Reagents, methods, and libraries for bead-based sequencing

Abstract

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.

Description

[0001] Cross References to Related Applications [0002] This application claims provisional applications USSN 60 / 649,294, filed February 1, 2005; USSN 60 / 656,599, filed February 25, 2005; USSN 60 / 673,749, filed April 21, 2005, July 15, 2005 Priority and benefit of USSN 60 / 699,541 filed September 30, 2005 and USSN 60 / 722,526 filed September 30, 2005, all of which are incorporated herein by reference. Background of the invention [0003] Nucleic acid sequencing technology is important in a variety of fields from basic research to clinical diagnosis. Results obtained from such techniques can include varying degrees of specific information. For example, useful information may include: determining whether the sequence of a specific polynucleotide differs from a reference polynucleotide, confirming the presence or absence of a specific polynucleotide sequence in a sample, determining partial sequence information such as identifying one or more nuclei within a polynucleotide Nucle...

AI Technical Summary

Problems solved by technology

Although electrophoretic separation is not required, pyrosequencing has a number of disadvantages that still limit its widespread application (Franca et al., Quarterly Reviews of Biophysics, 35(2):169-200, 2002)

Sequencing by hybridization has also been proposed as an alternative (US Patent 5,202,231; WO 99 / 60170; WO 00 / 56937; Drmanac et al., Advances in Biochemical Engineering / Biotechnology, 11:16-101, 2002), but has many disadvantages, including in distinguishing Errors may occur with highly similar sequences

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