Reagents, methods, and libraries for bead-based sequencing
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A technology of sequence and cutting agent, applied in the field of bead-based sequencing reagents and libraries, can solve the problems of limiting wide application, distinguishing errors of highly similar sequences, etc.
Inactive Publication Date: 2008-05-28
ADVANCED GENETIC ANALYSIS CORP
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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 propo
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Embodiment 1
[0379] Example 1: Efficient Cleavage and Ligation of Phosphorothioated Oligonucleotides
[0380] This example describes experiments showing efficient ligation and cleavage of extended oligonucleotides containing 3'-S phosphorothioate linkages.
[0381] Materials and methods
[0382] ligation sequencing method
[0383] Template Preparation: To evaluate the potential for sequencing by oligonucleotide ligation and cleavage cycles and to explore the effect of altering certain aspects of the method, two populations of model bead-based templates were prepared. In a preferred implementation, cycles of oligonucleotide ligation and cleavage extend the strand in the 3'→5' direction, as described in the Examples. Therefore, in order to evaluate the ligation efficiency, the 5' end of the pattern template was bound to beads, and the same binding region was designed at the 3' end. One set consisted of short (70 bp) oligonucleotides bound to streptavidin-coated magnetic beads (1 micron)...
Embodiment 2
[0397] Example 2: Efficient Cleavage and Ligation of Phosphorothioated Oligonucleotides Containing Nucleotides with Reduced Degeneracy
[0398] However, another consideration for probe length is the fidelity of the extended oligo and its impact on subsequent ligation efficiency. The fidelity of T4 DNA ligase has been shown to decrease rapidly after the 5th base after the junction (Luo et al., Nucleic Acids Res., 24:3071-3078 and 3079-3085, 1996). If a mismatch is introduced on the 5' side of the newly ligated junction, ligation efficiency can be reduced by depletion, however, without a phase shift or increase in background signal (a major hurdle encountered in polymerase-based sequencing by synthetic approaches). ).
[0399] Preferably, the probe set should be able to hybridize to any DNA sequence in order to resequence uncharacterized DNA. However, the complexity of labeled probe sets increases exponentially with the length and number of 4-fold degenerate bases. Furthermor...
Embodiment 3
[0404] Example 3: Fidelity of Probe Ligation
[0405] Bacterial NAD-dependent ligases such as Taq DNA ligase have been reported to have high sequence fidelity at the ligation, with essentially no nick-closing activity for mismatches on the 3' side but some tolerance for mismatches on the 5' side (Luo et al., Nucleic Acids Res., 24:3071-3078 and 3079-3085, 1996). On the other hand, T4 DNA ligase has been reported to be slightly less stringent, allowing mismatches at the 3'- and 5'-sides of the junction. Therefore, it was of interest to evaluate the fidelity of probe ligation with T4 DNA ligase compared to Taq DNA ligase in our system.
[0406] Using standard ABI sequencing techniques, we developed two methods to assess the sequence fidelity of ligated oligonucleotides. The first approach was designed to clone and sequence the ligation products. In this method, ligated extension products are ligated to adapter sequences, cloned and transformed into bacteria. Individual colon...
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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...
Claims
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