Methods of sample preparation

• US 9,968,901 B2
• Filed: 05/21/2013
• Issued: 05/15/2018
• Est. Priority Date: 05/21/2012
• Status: Active Grant

• Current Assignee: The Scripps Research Institute
• Original Assignee: The Scripps Research Institute
• Inventors: Head, Steven Robert, Ordoukhanian, Phillip T., Salomon, Daniel R.
• Primary Examiner(s): Zhang, Kaijiang
• Application Number:
  US14/549,411
• Publication Number:
  US 20150265995A1
• Time in Patent Office: 1,820 Days
• Field of Search: None
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/0059 Sequential processes
• CPC Class Codes: B01J 2219/00709 Type of synthesis
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2563/143 Magnetism, e.g. magnetic label
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 50/06 Biochemical methods, e.g. using enzymes or whole viable microorganisms

Abstract

The present disclosure provides methods, compositions, and kits for methods that can improve techniques nucleic acid analysis, and can allow for more reliable and accurate targeted, multiplexed, high throughput sequencing. The methods, compositions, and kits can be used for sequencing target loci of nucleic acid. The methods, compositions, and kits disclosed herein can be used for assisted de novo targeted sequencing. The methods, compositions, and kits disclosed herein can also be used for library labeling for de novo sequencing and phasing.

Claims

1. A sequencing library nucleic acid comprising:

a first nucleic acid strand comprising;

a 5′

sequence comprising at least 6 random bases contributed by a first strand donor primer that anneals to a nucleic acid sample sequence with at least one mismatch,

a 3′

sequence comprising a fragment of the nucleic acid sample sequence, wherein the fragment comprises at least 200 bp,

a 3′

terminal end that cannot support strand extension; and

at least one non-nucleic acid affinity tag bound at the 3′

terminal position of the first nucleic acid strand;

a second nucleic acid strand, comprising a second strand oligo comprising at least 6 random bases contributed by a second strand donor primer that anneals to the first nucleic acid strand with at least one mismatch,

wherein the second nucleic acid strand 3′

end is annealed to the first nucleic acid strand at an internal position on the first strand, so as to be configured to prime synthesis complementary to the first strand.

2. The composition sequencing library nucleic acid of claim 1, wherein the first nucleic acid strand is terminated by incorporation of a biotin-tagged ddNTP at the 3′

terminal position.

3. The composition sequencing library nucleic acid of claim 1, wherein the affinity tag comprises biotin bound to a dideoxy moiety at the 3′

end of the first nucleic acid strand, and wherein the biotin is bound to a streptavidin moiety.

4. The composition sequencing library nucleic acid of claim 1, wherein the second strand oligo comprises an adapter sequence region.

5. The sequencing library nucleic acid of claim 1, wherein the second strand oligo is shorter than the first strand.

6. The sequencing library nucleic acid of claim 1, wherein the second strand at least partially anneals within the 3′

half of the first strand.

7. The sequencing library nucleic acid of claim 1, wherein the second strand at least partially anneals within 20 bases of the first strand 3′

end.

8. The sequencing library nucleic acid of claim 1, wherein the second strand is extended via a DNA polymerase to form a complement of the 5′

end of the first strand.

9. The sequencing library nucleic acid of claim 8, wherein the DNA polymerase is a DNA polymerase having strand-displacement activity.