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. In certain embodiments the methods make use of extension probes containing an abasic residue or a damaged base and employ agents appropriate to cleave linkages between a nucleoside and an abasic residue and/or agents appropriate to remove a damaged base from a nucleic acid. 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 beads, which are immobilized in or on a semi-solid support. The invention further provides sets of labeled extension probes containing phosphorothiolate linkages or trigger residues that are suitable for use in the method. 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. The invention further provides efficient methods for preparing templates, particularly for performing sequencing multiple different templates in parallel. The invention also provides methods for performing ligation and cleavage. The invention also provides new libraries of nucleic acid fragments containing paired tags, and methods of preparing microparticles having multiple different templates (e.g., containing paired tags) attached thereto and of sequencing the templates individually. The invention also provides automated sequencing systems, flow cells, image processing methods, and computer-readable media that store computer-executable instructions (e.g., to perform the image-processing methods) and/or sequence information. In certain embodiments the sequence information is stored in a database.

1 - 172 . (canceled)  
     
     
         173 . A method for identifying a sequence of nucleotides in a template polynucleotide, 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 comprises a trigger residue;    (b) identifying one or more nucleotides of the polynucleotide;    (c) generating an extendable probe terminus by cleaving the oligonucleotide probe with a cleavage agent; and    (d) repeating steps (a), (b), and (c) until the sequence of nucleotides is determined.    
     
     
         174 . The method of  claim 173 , wherein the step of identifying includes detecting a label attached to the most recently ligated oligonucleotide probe.  
     
     
         175 . The method of  claim 173 , wherein the oligonucleotide probe comprises an abasic residue, a damaged base, or deoxyinosine.  
     
     
         176 . The method of  claim 173 , wherein the oligonucleotide probe comprises a damaged base, further comprising the step of removing the damaged base.  
     
     
         177 . The method of  claim 176 , wherein the step of removing comprises contacting the extended duplex with a DNA glycosylase.  
     
     
         178 . The method of  claim 173 , further comprising the step of generating an extendable probe terminus by cleaving the oligonucleotide probe with a cleavage agent.  
     
     
         179 . The method of  claim 178 , wherein the cleavage agent is selected from the group consisting of: AP endonucleases, Endo V, and periodate.  
     
     
         180 . The method of  claim 178 , wherein the cleavage agent is an AP endonuclease.  
     
     
         181 . The method of  claim 178 , wherein the cleavage agent is endonuclease VIII.  
     
     
         182 . The method of  claim 173 , wherein the extending step is performed in or on a semi-solid support.  
     
     
         183 . A method for determining a sequence of nucleotides in a template polynucleotide, the method comprising the steps of: 
 (a) providing a probe-template duplex comprising a probe hybridized to a template polynucleotide, the probe 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 the extension probe comprises 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.    
     
     
         184 . The method of  claim 183 , wherein the extension probe comprises an abasic residue, a damaged base, or deoxyinosine.  
     
     
         185 . The method of  claim 183 , wherein each extension probe has a non-extendable moiety at one terminus.  
     
     
         186 . The method of  claim 183 , wherein the step of identifying includes detecting a label attached to the most recently ligated extension probe.  
     
     
         187 . The method of  claim 183 , 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.  
     
     
         188 . The method of  claim 183 , further including a step of capping an extended oligonucleotide probe whenever no extension probe has ligated to the extendable terminus in the ligation step.  
     
     
         189 . The method of  claim 183 , wherein the generating step comprises cleaving the oligonucleotide probe with a cleavage agent selected from the group consisting of AP endonucleases and periodate.  
     
     
         190 . The method of  claim 189 , wherein the cleavage agent is an AP endonuclease.  
     
     
         191 . The method of  claim 189 , wherein the cleavage agent is endonuclease VIII.  
     
     
         192 . The method of  claim 183 , wherein the ligating and generating steps are performed in or on a semi-solid support.  
     
     
         193 . The method of  claim 183 , wherein step (a) includes providing, in separate aliquots, a plurality of distinct probe-template duplexes, each distinct duplex comprising an initializing oligonucleotide probe hybridized to a template polynucleotide, wherein the template polynucleotide in each duplex is the same, but the initializing oligonucleotide probe in each duplex is bound to a different sequence of the template polynucleotide; and steps (b) to (e) are carried out independently on each aliquot.  
     
     
         194 . The method of  claim 193  wherein for each aliquot, said extension oligonucleotide probe has a non-extendable moiety at one terminus.  
     
     
         195 . The method of  claim 194 , wherein for each aliquot, said step of identifying includes detecting a label attached to the most recently ligated extension probe.  
     
     
         196 . The method of  claim 194 , wherein for each aliquot, said 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.  
     
     
         197 . The method of  claim 194 , further including a step of capping an extended oligonucleotide probe whenever no extension probe has ligated to the extendable terminus in the ligation step.  
     
     
         198 . The method of  claim 194 , wherein the generating step comprises cleaving the oligonucleotide probe with a cleavage agent selected from the group consisting of: AP endonucleases and periodate.  
     
     
         199 . The method of  claim 198 , wherein the cleavage agent is an AP endonuclease.  
     
     
         200 . The method of  claim 198 , wherein the cleavage agent is endonuclease VIII.  
     
     
         201 . The method of  claim 194 , wherein the ligating and generating steps are performed in or on a semi-solid support.  
     
     
         202 . The method of  claim 183 , further comprising the steps of: (f) removing the ligated probes and the initializing oligonucleotide from the template; (g) repeating step (a) using a second oligonucleotide that is bound to a different sequence of the template polynucleotide; and (h) repeating steps (b) to (e).  
     
     
         203 . The method of  claim 202 , wherein the method is repeated a plurality of times using initializing oligonucleotides bound to different sequences of the template polynucleotide.  
     
     
         204 . The method of  claim 202 , wherein the removing step comprises contacting the ligated probes, initializing oligonucleotide, and template with a solution containing 1.0-3.0% SDS, 100-300 mM NaCl, and 5-15 mM sodium bisulfate (NaHSO4) in water.  
     
     
         205 . The method of  claim 202 , wherein the removing step comprises contacting the ligated probes, initializing oligonucleotide, and template with a solution containing about 2% SDS, about 200 mM NaCl, and about 10 mM sodium bisulfate (NaHSO4), e.g., 2% SDS, 200 mM NaCl, and 10 mM sodium bisulfate (NaHSO4).  
     
     
         206 . The method of  claim 203 , wherein the extension probes have a non-extendable moiety at one terminus.  
     
     
         207 . The method of  claim 203 , wherein for each repetition, the step of identifying includes detecting a label attached to the most recently ligated extension probe.  
     
     
         208 . The method of  claim 203 , wherein for each repetition, 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.  
     
     
         209 . The method of  claim 203 , further including a step of capping an extended oligonucleotide probe whenever no extension probe has ligated to the extendable terminus in the ligation step.  
     
     
         210 . The method of  claim 203 , wherein the generating step comprises cleaving the oligonucleotide probe with a cleavage agent selected from the group consisting of: AP endonucleases and periodate.  
     
     
         211 . The method of  claim 210 , wherein the cleavage agent is an AP endonuclease.  
     
     
         212 . The method of  claim 210 , wherein the cleavage agent is endonuclease VIII.  
     
     
         213 . The method of  claim 203 , wherein the ligating and generating steps are performed in or on a semi-solid support.  
     
     
         214 - 354 . (canceled)