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 - 354 . (canceled) 
     
     
         355 . 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 scissile internucleoside linkage. 
     
     
         356 . The collection of distinguishably labeled oligonucleotide probe families of  claim 355 , wherein each probe comprises a terminus that is not extendable by ligase. 
     
     
         357 . The collection of distinguishably labeled oligonucleotide probe families of  claim 355 , 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. 
     
     
         358 . The collection of distinguishably labeled oligonucleotide probe families of  claim 355 , wherein the scissile linkage is a phosphorothiolate linkage. 
     
     
         359 . The collection of distinguishably labeled oligonucleotide probe families of  claim 355 , wherein the collection comprises 2 probe families. 
     
     
         360 . The collection of distinguishably labeled oligonucleotide probe families of  claim 355 , wherein the collection comprises 3 probe families. 
     
     
         361 . The collection of distinguishably labeled oligonucleotide probe families of  claim 355 , wherein the collection comprises more than 4 probe families. 
     
     
         362 . The collection of distinguishably labeled oligonucleotide probe families of  claim 355 , wherein the probes comprise a detectable moiety that is attached by a cleavable linker, is photobleachable, or both. 
     
     
         363 . A collection of at least two distinguishably labeled oligonucleotide probe families, wherein the oligonucleotide probes in each probe family have the structure 5′-(X) j (N) k N B -3′ or 3′-(X) j (N) k N B -5′, wherein N represents any nucleoside or an abasic residue, N B  represents a moiety that is not extendable by ligase, (X) j  is a constrained portion of the probe in which each X represents a nucleoside or abasic residue, with the proviso that X l  represents a nucleotide, and nucleosides in (X) j  are identical or different but are not independently selected, each X is at least 2-fold degenerate, j is between 2 and 5, k is between 1 and 100, inclusive, each probe comprises a detectable moiety at a position other than the nucleoside in (X) j  which is at the probe terminus, and wherein probes in each probe family comprise the same label and probes in different probe families comprise different distinguishable labels. 
     
     
         364 . The collection of distinguishably labeled encoded oligonucleotide probe families of  claim 363 , wherein at least one internucleoside linkage is a scissile linkage. 
     
     
         365 . The collection of distinguishably labeled oligonucleotide probe families of  claim 363 , wherein the scissile linkage is a phosphorothiolate linkage. 
     
     
         366 . The collection of distinguishably labeled oligonucleotide probe families of  claim 363 , wherein the set consists of four probe families, wherein the oligonucleotide probes in each probe family have the structure 5′-(XY)(N) k N B *-3′ or 3′-(XY)(N) k N B *-5′, wherein N represents any nucleoside, Ns represents a moiety that is not extendable by ligase, * represents a detectable moiety, XY is a constrained portion of the probe in which X and Y represent nucleosides that are identical or different but are not independently selected, X and Y are at least 2-fold degenerate, at least one internucleoside linkage is a scissile linkage, and k is between 1 and 100, inclusive, with the proviso that a detectable moiety may be present on any nucleoside of (N) k  or on Y instead of, or in addition to, N B . 
     
     
         367 . The collection of distinguishably labeled oligonucleotide probe families of  claim 366 , wherein the scissile linkage is a phosphorothiolate linkage. 
     
     
         368 . The collection of distinguishably labeled oligonucleotide probe families of  claim 366 , wherein the detectable moiety is attached by a cleavable linker, is photobleachable, or both. 
     
     
         369 . The collection of distinguishably labeled probe families of  claim 366 , wherein oligonucleotide probes having different sequences for the constrained portion of the probe are assigned to first, second, third, and fourth probe families according to one of the 24 encodings set forth in Table 1. 
     
     
         370 . A collection of at least two distinguishably labeled oligonucleotide probe families, wherein the oligonucleotide probes in each probe family have the structure 5′-(X) j (N) k N B -3′ or 3′-(X) j (N) k N B -5′, wherein N represents any nucleoside or an abasic residue, N B  represents a moiety that is not extendable by ligase, (X) j  is a constrained portion of the probe in which each X represents a nucleoside or abasic residue, with the proviso that X l  represents a nucleotide, and nucleosides in (X) j  are identical or different but are not independently selected, each X is at least 2-fold degenerate, j is between 2 and 5, k is between 1 and 100, inclusive, each probe comprises a detectable moiety at a position other than the nucleoside in (X) j  which is at the probe terminus, and wherein probes in each probe family comprise the same label and probes in different probe families comprise different distinguishable labels. 
     
     
         371 . The collection of distinguishably labele,d encoded oligonucleotide probe families of  claim 370 , wherein at least one internucleoside linkage is a scissile linkage. 
     
     
         372 . The collection of distinguishably labeled oligonucleotide probe families of  claim 370 , wherein the scissile linkage is between a nucleoside and an abasic residue. 
     
     
         373 . The collection of distinguishably labeled encoded oligonucleotide probe families of  claim 370 , wherein the oligonucleotide probes comprise a trigger residue. 
     
     
         374 . The collection of distinguishably labeled oligonucleotide probe families of  claim 370 , wherein the detectable moiety is attached by a cleavable linker, is photobleachable, or both. 
     
     
         375 . The collection of distinguishably labeled oligonucleotide probe families of  claim 374 , wherein the cleavable linker comprises a disulfide bond. 
     
     
         376 . The collection of distinguishably labeled encoded oligonucleotide probe families of claim  26 , wherein the oligonucleotide probes comprise a trigger residue. 
     
     
         377 . The collection of distinguishably labeled probe families of  claim 370 , wherein oligonucleotide probes having different sequences for the constrained portion of the probe are assigned to first, second, third, and fourth probe families according to one of the 24 encodings set forth in Table 1. 
     
     
         378 . A kit comprising a collection of at least two distinguishably labeled oligonucleotide probe families, wherein the probes comprise a phosphorothiolate linkage. 
     
     
         379 . The kit of  claim 378 , further comprising at least one item selected from the group consisting of: a ligase, an agent capable of cleaving the phosphorothiate linkage, a phosphatase, a polymerase, a support, a buffer, a thermostable polymerase, nucleotides, reagents for preparing an emulsion, and reagents for preparing a gel.