PAIRED-END READS IN SEQUENCING BY SYNTHESIS
First Claim
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1. A method for generating and/or using paired reads, the method comprising:
- a) providing a nucleic acid template;
b) conducting a sequencing-by-synthesis reaction to obtain a first read of the template;
c) synthesizing a spacer of a defined length; and
d) conducting a sequencing-by-synthesis reaction 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., 15-35 bases), 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.
125 Citations
28 Claims
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1. A method for generating and/or using paired reads, the method comprising:
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a) providing a nucleic acid template; b) conducting a sequencing-by-synthesis reaction to obtain a first read of the template; c) synthesizing a spacer of a defined length; and d) conducting a sequencing-by-synthesis reaction 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, 27)
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26. A method for generating and using paired reads, the method comprising:
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a) providing individually optically resolvable nucleic acid templates anchored to a solid support; b) conducting a sequencing-by-synthesis reaction to obtain a first read of a template, said first read consisting of 20-150 bases; c) synthesizing a spacer of 20-1000 bases; d) conducting a sequencing-by-synthesis reaction 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; and e) mapping the first and second reads onto a reference sequence. - View Dependent Claims (28)
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Specification