Reagents, Methods, and Libraries for Bead-Based Sequencing

Abstract

The present invention provides methods for determining a nucleic acid sequence by performing successive cycles of duplex extension along a single stranded template. The cycles comprise steps of extension, ligation, and, preferably, cleavage. In certain embodiments the methods make use of extension probes containing phosphorothiolate linkages and employ agents appropriate to cleave such linkages. The invention provides methods of determining information about a sequence using at least two distinguishably labeled probe families. In certain embodiments the methods acquire less than 2 bits of information from each of a plurality of nucleotides in the template in each cycle. In certain embodiments the sequencing reactions are performed on templates attached to immobilized beads. The invention further provides sets of labeled extension probes containing phosphorothiolate linkages. In addition, the invention includes performing multiple sequencing reactions on a single template by removing initializing oligonucleotides and extended strands and performing subsequent reactions using different initializing oligonucleotides.

1 - 213 . (canceled) 
     
     
         214 . A method for identifying a sequence of nucleotides in a template polynucleotide attached to a support at a point of attachment, the method comprising the steps of:
 (a) extending an initializing oligonucleotide along the template polynucleotide by ligating an oligonucleotide probe thereto to form an extended duplex, wherein the oligonucleotide probe contains a trigger residue and wherein extension proceeds along the template towards its point of attachment to the support;   (b) identifying one or more nucleotides of the polynucleotide; and   (c) repeating steps (a) and (b) until the sequence of nucleotides is determined.   
     
     
         215 . The method of  claim 214 , wherein the step of identifying includes detecting a label attached to the most recently ligated extension probe. 
     
     
         216 . The method of  claim 214 , wherein the generating step comprises cleaving the oligonucleotide probe with a cleavage agent selected from the group consisting of: AP endonucleases, Endo V, and periodate. 
     
     
         217 - 218 . (canceled) 
     
     
         219 . The method of  claim 216 , wherein the ligating and generating steps are performed in or on a semi-solid support. 
     
     
         220 . A method for determining a sequence of nucleotides in a template polynucleotide attached to a support at a point of attachment, the method comprising the steps of:
 (a) providing a probe-template duplex comprising a probe hybridized to a template polynucleotide, the primer having an extendable terminus;   (b) ligating an extension oligonucleotide probe to said extendable terminus to form an extended duplex containing an extended oligonucleotide probe wherein   
       extension proceeds along the template towards its point of attachment to the support and wherein the oligonucleotide probe contains a trigger residue;
 (c) identifying, in the extended duplex, at least one nucleotide in the template polynucleotide that is either (1) complementary to the just-ligated extension probe or (2) a nucleotide residue in the template polynucleotide which is immediately downstream of the extended oligonucleotide probe; 
 (d) generating an extendable terminus on the extended oligonucleotide probe, if an extendable terminus is not already present, such that the terminus generated is different from the terminus to which the last extension probe was ligated; and 
 (e) repeating steps (b), (c) and (d) until a sequence of nucleotides in the template polynucleotide is determined. 
 
     
     
         221 . The method of  claim 220 , wherein each extension probe has a non-extendable moiety at one terminus. 
     
     
         222 . The method of  claim 220 , wherein the step of identifying includes detecting a label attached to the most recently ligated extension probe. 
     
     
         223 . The method of  claim 220 , wherein the step of identifying includes removing said non-extendable moiety and extending said extended oligonucleotide probe with a nucleic acid polymerase in the presence of one or more labeled chain-terminating nucleoside triphosphates. 
     
     
         224 . The method of  claim 220 , further including a step of capping an extended oligonucleotide probe whenever no extension probe has ligated to the extendable terminus in the ligation step. 
     
     
         225 . The method of  claim 220 , wherein the generating step comprises cleaving the oligonucleotide probe with a cleavage agent. 
     
     
         226 . The method of  claim 225 , wherein the cleavage agent is selected from the group consisting of AP endonucleases, EndoV, and periodate. 
     
     
         227 - 230 . (canceled) 
     
     
         231 . The method of  claim 220 , wherein the ligating and generating steps are performed in or on a semi-solid support. 
     
     
         232 . The method of  claim 220 , wherein the template is attached to a microparticle that is attached to a substantially planar, rigid substrate. 
     
     
         233 - 283 . (canceled) 
     
     
         284 . A collection of at least two distinguishably labeled oligonucleotide probe families, wherein probes in each probe family comprise a constrained portion and an unconstrained portion, each position in the constrained portion is at least 2-fold degenerate, and probes in each family comprise a trigger residue. 
     
     
         285 . The collection of distinguishably labeled oligonucleotide probe families of  claim 284 , wherein each probe comprises a terminus that is not extendable by ligase. 
     
     
         286 . The collection of distinguishably labeled oligonucleotide probe families of  claim 284 , wherein each probe comprises a terminus that is not extendable by ligase, and each probe comprises a detectable moiety at a position between the scissile linkage and the terminus that is not extendable by ligase. 
     
     
         287 . The collection of distinguishably labeled oligonucleotide probe families of  claim 284 , wherein the collection comprises 2 probe families. 
     
     
         288 . The collection of distinguishably labeled oligonucleotide probe families of  claim 284 , wherein the collection comprises 3 probe families. 
     
     
         289 . The collection of distinguishably labeled oligonucleotide probe families of  claim 284 , wherein the collection comprises 4 probe families. 
     
     
         290 - 354 . (canceled)