Method for sequencing a polynucleotide template

1. A method of preparing a library of nucleic acid fragments for sequence analysis, comprising:

(a) cleaving a target nucleic acid to produce a plurality of double-stranded cleavage products, wherein the target nucleic acid comprises human genomic DNA;

(b) ligating a first universal adapter to the double-stranded cleavage products to form a plurality of double-stranded templates having known ends, wherein the first universal adapter comprises a region of double-stranded sequence for ligation to the double-stranded cleavage products, and a first universal adapter sequence;

(c) denaturing the double-stranded templates to produce a plurality of single-stranded templates comprising the first universal adapter sequence;

(d) annealing the single-stranded templates to a population of complementary primer nucleic acids attached to a solid support;

(e) performing a defined number of at least 50 incremental extension cycles, each extension cycle comprising:

(i) extending each of the primer nucleic acids by incorporating an unlabeled nucleotide complementary to the annealed template and comprising a reversible blocking moiety; and

(ii) contacting the incorporated nucleotide with a deblocking agent to remove the blocking moiety;

thereby producing a population of extended primers comprising different portions of the target nucleic acid, wherein the individual extended primers in the population comprise a defined length that is correlated with the number of incremental extension cycles;

(f) subjecting the annealed templates to denaturing conditions to remove the templates from the solid support; and

(g) ligating a second universal adapter sequence to the 3′ ends of the extended primers to produce a population of immobilized nucleic acid fragments having universal adapter sequences at both ends;

thereby preparing a library of nucleic acid fragments for sequence analysis.

2. The method of claim 1 , wherein individual cycles in the incremental extension cycles comprise a wash step to remove an unreacted reagent.

3. The method according to claim 1 , wherein the primer nucleic acids attached to the solid support comprise a cleavable linkage.

4. The method of claim 1 , further comprising:

(h) removing the nucleic acid fragments having a defined length and universal adapter sequences at both ends from the solid support.

5. The method of claim 1 , wherein the defined length of the immobilized nucleic acid fragments is such that at least 50% of the immobilized nucleic acid fragments have a defined length that varies by less than 5 nucleotides.

6. The method of claim 1 wherein the defined length is between 200-1000 bases.

7. The method of claim 1 , wherein the number of incremental extension cycles performed in step (e) is between 100-1000 cycles.

8. The method of claim 1 , wherein the first universal adapter comprises a region of single-stranded sequence comprising the first universal adapter sequence.

9. A method of preparing a library of nucleic acid fragments for sequence analysis, comprising:

(a) cleaving a target nucleic acid to produce a plurality of double-stranded cleavage products, wherein the target nucleic acid comprises human genomic DNA;

(b) ligating a single-stranded first universal adapter to the 3′ ends of the double-stranded cleavage products to form a plurality of double-stranded templates having known 3′ ends, wherein the first universal adapter comprises a first universal adapter sequence;

(c) denaturing the double-stranded templates to produce a plurality of single-stranded templates comprising the first universal adapter sequence;

(d) annealing the single-stranded templates to a population of complementary primer nucleic acids attached to a solid support;

(e) performing a defined number of at least 50 incremental extension cycles, each extension cycle comprising:

(i) extending each of the primer nucleic acids by incorporating an unlabeled nucleotide complementary to the annealed template and comprising a reversible blocking moiety; and

(ii) contacting the incorporated nucleotide with a deblocking agent to remove the blocking moiety;

thereby producing a population of extended primers comprising different portions of the target nucleic acid, wherein the individual extended primers in the population comprise a defined length that is correlated with the number of incremental extension cycles;

(f) subjecting the annealed templates to denaturing conditions to remove the templates from the solid support; and

(g) ligating a second universal adapter sequence to the 3′ ends of the extended primers to produce a population of immobilized nucleic acid fragments having universal adapter sequences at both ends;

thereby preparing a library of nucleic acid fragments for sequence analysis.

10. The method of claim 9 , wherein individual cycles in the incremental extension cycles comprise a wash step to remove an unreacted reagent.

11. The method of claim 9 , wherein the primer nucleic acids attached to the solid support comprise a cleavable linkage.

12. The method of claim 9 , further comprising:

(h) removing the nucleic acid fragments having a defined length and universal adapter sequences at both ends from the solid support.

13. The method of claim 9 , wherein the defined length of the immobilized nucleic acid fragments is such that at least 50% of the immobilized nucleic acid fragments have a defined length that varies by less than 5 nucleotides.

14. The method of claim 9 , wherein the defined length is between 200-1000 bases.

15. The method of claim 9 , wherein the number of incremental extension cycles is between 100-1000 cycles.