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.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to and the benefit of provisional applications U.S. Ser. No. 60 / 649,294, filed Feb. 1, 2005; U.S. Ser. No. 60 / 656,599, filed Feb. 25, 2005; U.S. Ser. No. 60 / 673,749, filed Apr. 21, 2005, U.S. Ser. No. 60 / 699,541, filed Jul. 15, 2005, and U.S. Ser. No. 60 / 722,526, filed Sep. 30, 2005, all of which are herein incorporated by reference.BACKGROUND OF THE INVENTION [0002] Nucleic acid sequencing techniques are of major importance in a wide variety of fields ranging from basic research to clinical diagnosis. The results available from such technologies can include information of varying degrees of specificity. For example, useful information can consist of determining whether a particular polynucleotide differs in sequence from a reference polynucleotide, confirming the presence of a particular polynucleotide sequence in a sample, determining partial sequence information such as the identity of one or more nuc...

AI Technical Summary

Benefits of technology

[0010] The present invention provides new and improved sequencing methods that avoid the necessity for performing fragment separation and also in certain embodiments avoid the need to use polymerase enzymes. An alternative to the methods discussed in the Background is described in U.S. Pat. Nos. 5,740,341 and 6,306,597, to Macevicz. The methods are based on repeated cycles of duplex extension along a single-stranded template. In preferred embodiments of these methods a nucleotide is identified in each cycle. The present invention provides improvements to these methods. The improvements allow efficient implementation of the methods and are particularly suited for high throughput sequencing. In addition, the invention provides methods for sequence determination that involve repeated cycles of duplex extension along a single-stranded template but do not involve identification of any individual nucleotide during each cycle.

Problems solved by technology

Although currently available sequencing technologies have allowed the achievement of major landmarks such as the sequencing of a number of complete genomes, these techniques have a number of disadvantages, and considerable need for improvement remains in a number of areas.

However, this step has proven to be a major bottleneck limiting both the speed and accuracy of sequencing in many contexts.

For example, CAE still requires a time-consuming separation step and still involves discrimination based on size, which can be inaccurate.

However, it has proven difficult to identify reversible terminators that can be incorporated by polymerase with high efficiency, probably due to the fact that given the small size of a nucleotide, modifications that affect the ability of the nucleotide to act as a terminator also affect its incorporation into a growing polynucleotide strand.

While avoiding the need for electrophoretic separation, pyrosequencing suffers from a large number of drawbacks that have as yet limited its widespread applicability (Franca, et al., Quarterly Reviews of Biophysics, 35(2):169-200, 2002).

Sequencing by hybridization has also been proposed as an alternative (U.S. Pat. No. 5,202,231; WO 99 / 60170; WO 00 / 56937; Drmanac, et al., Advances in Biochemical Engineering / Biotechnology, 77:76-101, 2002) but has a number of disadvantages including the potential for error in discriminating between highly similar sequences.

However, various technical hurdles remain to be overcome before realization of this potential (Stephan, et al., 2001).

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