
         7 . The method of  claim 1 , wherein the sequence representation comprises haplotype information based at least in part on assembling sequence reads from indexed template nucleic acid fragments. 
     
     
         8 . The method of  claim 1 , wherein another vessel of the plurality of vessels comprises indexed template nucleic acid fragments comprising the first index and a second index. 
     
     
         9 . The method of  claim 1 , wherein the second index is associated with the vessel. 
     
     
         10 . A library of template nucleic acids, comprising:
 a tagmented template nucleic acid comprising a plurality of transposons inserted into a template nucleic acid using transposomes, wherein at least some of the transposomes each comprise a first transposon sequence and a second transposon sequence noncontiguous with said first transposon sequence and a transposase, and wherein the template nucleic acid is derived from a single cell.   
     
     
         11 . The library of  claim 10 , wherein the tagmented template nucleic acid from the cell is distributed into a vessel and assigned a first index. 
     
     
         12 . The library of  claim 11 , wherein the first index identifies the sample. 
     
     
         13 . The library of  claim 12 , wherein the first index sequence is introduced using ligation. 
     
     
         14 . The library of  claim 12 , wherein the index sequence is inserted into the template nucleic acid using transposomes. 
     
     
         15 . The library of  claim 10 , wherein the template nucleic acid is a rolling circle amplification product. 
     
     
         16 . The library of  claim 10 , wherein the template nucleic acid is distributed in a well, on a microparticle or bead, or microfluidic device. 
     
     
         17 . The library of  claim 10 , wherein the tagmented template nucleic acid is associated with or coupled to transposases of the transposomes. 
     
     
         18 . A method of obtaining sequence information from a single cell, comprising obtaining a template nucleic acid of a cell comprising a plurality of transposons inserted into said target nucleic acid such that the contiguity of the template is retained, compartmentalizing the nucleic acid of the cell comprising the plurality of inserted transposons into a plurality of vessels, generating compartment-specific indexed libraries from the transposed nucleic acid targets and obtaining single cell sequence information from the template nucleic acids from a plurality of vessels. 
     
     
         19 . The method of  claim 18 , wherein generating the compartment-specific index is performed using transposition or ligation. 
     
     
         20 . The method of  claim 18 , wherein prior to generating the indexed template nucleic acid fragments, the incorporation of a universal primer site using transposition or ligation is used. 
     
     
         21 . The method of  claim 18 , wherein generating the indexed template nucleic acids fragments comprises (a) compartmentalizing the target nucleic acid of cells into a plurality of first vessels; (b) providing a first index to the target nucleic acid of each first vessel, thereby obtaining a first indexed nucleic acid; (c) combining the first indexed nucleic acids; (d) compartmentalizing the first indexed template nucleic acids into a plurality of second vessels; and (e) providing a second index to the first indexed template nucleic of each second vessel, thereby obtaining a second indexed nucleic acid. 
     
     
         22 . The method of  claim 21 , wherein the indexed template nucleic acids are generated by undergoing at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more rounds of compartmentalizing and indexing. 
     
     
         23 . The method of  claim 21 , wherein providing the first index comprises contacting the template nucleic acid with a plurality of transposomes each comprising a transposase and a transposon sequence comprising the first index under conditions such that at least some of the transposon sequences insert into the template nucleic acid. 
     
     
         24 . The method of  claim 21 , wherein the first index is introduced using ligation.