Method for sequencing a polynucleotide template
First Claim
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1. A method for producing a library of 3′
- and 5′
modified nucleic acids suitable for paired end sequencing comprising;
a. fragmenting a primary double stranded nucleic acid target sequence to produce a first population of linear nucleic acid target fragments with blunt ends;
b. self-ligating the blunt ends of the linear nucleic acid target fragments to form circular constructs;
c. treating the circular constructs to reduce the ratio of linear fragments to circular constructs;
d. fragmenting the circular constructs to produce a second population of linear fragments, a percentage of which comprises two ends from each of the first population of linear target fragments;
e. ligating adapter sequences to the 3′ and
5′
ends of the second population of linear fragments to produce a library of 3′ and
5′
modified nucleic acids suitable for paired end sequencing.
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Abstract
The invention relates to methods for pairwise sequencing of a double-stranded polynucleotide template, which methods result in the sequential determination of nucleotide sequences in two distinct and separate regions of the polynucleotide template.
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20 Claims
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1. A method for producing a library of 3′
- and 5′
modified nucleic acids suitable for paired end sequencing comprising;a. fragmenting a primary double stranded nucleic acid target sequence to produce a first population of linear nucleic acid target fragments with blunt ends; b. self-ligating the blunt ends of the linear nucleic acid target fragments to form circular constructs; c. treating the circular constructs to reduce the ratio of linear fragments to circular constructs; d. fragmenting the circular constructs to produce a second population of linear fragments, a percentage of which comprises two ends from each of the first population of linear target fragments; e. ligating adapter sequences to the 3′ and
5′
ends of the second population of linear fragments to produce a library of 3′ and
5′
modified nucleic acids suitable for paired end sequencing. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)
- and 5′
Specification