Normalized iterative barcoding and sequencing of dna collections

Abstract

The present invention features, inter alia, compositions and methods for preparing, from a plurality of original, nucleic acid-containing samples, a unified library of linear, non-selectively amplified DNA fragments in which the proportional representation of the fragments from each of the plurality of original samples is normalized and the library is created in a highly parallelized, pool-based fashion. The invention is particularly useful for preparing libraries in which specific information is encoded that allows shorter sequencing reads derived from high-throughput sequencing of the library to be analyzed or assembled into longer scale sequences that are fully traceable to an original, nucleic acid-containing sample within a potentially very large collection of samples. The compositions of the invention encompass the various constructs described herein, which may be variously packaged with one or more additional reagents useful in the present methods and instructions for use.

1 . A method of obtaining an iteratively tagged library of DNA fragments from a plurality of samples, the method comprising:
 (a) contacting target nucleic acid molecules in first and second samples of the plurality with, respectively, uniform and limiting amounts of first and second constructs each comprising an identifiable sample sequence tag, wherein the sample sequence tag in the first construct differs from the sample sequence tag in the second construct and the contacting occurs for a duration and under conditions in which the sample sequence tag in the first construct is integrated into the nucleic acid molecules in the first sample and the sample sequence tag in the second construct is integrated into the nucleic acid molecules in the second sample;   (b) pooling the first and second samples to yield a pool of sample-tagged nucleic acid molecules;   (c) contacting the pool of sample-tagged nucleic acid molecules with a third construct comprising a pool tag, wherein the contacting occurs for a duration and under conditions in which the pool tag is integrated into the nucleic acid molecules of the pool at a prescribed frequency per nucleotide, thereby generating a pool of sample-tagged, pool-tagged nucleic acid molecules; and   (d) amplifying the sample-tagged, pool-tagged nucleic acid molecules with oligonucleotides comprising (i) a nucleic acid sequence complementary to a nucleic acid sequence in the sample-tagged, pool-tagged nucleic acid molecules and (ii) a nucleic acid sequence suitable for next generation sequencing, thereby generating an iteratively tagged library of DNA fragments.   
     
     
         2 . The method of  claim 1 , wherein the pool tag comprises an identifiable sequence tag. 
     
     
         3 . The method of  claim 1 , wherein at least one sample in the plurality of samples comprises genomic DNA. 
     
     
         4 . The method of  claim 1 , wherein at least one sample in the plurality of samples comprises cDNA, synthetic DNA, or DNA found naturally in a virus, bacterium, yeast, fungus, protozoan, insect, fish, avian, mammal, or plant. 
     
     
         5 . The method of  claim 1 , wherein the plurality of samples comprises 2-9,600 samples. 
     
     
         6 . The method of  claim 1 , wherein the first and second constructs comprise an adapter that is subsequently ligated to the target nucleic acid molecules. 
     
     
         7 . The method of  claim 1 , wherein the first and/or second constructs comprise a transposase, or a biologically active variant thereof, that subsequently introduces the identifiable sample sequence tag into the target nucleic acid molecules. 
     
     
         8 . The method of  claim 7 , wherein, in the conditions in which the sample sequence tag is integrated into the target nucleic acid molecules, the amount(s) of the sample sequence tag(s) is/are at least two-fold lower than the amount(s) of the pool tag(s) subsequently used to contact the pool of sample-tagged nucleic acid molecules. 
     
     
         9 . The method of  claim 1 , wherein the third construct comprises an adapter that is subsequently ligated to the sample-tagged nucleic acid molecules. 
     
     
         10 . The method of  claim 1 , wherein the third construct comprises a transposase, or a biologically active variant thereof, that subsequently introduces the pool tag into the sample-tagged nucleic acid molecules. 
     
     
         11 . The method of  claim 1 , wherein the first, second, or third constructs, independently, comprise a moiety that specifically binds a capture agent. 
     
     
         12 . The method of  claim 11 , wherein the moiety that specifically binds a capture agent is a nucleic acid sequence that specifically binds a protein or nucleic acid sequence within the capture moiety. 
     
     
         13 . The method of  claim 11 , wherein the moiety that specifically binds a capture agent is a moiety that has been biotinylated or digoxigenylated. 
     
     
         14 . The method of  claim 11 , further comprising the step of normalizing a population of sample-tagged and/or pool-tagged nucleic acid molecules by exposing the population to a limited amount of the capture agent. 
     
     
         15 . The method of  claim 1 , wherein the pool tag is integrated into the pooled, sample-tagged nucleic acid molecules at a prescribed frequency per nucleotide. 
     
     
         16 . The method of  claim 15 , wherein the prescribed frequency yields a pool tag between about 100-20,000 nucleotides, on average, away from each sample sequence tag on the sample-tagged nucleic acid molecules in the pool. 
     
     
         17 . The method of  claim 1 , further comprising subjecting the iteratively tagged library of DNA fragments to next generation sequencing. 
     
     
         18 . The method of  claim 1 , wherein the method results in a normalized library. 
     
     
         19 . A kit comprising:
 (a) a plurality of constructs comprising an identifiable sample sequence tag, wherein the identifiable sample sequence tag in a first construct of the plurality is distinct from the identifiable sample sequence tag in the second construct; and   (b) instructions for use.   
     
     
         20 . The kit of  claim 19 , further comprising a pool-tagging reagent for adding an identifiable sequence tag to one or more nucleic acids in a pool of sample-tagged nucleic acids and/or a reagent for amplifying a sample-tagged and/or pool-tagged nucleic acid molecule. 
     
     
         21 . (canceled)