Methods and compositions for sequencing

Abstract

Methods, compositions and kits are provided herein for insertional modification of nucleic acids by, for example transposase-mediated covalent insertion of insertion sequence into a sample nucleic acid molecule. Using sequence of the insertion to direct amplification of adjacent nucleic acid sequence, and using bar codes to map amplified sequence to partitions, one can map sample nucleic acid sequence to single molecules of the nucleic acid sample that are derived directly from the sample nucleic acid molecule.

What we claim is: 
     
         1 . A genomic nucleic acid library comprising a plurality of RNA molecules transcribed from an RNA promoter population, said plurality of RNA molecules each comprising a first end comprising an insert sequence and a second end comprising genomic nucleic acid sample sequence, wherein at least 90% of said genomic nucleic acid sample is represented in said plurality of RNA molecules, wherein said insert sequence comprises a mosaic end (ME) border, and wherein said genomic nucleic acid sample is isolated from a single cell. 
     
     
         2 . The library of  claim 1 , wherein the plurality of RNA molecules is generated directly from the genomic nucleic acid sample. 
     
     
         3 . The library of  claim 1 , wherein said sample is amplified at least 100× relative to said genomic sample. 
     
     
         4 . The library of  claim 3 , wherein at least 85% of said amplified sample is present at a level that is no more than 4× of a mean amplification level. 
     
     
         5 . The library of  claim 1 , wherein said RNA promoter population comprises T7 promoter sequence. 
     
     
         6 . The library of  claim 1 , wherein the insert sequence comprises an RNA promoter. 
     
     
         7 . The library of  claim 1 , wherein the insert sequence comprises a first ME border, an RNA promoter, and a second ME border. 
     
     
         8 . The library of  claim 6 , wherein said genomic nucleic acid sample is treated to insert a nucleic acid encoding said RNA promoter and comprising said insert sequence into said genomic nucleic acid sample at a density of from one insert every 500 bases to one insert every 5 kb. 
     
     
         9 . The library of  claim 8 , wherein said genomic nucleic acid sample is contacted to a transposase. 
     
     
         10 . The library of  claim 7 , wherein said transposase is Tn5. 
     
     
         11 . A genomic nucleic acid library comprising a plurality of nucleic acid molecules, wherein the nucleic acid molecules are generated directly from the genomic nucleic acid sample, such that no nucleic acid molecule serves as a template for a second nucleic acid molecule. 
     
     
         12 . The library of  claim 11 , wherein said nucleic acid molecules comprise RNA molecules. 
     
     
         13 . The library of  claim 11 , wherein at least 90% of said genomic nucleic acid sample is represented in said library. 
     
     
         14 . The library of  claim 11 , wherein said sample is amplified at least 1000× relative to said genomic sample. 
     
     
         15 . The library of  claim 14 , wherein at least 85% of said amplified sample is present at a level that is no more than 4× of a mean amplification level. 
     
     
         16 . The library of  claim 11 , wherein said RNA promoter sequence comprises T7 promoter sequence. 
     
     
         17 . The library of  claim 11 , wherein the nucleic acid molecule comprises an exogenous promoter inserted at an average density of at least 1 insertion per 5 kb. 
     
     
         18 . The library of  claim 17 , wherein the nucleic acid sample comprises a plurality of multi-insert nucleic acids comprising exogenous insert sequence and isolated genomic nucleic acid sample sequence. 
     
     
         19 . The library of  claim 18 , wherein the exogenous insert sequence has been integrated into a target nucleic acid sequence and comprises at least an adapter sequence and an RNA promoter sequence flanked by nucleic integrase recognition sites.