Enhanced Sequencing by Hybridization Using Pools of Probes

Abstract

The invention provides methods for sequencing by hybridization (SBH) using pools of probes that allow greater efficiency in conducting SBH by reducing the number of separate measurements of hybridization signals required to identify each particular nucleotide in a target nucleic acid sequence. The invention also provides pools and set of pools of probes, as well as method of generating pools of probes.

1 . A complete or almost complete set of oligonucleotides of known sequence of given number of informative bases that are prepared individually in independent synthesis reactions and then mixed in a set of pools wherein oligonculeotides in a pool are i) not labeled or labeled with the same label and ii) differ at more than one informative position. 
     
     
         2 . The set of  claim 1  wherein substantively each oligonucleotide is added in a single pool. 
     
     
         3 . The set of  claim 1  wherein a pool have less oligonculeotides than a pool of the same consensus sequence prepared by single synthesis using two or more mixed nucleotides at positions that differ at least between two oligonucleotides. 
     
     
         4 . The set of  claim 1  wherein each prepared oligonucleotide has one or more degenerated or universal bases. 
     
     
         5 . The set of  claim 1  wherein pools labeled with different labels are mixed together. 
     
     
         6 . A set of oligonucleotides of known informative sequence arranged in two or more pools wherein oligonucleotides with minimal relatedness or maximal difference in the informative sequences are predominately included in the same pool, and wherein none, some or all of said pools are combined when oligonucleotides in different pools are labeled with different labels. 
     
     
         7 . The set of  claim 6  wherein pools are prepared by mixing individually prepared oligonucleotides or smaller pools of oligonucleotides. 
     
     
         8 . The set of  claim 6  wherein said oligonucleotides have two informative bases. 
     
     
         9 . The set of  claim 6  wherein at least some of said probe pools if labeled with different label are combined in a mix.