Parallel Methods For Insertional Mutagenesis

Abstract

The present invention provides parallel methods for identifying the locations of insertion elements that are distributed at different sites in the genome among a collection of cells.

1 . A parallel method for locating insertion elements in a collection of cells, comprising;
 a) integrating insertion elements into host cells to produce a collection of the cells comprising the insertion elements at a plurality of locations in the genomes of the cells wherein the insertion elements are flanked by junctions comprising host DNA;   b) preparing a pool of polynucleotides comprising the junctions from a plurality of the locations;   c) identifying sequences from the host DNA at the junctions by sequencing the pool of polynucleotides in parallel to locate the insertion elements.   
     
     
         2 . The method of  claim 1 , further comprising isolating a cell from the collection of cells such that the cell comprises at least one of the insertion elements located in step c). 
     
     
         3 . The method of  claim 1 , wherein the host cells are eukaryotic cells. 
     
     
         4 . The method of  claim 1 , wherein the insertion elements are derived from transposable elements. 
     
     
         5 . The method of  claim 1 , wherein the insertion elements are derived from viruses. 
     
     
         6 . The method of  claim 1 , wherein the pool comprises the junctions from at least 100 distinct locations. 
     
     
         7 . The method of  claim 1 , wherein the pool comprises the junctions from at least 1000 distinct locations. 
     
     
         8 . The method of  claim 1 , wherein the polynucleotides comprise sample tags. 
     
     
         9 . The method of  claim 8 , wherein sequencing the pool of polynucleotides identifies the sequences from the junctions and sequences from the sample tags. 
     
     
         10 . The method of  claim 9 , wherein the insertion elements comprise the sample tags. 
     
     
         11 . The method of  claim 9 , wherein the sample tags are genomic tags. 
     
     
         12 . The method of  claim 9 , wherein the collection of cells comprises a plurality of separate, clonal populations of cells, and further comprising identifying at least one clonal population that contains at least one of the located insertion elements by associating at least one of the sample tags with the clonal population. 
     
     
         13 . The method of  claim 12 , wherein the step of associating comprises amplifying the sample tags to produce tagged amplicons, and hybridizing the amplicons to an array comprising tag complements.