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 producing cells containing located insertion elements, comprising;
 a) producing cells comprising insertion elements integrated into a plurality of locations;   b) preparing a library of polynucleotide clones comprising sample-tagged junctions from the insertion elements;   c) carrying out a mapping reaction on the library to generate tagged reaction products; and   d) identifying the locations of the insertion elements by associating the reaction products with an array of tag complements to deconvolute maps of the junctions.   
     
     
         2 . The method of  claim 1 , wherein the library comprises a pool of the polynucleotide clones. 
     
     
         3 . The method of  claim 1 , wherein the insertion elements comprise sample tags. 
     
     
         4 . The method of  claim 1 , wherein the sample-tagged junctions comprise genomic tags. 
     
     
         5 . The method of  claim 1 , wherein the sample-tagged junctions comprise adapter tags. 
     
     
         6 . The method of  claim 1 , further comprising maintaining separate clonal populations of cells wherein each clonal population comprises a subset of the insertion elements, and identifying the subset in each clonal population. 
     
     
         7 . The method of  claim 6 , wherein the cells comprise the sample-tagged junctions and the step of identifying the subset comprises pooling the clonal populations to generate a plurality of subpools, amplifying polynucleotides from each subpool to generate tagged amplicons, and hybridizing the amplicons to an array of the tag complements. 
     
     
         8 . The method of  claim 6 , wherein the locations comprise sequences from the junctions, and the step of identifying the subset comprises pooling the clonal populations to generate a plurality of subpools; amplifying polynucleotides from each subpool to generate amplicons comprising the junctions; and hybridizing the amplicons to an array comprising polynucleotides that are complementary to the junction sequences. 
     
     
         9 . The method of  claim 1 , further comprising before step (c) hybridizing the clones to the tag complements to generate an array comprising tagged clones. 
     
     
         10 . The method of  claim 9 , wherein the mapping reaction is an array sequencing reaction. 
     
     
         11 . The method of  claim 1 , wherein the step of associating (d) comprises hybridizing the tagged reaction products to tag complements. 
     
     
         12 . The method of  claim 11 , further comprising separating the tagged reaction products by size and collecting fractions of the separated products before the hybridization step. 
     
     
         13 . The method of  claim 1 , wherein the mapping reaction comprises a sequencing method. 
     
     
         14 . A parallel method for producing cells containing located insertion elements, comprising;
 a) producing a collection of cells comprising insertion elements integrated into a plurality of locations;   b) preparing a pool of polynucleotides comprising a plurality of junctions from the insertion elements; and   d) identifying sequences from the junctions by sequencing the polynucleotides in parallel to locate the insertion elements.   
     
     
         15 . The method of  claim 14 , wherein the polynucleotides comprise sample tags. 
     
     
         16 . The method of  claim 14 , wherein the junctions comprise sample tags and the sequences from the junctions comprise sequences from the sample tags. 
     
     
         17 . The method of  claim 16 , wherein the sample tags are adapter tags. 
     
     
         18 . The method of  claim 16 , wherein the sample tags are genomic tags. 
     
     
         19 . The method of  claim 16 , 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. 
     
     
         20 . The method of  claim 19 , wherein the step of associating comprises amplifying the sample tags to produce tagged amplicons, and hybridizing the amplicons to an array comprising tag complements.