The present invention provides a method for determining the sequence of a
nucleic acid by successive cycles of duplex extension along a single-stranded template. This cycle includes the steps of: extension,
ligation, preferably cleavage. In certain embodiments, the method utilizes an extension probe containing a phosphorothioate linkage and utilizes a substance suitable for cleaving such linkage. In certain embodiments, the method utilizes extension probes containing abasic residues or damaged bases, and utilizes substances suitable for cleaving linkages between nucleosides and abasic residues and / or suitable for Removes substances that damage bases in nucleic acids. The present invention provides methods for determining sequence information using at least two differentially labeled probe families. In certain embodiments, the method obtains less than 2 bits of information per cycle from each of the plurality of nucleotides of the template. In certain embodiments, the sequencing reaction is performed on templates attached to beads immobilized in or on a semi-
solid support. The invention also provides labeled extension probe sets containing phosphorothioate linking or priming residues suitable for use in this method. In addition, the present invention includes removing the starting
oligonucleotide and the extending strand and performing subsequent reactions with different starting oligonucleotides, thereby performing multiple sequencing reactions on one template. The invention also provides an
effective method for preparing templates, especially for parallel sequencing of multiple different templates. The invention also provides methods for performing
ligation and cleavage. The invention also provides novel libraries of
nucleic acid fragments containing paired tags, methods for preparing microparticles to which multiple different templates (eg, containing paired tags) are attached, and for individually sequencing these templates. The invention also provides
automated sequencing systems, flow chambers,
image processing methods, and computer-readable media storing computer-
executable instructions (such as performing
image processing methods) and / or sequence information. In certain embodiments, sequence information is stored in a
database.