Paired-end reads in sequencing by synthesis
DC CAFCFirst Claim
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1. A method for generating paired reads from a strand of a nucleic acid duplex, the method comprising:
- a) providing a nucleic acid template attached, either directly or indirectly, to a solid support;
b) conducting a sequencing-by-synthesis reaction using detectably labeled nucleotides to obtain a first read of the template;
c) synthesizing a spacer of a defined length; and
d) conducting a sequencing-by-synthesis reaction using detectably labeled nucleotides to obtain a second read of the template, said second read being separated from the first read by the spacer.
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Abstract
The disclosure provides methods of generating paired reads in sequencing-by-synthesis process, particularly, in systems with relatively short read lengths (e.g., 15-35bases), such as for example, in single molecule sequencing by synthesis. Several implementations of the methods are provided. Of particular advantage are the methods that permit re-sequencing of the template, which yields lower error rates. The invention further provides methods of using paired reads, for example, for positioning them over repeats or for assembly into large sequences, including whole genome assembly.
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32 Claims
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1. A method for generating paired reads from a strand of a nucleic acid duplex, the method comprising:
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a) providing a nucleic acid template attached, either directly or indirectly, to a solid support; b) conducting a sequencing-by-synthesis reaction using detectably labeled nucleotides to obtain a first read of the template; c) synthesizing a spacer of a defined length; and d) conducting a sequencing-by-synthesis reaction using detectably labeled nucleotides to obtain a second read of the template, said second read being separated from the first read by the spacer. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29)
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30. A method for generating paired reads from a strand of a nucleic acid duplex, the method comprising:
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a) providing individually optically resolvable nucleic acid templates anchored, directly or indirectly, to a solid support; b) conducting a sequencing-by-synthesis reaction using detectably labeled nucleotides to obtain a first read of a template, said first read consisting of 20-150 bases; c) synthesizing a spacer of 20-1000 bases; and d) conducting a sequencing-by-synthesis reaction using detectably labeled nucleotides to obtain a second read of the template, said second read consisting of 20-150 bases and separated from the first read by the spacer. - View Dependent Claims (31, 32)
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