Multitag sequencing ecogenomics analysis

Abstract

Embodiments of the invention herein described relate to multiplex polynucleotide sequence analysis without the use of size separation methods or blotting. In certain particulars the invention relates to multiplex sequencing using massively parallel sequencing methods, such as pyrosequencing methods and sequencing by synthesis. The invention provides increased throughput, increased accuracy of enumerating sample components, and the ability to analyze greater numbers of samples simultaneously or serially on presently available systems, as well as others yet to be developed. In certain of its embodiments the invention relates to the analysis of complex microbial communities, particularly to in-depth analysis thereof in large numbers of samples.

1 .- 26 . (canceled) 
     
     
         27 . A kit comprising at least five forward and reverse primer pairs, wherein each forward and reverse primer comprises, in 5′ to 3′ order: a priming sequence, a tag sequence of from 4 to 36 nucleotides in length, and a probe sequence targeting a genetic region for amplification, wherein:
 the priming sequence is the same between the primer pairs, with the proviso that forward and reverse primers may have the same or different priming sequences; 
 the tag sequences in each primer pair are unique so as to identify the primer pair used in an amplification reaction, wherein the tag sequences differ by at least two nucleotides; and 
 the probe sequences between the primer pairs are the same, and hybridize 3′ to a variable genetic region selected from: a 16S rRNA variable sequence, an 18S rRNA variable sequence, and an ITS variable sequence, so as to amplify a population of variable sequences in a sample. 
 
     
     
         28 . The kit of  claim 27 , wherein the genetic region varies between species. 
     
     
         29 . The kit of  claim 27 , wherein the genetic region varies within a species. 
     
     
         30 . The kit of  claim 27 , further comprising a primer complementary to and effective for elongation from the priming sequence. 
     
     
         31 . The kit of  claim 27 , wherein the kit comprises from 10 to 25 primer pairs. 
     
     
         32 . The kit of  claim 27 , wherein the kit comprises from 20 to 50 primer pairs. 
     
     
         33 . The kit of  claim 27 , wherein the kit comprises from 50 to 150 primers pairs. 
     
     
         34 . The kit of  claim 27 , wherein the kit comprises from 100 to 500 primers pairs. 
     
     
         35 . The kit of  claim 27 , wherein the tag sequences are 4 nucleotides in length. 
     
     
         36 . The kit of  claim 27 , wherein the tag sequences are 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides in length. 
     
     
         37 . The kit of  claim 36 , wherein the tag sequences are 6 nucleotides in length. 
     
     
         38 . The kit of  claim 36 , wherein the tag sequences are 7 nucleotides in length. 
     
     
         39 . The kit of  claim 36 , wherein the tag sequences are 8 nucleotides in length. 
     
     
         40 . The kit of  claim 27 , wherein each of the primer pairs is disposed separately from one another in a microtiter plate. 
     
     
         41 . The kit of  claim 27 , wherein the tag sequences in a primer pair are the same.