METHODS AND COMPOSITIONS FOR ENRICHMENT OF TARGET POLYNUCLEOTIDES

• US 20140162278A1
• Filed: 12/10/2013
• Published: 06/12/2014
• Est. Priority Date: 07/17/2012
• Status: Abandoned Application

• Current Assignee: Myriad Women's Health Incorporated (Myriad Genetics, Inc.)
• Original Assignee: Counsyl Incorporated (Myriad Genetics, Inc.)
• Inventors: Richards, Hunter, Evans, Eric, Srinivasan, Subramaniam, Chu, Clement, Srinivasan, Balaji
• Application Number:
  US14/102,462
• Publication Number:
  US 20140162278A1
• Time in Patent Office: Days
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/543 characterised by the use of two or more capture oligonucleotide primers in concert, e.g. bridge amplification

Abstract

The invention provides methods, apparatuses, and compositions for high-throughput amplification sequencing of specific target sequences in one or more samples. In some aspects, barcode-tagged polynucleotides are sequenced simultaneously and sample sources are identified on the basis of barcode sequences. In some aspects, sequencing data are used to determine one or more genotypes at one or more loci comprising a causal genetic variant.

Claims

1. A method of enriching a plurality of different target polynucleotides in a sample, the method comprising:

(a) joining an adapter oligonucleotide to each of the target polynucleotides, wherein the adapter oligonucleotide comprises sequence Y;

(b) hybridizing a plurality of different oligonucleotide primers to adapted target polynucleotides, wherein each oligonucleotide primer comprises sequence Z and sequence W;

wherein sequence Z is common among all oligonucleotide primers; and

further wherein sequence W is different for each different oligonucleotide primer, is positioned at the 3′

end of each oligonucleotide primer, and is complementary to a sequence comprising a causal genetic variant or a sequence within 200 nucleotides of a causal genetic variant;

(c) in an extension reaction, extending the oligonucleotide primers along the adapted target polynucleotides to produce extended primers comprising sequence Z and sequence Y′

, wherein sequence Y′

is complementary to sequence Y; and

(d) exponentially amplifying the purified extension products using a pair of amplification primers comprising (i) a first amplification primer comprising sequence V and sequence Z, wherein sequence Z is positioned at the 3′

end of the first amplification primer; and

(ii) a second amplification primer comprising sequence X and sequence Y, wherein sequence Y is positioned at the 3′

end of the second amplification primer;

wherein sequences W, Y, and Z are different sequences and comprise 5 or more nucleotides each.

2. The method of claim 1, wherein each oligonucleotide primer comprises a first binding partner.

3. The method of claim 2, wherein the method further comprises, before step (d), exposing the extended primers to a solid surface comprising a second binding partner that binds to the first binding partner, thereby purifying the extended primers away from one or more components of the extension reaction.

4. The method of claim 1, wherein the plurality of oligonucleotide primers comprises at least about 100 different oligonucleotide primers each comprising a different sequence W.

5. The method of claim 1, wherein sequence W of one or more of the plurality of oligonucleotide primers comprises a sequence selected from the group consisting of SEQ ID NOs 22-121.

6. The method of claim 1, wherein the target polynucleotides comprise fragmented polynucleotides.

7. The method of claim 6, wherein the fragmented polynucleotides have a median length between 200 and 1000 base pairs.

8. The method of claim 6, wherein the fragmented polynucleotides are treated to produce blunt ends or to have a defined overhang prior to step (a).

9. The method of claim 8, wherein the defined overhang consists of an adenine.

10. The method of claim 3, wherein the first binding partner and the second binding partner are members of a binding pair.

11. The method of claim 10, wherein the binding pair is streptavidin and biotin.

12. The method of claim 3, wherein the solid surface is a bead.

13. The method of claim 12, wherein the bead is responsive to a magnetic field.

14. The method of claim 13, wherein the purifying step comprises application of a magnetic field to purify the beads.

15. The method of claim 3, wherein the extended primers are purified away from the target polynucleotides.

16. The method of claim 1, further comprising sequencing the products of step (d).

17. The method of claim 16, wherein sequencing comprises amplifying the products of step (d) by bridge amplification with bound oligonucleotides attached to a solid support to produce double-stranded bridge polynucleotides;

cleaving one strand of a bridge polynucleotide at a cleavage site in a bound oligonucleotide;

denaturing the cleaved bridge polynucleotide to produce a free single-stranded polynucleotide comprising a target sequence attached to the solid support; and

sequencing the target sequence by extending a sequencing primer hybridized to at least a portion of one or more sequences added during one or more of steps (a), (c), or (d).

18. The method of claim 16, wherein sequencing comprises amplifying the products of step (d) by extension of a bound primer on a solid support to produce bound templates, hybridizing a sequencing primer to a bound template, extending the sequencing primer, and identifying nucleotides added by extension of the sequencing primer.

19. The method of claim 1, wherein the plurality of different oligonucleotide primers further comprises additional oligonucleotide primers comprising sequence Z and sequence W, wherein sequence W is different for each different additional oligonucleotide primer, is at the 3′

end of each additional oligonucleotide primer, and is complementary to a sequence comprising a non-subject sequence or a sequence within 200 nucleotides of a non-subject sequence.

20. A method of enriching a plurality of different target polynucleotides in a sample, the method comprising:

(a) hybridizing a plurality of different oligonucleotide primers to the target polynucleotides, wherein each oligonucleotide primer comprises sequence Z and sequence W;

wherein sequence Z is common among all oligonucleotide primers; and

further wherein sequence W is different for each different oligonucleotide primer, is positioned at the 3′

end of each oligonucleotide primer, and is complementary to a sequence comprising a causal genetic variant or a sequence within 200 nucleotides of a causal genetic variant;

(b) in an extension reaction, extending the oligonucleotide primers along the target polynucleotides to produce extended primers;

(c) joining an adapter oligonucleotide to each extended primer, wherein the adapter oligonucleotide comprises sequence Y′

, and further wherein sequence Y′

is the complement of a sequence Y; and

(d) exponentially amplifying the purified extension products using a pair of amplification primers comprising (i) a first amplification primer comprising sequence V and sequence Z, wherein sequence Z is positioned at the 3′

end of the first amplification primer; and

(ii) a second amplification primer comprising sequence X and sequence Y, wherein sequence Y is positioned at the 3′

end of the second amplification primer;

wherein sequences W, Y, and Z are different sequences and comprise 5 or more nucleotides each.

21. The method of claim 20, wherein each oligonucleotide primer comprises a first binding partner.

22. The method of claim 21, wherein the method further comprises, before step (d), exposing the extended primers to a solid surface comprising a second binding partner that binds to the first binding partner, thereby purifying the extended primers away from one or more components of the extension reaction.

23. The method of claim 20, wherein the plurality of oligonucleotide primers comprises at least about 100 different oligonucleotide primers each comprising a different sequence W.

24. The method of claim 20, wherein sequence W of one or more of the plurality of oligonucleotide primers comprises a sequence selected from the group consisting of SEQ ID NOs 22-121.

25. The method of claim 20, wherein the target polynucleotides comprise fragmented polynucleotides.

26. The method of claim 25, wherein the fragmented polynucleotides have a median length between 200 and 1000 base pairs.

27. The method of claim 20, wherein step (b) further comprises treating the extended primers and the target polynucleotides to which they are hybridized to produce blunt ends or to have a defined overhang prior to step (c).

28. The method of claim 27, wherein the defined overhang consists of an adenine.

29. The method of claim 22, wherein the first binding partner and the second binding partner are members of a binding pair.

30. The method of claim 29, wherein the binding pair is streptavidin and biotin.

31. The method of claim 22, wherein the solid surface is a bead.

32. The method of claim 31, wherein the bead is responsive to a magnetic field.

33. The method of claim 32, wherein the purifying step comprises application of a magnetic field to purify the beads.

34. The method of claim 22, wherein the extended primers are purified away from the target polynucleotides.

35. The method of claim 20, further comprising sequencing the products of step (d).

36. The method of claim 35, wherein sequencing comprises amplifying the products of step (d) by bridge amplification with bound oligonucleotides attached to a solid support to produce double-stranded bridge polynucleotides;

cleaving one strand of a bridge polynucleotide at a cleavage site in a bound oligonucleotide;

denaturing the cleaved bridge polynucleotide to produce a free single-stranded polynucleotide comprising a target sequence attached to the solid support; and

sequencing the target sequence by extending a sequencing primer hybridized to at least a portion of one or more sequences added during one or more of steps (b), (c), or (d).

37. The method of claim 35, wherein sequencing comprises amplifying the products of step (d) by extension of a bound primer on a solid support to produce bound templates, hybridizing a sequencing primer to a bound template, extending the sequencing primer, and identifying nucleotides added by extension of the sequencing primer.

38. The method of claim 20, wherein the plurality of different oligonucleotide primers further comprises additional oligonucleotide primers comprising sequence Z and sequence W, wherein sequence W is different for each different additional oligonucleotide primer, is at the 3′

end of each additional oligonucleotide primer, and is complementary to a sequence comprising a non-subject sequence or a sequence within 200 nucleotides of a non-subject sequence.